IL-1 BINDING PROTEINS AND PHARMACEUTICAL COMPOSITION INCLUDING SUCH BINDING PROTEINS

专利摘要:
IL-1 binding proteins. proteins that bind to il-1(alpha) and il-1(beta) are described, along with their use in compositions and methods to treat, prevent and diagnose disorders related to il-1 and to detect il-1(alpha) and il-1(beta) in cells, tissues, samples and compositions.
公开号:BR112012031071B1
申请号:R112012031071-2
申请日:2011-05-13
公开日:2021-08-10
发明作者:Chung-Ming Hsieh；Tariq Ghayur；Chegbin Wu；Dominic J. Ambrosi
申请人:Abbvie Inc；
IPC主号:

专利说明:

Cross-reference to related patent applications
[001]This patent application claims the priority benefit thereof to US Provisional Patent Application No. 61/334 917, filed May 14, 2010, and US Provisional Patent Application No. 61/425 701, filed May 21, December 2010, the contents of which are incorporated herein in their entirety by reference in this patent application. field of invention
[002] The present invention relates to IL-1 binding proteins and specifically to their uses in the prevention and/or treatment of acute and chronic immunological diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, multiple sclerosis and other diseases autoimmune. Background of the invention
[003] Cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), are molecules produced by a variety of cells, such as monocytes and macrophages, which are mediators of inflammatory processes. Interleukin-1 is a cytokine with a wide range of biological and physiological effects, including fever, prostaglandin synthesis (in, for example, fibroblasts, muscle cells and endothelial cells), T lymphocyte activation, and interleukin-2 production.
[004] The original members of the IL-1 superfamily are IL-1α, IL-1β and the IL-1 Receptor antagonist (IL-1Ra, IL-1RA, IL-1ra, IL-1Rα). IL-1α and IL-1β are pro-inflammatory cytokines involved in the immune defense against infection. IL-1Rα is a molecule that competes with IL-1α and IL-1β for binding to the receptor, blocking their role in immune activation. In recent years, the addition of other molecules to the IL-1 superfamily has been seen, including IL-18 (see Dinarello et al., FASEB J., 8(15):1314-3225 (1994); Huising et al., Dev. Comp. Immunol., 28(5):395-413 (2004)) and six more genes with structural homology to IL-1α, IL-1β or IL-1RA. These last six members are termed IL1F5, IL1F6, IL1F7, IL1F8, IL1F9 and IL1F10. Accordingly, IL-1α, IL-1β and IL-1RA have been renamed IL-1F1, IL-1F2 and IL-1F3, respectively (see Sims et al., Trends Immunol., 22(10): 536-537 (2001) ); Dunn et al., Trends Immunol., 22(10): 533-536 (2001)). A purported additional member of the IL-1 family has been described and named IL-33 or IL-1F11, although this name is not officially accepted in the HGNC gene family nomenclature database.
[005] Both IL-1α and IL-1β are produced by macrophages, monocytes and dendritic cells. Both are an important part of the body's inflammatory response to infection. These cytokines increase the expression of adhesion factors in endothelial cells in order to enable the transmigration of leukocytes, cells that fight pathogens, to sites of infection and re-establish the thermoregulatory center of the hypothalamus, leading to an increase in body temperature that is expressed in the form of. IL-1 is therefore called endogenous pyrogen. Increased body temperature helps the body's immune system fight infection. IL-1 is also important in the regulation of hemopoiesis. IL-1β production in peripheral tissues has also been associated with the hyperalgesia (increased sensitivity to pain) that accompanies fever (Morgan et al., Brain Res., 1022(1-2):96100 (2004)). Most of the time, these two forms of IL-1 bind to the same cell receptor. This receptor is composed of two related, but not identical, subunits that transmit intracellular signals through a pathway that is mostly shared with certain other receptors. These include the Toll family of innate immune receptors and the receptor for IL-18. IL-1α and IL-1β also have similar biological properties, including fever induction, slow wave sleep and neutrophilia, T and B lymphocyte activation, fibroblast proliferation, cytotoxicity to certain cells, collagenase induction, protein synthesis acute phase liver disease and increased production of colony and collagen stimulating factors.
[006]cDNAs encoding the two distinct forms of IL-1 were isolated and expressed; these cDNAs represent two different gene products, termed IL-1β (Auron et al., Proc. Natl. Acad. Sci. USA, 81: 7907-7911 (1984)) and IL-1α (Lomedico et al., Nature, 312 : 458-462 (1984)). IL-1β is the predominant form produced by human monocytes at both the mRNA and protein levels. The two forms of human IL-1 share only 26% amino acid homology. Despite their distinct polypeptide sequences, the two forms of IL-1 have structural similarities (Auron et al., J. Mol. Cell Immunol., 2: 169-177 (1985)), in the sense that amino acid homology is low. restricted to differentiated regions of the IL-1 molecule.
[007]IL-1α and IL-1β are produced in the form of precursor peptides. In other words, both are made up of a long protein that is then processed, releasing a shorter active molecule, which is called the mature protein. Mature IL-1β, for example, is released from Pr0-IL-1β after cleavage by a particular member of the protein family of caspases, called caspase-1 or interleukin-1 converting enzyme (ICE). The three-dimensional structure of the mature forms of each member of the human IL-1 superfamily is composed of 12 - 14 β-strands that produce a barrel-shaped protein.
[008] Although a variety of antibodies against IL-1 have been described in the last two decades of work since the discovery of this fundamental pro-inflammatory cytokine, there is still a need to improve antibodies that are capable of effectively mediating or neutralizing the activity of IL- 1 in the inflammatory response and in autoimmune disorders and for use in detecting IL-1β in samples and tissues. Invention Summary
[009] This invention to proteins that bind to human IL-1α and IL-1β. The binding proteins of the invention include, among others, antibodies, antigen-binding portions thereof, and multivalent, multispecific binding proteins such as DVD-Ig™ binding proteins that are capable of binding to IL-1α and IL- 1β human. The invention also provides methods for producing and using the IL-1α and IL-1β binding proteins described herein, as well as various compositions that can be used in methods to detect IL-1α and IL-1β in a sample or in methods to treat or prevent a disorder in an individual that is associated or suspected to be associated with IL-1 activity.
[010] In one embodiment, the invention provides a binding protein comprising a first and a second polypeptide chain, wherein said first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, where: VD1 is a first heavy chain variable domain; VD2 is a second heavy chain variable domain; C is a heavy chain constant domain; X1 is a linker with the proviso that it is not CH1; X2 is an Fc region; and n is independently 0 or 1; and wherein said second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a first light chain variable domain; VD2 is a second light chain variable domain; C is a light chain constant domain; X1 is a linker with the proviso that it is not CH1; X2 does not comprise an Fc region; and n is independently 0 or 1; wherein, in said first polypeptide chain, VD1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-148, 196, 198, 200, 202, 204, 206, 208 and 210; and VD2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213 and 227; wherein, in said second polypeptide chain, VD1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 149-189, 197, 199, 201, 203, 205, 207, 209 and 211; and VD2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 216 and 229; and wherein the binding protein binds to human IL-1β and human IL-1a.
[011] In one embodiment, a binding protein described above comprises a first polypeptide chain that contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 212, 217, 226, 230, 232, 234 and 236.
[012] In another embodiment, a binding protein described above comprises a second polypeptide chain that contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 215, 218, 228, 231, 233, 235 and 237.
[013] In another aspect of the invention, a binding protein comprises a first and a second polypeptide chain, wherein said first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein : VD1 is a first heavy chain variable domain; VD2 is a second heavy chain variable domain; C is a heavy chain constant domain; X1 is a linker with the proviso that it is not CH1; X2 is an Fc region; and n is independently 0 or 1; and wherein said second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a first light chain variable domain; VD2 is a second light chain variable domain; C is a light chain constant domain; XI is a linker with the proviso that it is not CH1; X2 does not comprise an Fc region; and n is independently 0 or 1; wherein, in said first polypeptide chain, VD1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213 and 227; and VD2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-148, 196, 198, 200, 202, 204, 206, 208 and 210; wherein, in said second polypeptide chain, VD1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 216 and 229; and VD2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 149-189, 197, 199, 201, 203, 205, 207, 209 and 211; and wherein the binding protein binds to human IL-1β and human IL-1a.
[014] In another embodiment, a binding protein described above comprises a first polypeptide chain that contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 219 and 221.
[015] In another embodiment, a binding protein described above comprises a second polypeptide chain that contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 220 and 222.
[016] In another aspect, the invention provides a binding protein described above, wherein: when said first polypeptide chain includes the amino acid sequence of SEQ ID NO:212, then said second polypeptide chain includes a selected amino acid sequence from the group consisting of SEQ ID NOs: 215, 228, 231, 233 and 235; when said first polypeptide chain includes the amino acid sequence of SEQ ID NO:217, then said second polypeptide chain includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 218 and 237; when said first polypeptide chain includes the amino acid sequence of SEQ ID NO:219, then said second polypeptide chain includes the amino acid sequence of SEQ ID NO:220; when said first polypeptide chain includes the amino acid sequence of SEQ ID NO:221, then said second polypeptide chain includes SEQ ID NO:222; when said first polypeptide chain includes the amino acid sequence of SEQ ID NO:226, then said second polypeptide chain includes an amino acid sequence selected from the group consisting of SEQ ID NOs:228, 215, 231, 233 and 235; when said first polypeptide chain includes the amino acid sequence of SEQ ID NO:230, then said second polypeptide chain includes an amino acid sequence selected from the group consisting of SEQ ID NOs:231, 215, 228, 233 and 235; when said first polypeptide chain includes the amino acid sequence of SEQ ID NO:232, then said second polypeptide chain includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 233, 215, 228, 231 and 235; when said first polypeptide chain includes an amino acid sequence of SEQ ID NO:234, then said second polypeptide chain includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 235, 215, 228, 231 and 233; and when said first polypeptide chain includes the amino acid sequence of SEQ ID NO:236, then said second polypeptide chain includes SEQ ID NO:237.
[017] In another aspect, the invention provides a protein described above, wherein: said first polypeptide chain includes SEQ ID NO:212 and said second polypeptide chain includes SEQ ID NO:215; or said first polypeptide chain includes SEQ ID NO:217 and said second polypeptide chain includes SEQ ID NO:218; or said first polypeptide chain includes SEQ ID NO:219 and said second polypeptide chain includes SEQ ID NO:220; or said first polypeptide chain includes SEQ ID NO:221 and said second polypeptide chain includes SEQ ID NO:222; or said first polypeptide chain includes SEQ ID NO:226 and said second polypeptide chain includes SEQ ID NO:228; or said first polypeptide chain includes SEQ ID NO:230 and said second polypeptide chain includes SEQ ID NO:231; or said first polypeptide chain includes SEQ ID NO:232 and said second polypeptide chain includes SEQ ID NO:233; or said first polypeptide chain includes SEQ ID NO:234 and said second polypeptide chain includes SEQ ID NO:235; or said first polypeptide chain includes SEQ ID NO:236 and said second polypeptide chain includes SEQ ID NO:237. In one embodiment, a binding protein of the invention described above comprises two first polypeptide chains and two second polypeptide chains.
[018] In another aspect, in a binding protein described above, X1 or X2 is an amino acid sequence selected from the group consisting of SEQ ID NOs:26-57, 233, 224 and 225.
[019] In another embodiment, the invention provides a binding protein described above, wherein the Fc region is selected from the group consisting of a native sequence Fc region and a variant sequence Fc region. In another embodiment, the Fc region is selected from the group consisting of an Fc region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.
[020] In another embodiment, the invention provides a conjugated binding protein which includes a binding protein described above and which further comprises an agent. These agents include, but are not limited to, immunoadhesion molecule, imaging agent, therapeutic agent, and cytotoxic agent. Preferred imaging agents include, but are not limited to, radiolabel, enzyme, fluorescent label, luminescent label, bioluminescent label, magnetic label and biotin. Preferred radiolabels include, among others, 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho and 153Sm. A therapeutic or cytotoxic agent includes, but is not limited to, anti-metabolite, alkylating agent, antibiotic, growth factor, cytokine, anti-angiogenic agent, anti-mitotic agent, anthracycline, toxin, and apoptotic agent.
[021] The invention also provides a binding protein containing an antigen-binding domain, wherein the binding protein is capable of binding to human IL-1β, and the antigen-binding domain comprises six CDRs, i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 as defined below: CDR-H1: Xi-YDMS (SEQ ID NO:190), wherein; XII is S, K or R; CDR-H2: Y-X2-S-X4-G-G-X7-G-T-Y-Y-P-D-X14-X15-K-G (SEQ ID NO:191), wherein; X2 is I or V; X4 is S or H; X7 is G or A; X14 is T or S; and X15 is V or A; CDR-H3: G-G-V-X4-K-G-X7-F-D-X10 (SEQ ID NO: 192), wherein; X4 is T or Y; X7 is Y or C; and X10 is V, E, L, M, Q or Y; CDR-L1: R-A-S-G-N-I-X7-X8-X9-L-X11 (SEQ ID NO:193), wherein; X7 is H, Y or W; X8 is N, G, T, Q, E, H, D or K; X9 is Y or W; and XIII is T, A or N; CDR-L2: X1-A-K-X4-L-X6-X7 (SEQ ID NO:194), wherein; X1 is N, Q or D; X4 is T, N, I, E or S; X6 is A, M or E; and X7 is D, E, S or A; and CDR-L3: Q-X2-F-W-X5-X6-P-X8-X9 (SEQ ID NO:195), wherein; X2 is H or Q; X5 is S, N, T, K, R or M; X6 is I or L; X8 is Y or A; and X9 is T, I and N; Except when CDR-H1 is S-Y-D-M-S (SEQ ID NO:17), then: CDR-H2 cannot be Y-I-S-S-G-G-G-G-T-Y-Y-P-D-T-V-K-G (SEQ ID NO:18); CDR-H3 cannot be G-G-V-T-K-G-Y-F-D-V (SEQ ID NO:19); CDR-L1 cannot be R-A-S-G-N-I-H-N-Y-L-T (SEQ ID NO:20); CDR-L2 cannot be N-A-K-T-L-A-D (SEQ ID NO:21); and CDR-L3 cannot be Q-H-F-W-S-I-P-Y-T (SEQ ID NO:22).
[022] In one embodiment, an isolated binding protein described above contains at least one CDR that includes an amino acid sequence selected from the group consisting of CDR sequences consisting of:












[023] In another embodiment, a binding protein described above contains at least three CDRs, wherein the three CDRs comprise a set of CDRs selected from the group of CDR sets consisting of:
























[024]In one embodiment, a binding protein described above contains CDRs from two sets of CDRs selected from the above group of CDR sets.
[025] In another embodiment, the invention provides a binding protein containing CDRs from two sets of CDRs from the above group, in which the two sets of CDRs are selected from the group consisting of:





[026] In another embodiment, a binding protein described above further comprises a human acceptor framework. Preferably, the human framework contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-10, 13-16, 25, 240-316 and 317-381. In one embodiment, a binding protein of the invention comprises a human framework sequence selected from the group consisting of SEQ ID NOS:7-10 and 13-16.
[027] The binding proteins of the invention include those comprising a human acceptor framework containing at least one amino acid substitution in the Framework Region, wherein the amino acid sequence of the framework is at least 65% identical to the sequence of said human acceptor framework and includes at least 70 amino acid residues identical to those of said human acceptor framework.
[028] In another embodiment, a binding protein of the invention comprises a human acceptor framework, wherein said acceptor framework includes at least one amino acid substitution in the framework region at a fundamental residue, said fundamental residue selected from the group consisting of residue adjacent to a CDR; glycosylation site residue; rare residue; residue capable of interacting with human IL-1β; residue capable of interacting with a CDR; canonical residue; contact residue between the heavy chain variable region and the light chain variable region; residue inserted in a Vernier zone; and residue in a region that overlaps between a heavy chain variable CDR1 according to the Chothia definition and a first heavy chain scaffold according to the Kabat definition. In an exemplary embodiment, a binding protein of the invention comprises a key residue, wherein said key residue is selected from the group consisting of: 2H, 4H, 24H, 26H, 27H, 29H, 34H, 35H, 37H, 39H , 44H, 45H, 47H, 48H, 49H, 50H, 51H, 58H, 59H, 60H, 63H, 67H, 69H, 71H, 73H, 76H, 78H, 91H, 93H, 94H, 2L, 4L, 25L, 29L, 27bL , 33L, 34L, 36L, 38L, 43L, 44L, 46L, 47L, 48L, 49L, 55L, 58L, 62L, 64L, 71L, 87L, 89L, 90L, 91L, 94L, 95L (all according to the numbering of Kabat). An exemplary subset of these residues for the humanization of a binding protein of the invention consists of 27H, 48H, 67H, 69H, 93H, 36L, 43L, 46L, 47L, 49L, 58L, 71L and 87L.
[029] In another embodiment, a binding protein of the invention comprises a human consensus variable domain.
[030] In one embodiment, an IL-1β binding protein of the invention comprises at least one variable domain containing an amino acid sequence selected from the group consisting of SEQ ID NOs:60-189.
[031] In another embodiment, a binding protein of the invention comprises at least one heavy chain variable region (or domain) (VH) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-148, 196 , 198, 200, 202, 204, 206, 208 and 210.
[032] In another embodiment, a binding protein according to the invention comprises at least one light chain variable region (or domain) (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs:149- 189.
[033] In yet another embodiment, a binding protein according to the invention comprises at least one VH region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-148, 196, 198, 200, 202 , 204, 206, 208 and 210 and at least one VL region containing an amino acid sequence selected from the group consisting of SEQ ID NOs:149-189, 197, 199, 201, 203, 205, 207, 209 and 211.
[034] In one embodiment, a binding protein according to the invention comprises two variable domains, wherein the two variable domains contain amino acid sequences selected from the group consisting of:


[035] In another embodiment, an IL-1β binding protein described herein is selected from the group consisting of: immunoglobulin molecule, scFv, monoclonal antibody, humanized antibody, chimeric antibody, humanized antibody, Fab fragment, Fab' fragment , F(ab')2, Fv and disulfide-linked FV.
[036] In one aspect of the invention, a binding protein described herein is capable of modulating a biological function of IL-1. In another aspect, a binding protein described herein is capable of neutralizing IL-1.
[037] In one embodiment, a binding protein described herein has a constant for the rate of association (Kon) to IL-1β selected from the group consisting of: at least around 102M-1s-1; at least around 103M-1s-1; at least around 104M-1s-1; at least around 105M-1s-1; and at least around 106M-1s-1; as measured by surface plasmon resonance.
[038] In another modality, a binding protein described here has a constant for the dissociation rate (Koff) of IL-1β selected from the group consisting of: at most around 10-3s-1; at most around 10-4s-1; at most around 10-5s-1; and at most around 10-6s-1, as measured by surface plasmon resonance.
[039] In another modality, a binding protein described here has a dissociation constant (KD) of IL-1β selected from the group consisting of: at most around 10-7 M; at most around 10-8 M; at most around 10-9 M; at most around 10-10 M; at most around 10-11 M; at most around 10-12 M; and at most 10-13 M.
[040] In one aspect, the invention provides a constructed binding protein which consists of a binding protein described herein and which further comprises a linker or an immunoglobulin constant domain. In one embodiment, the constructed binding protein comprises a binding protein, wherein the binding protein is selected from the group consisting of: immunoglobulin molecule, disulfide-linked Fv, monoclonal antibody, scFv, chimeric antibody, grafted antibody CDR, diabody, humanized antibody, multispecific antibody, Fab, dual specific antibody, Fab', bispecific antibody and F(ab')2, DVD-Ig™ and Fv.
[041] In one embodiment, a constructed binding protein comprises an immunoglobulin heavy chain constant domain, selected from the group consisting of human IgM constant domain, human IgG4 constant domain, human IgG1 constant domain, human IgG1 constant domain. Human IgE, human IgG2 constant domain, human IgG3 constant domain, and human IgA constant domain.
[042] In another embodiment, a binding protein constructed comprises an immunoglobulin constant domain having an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
[043] The invention also provides a conjugated binding protein comprising a constructed binding protein and additionally an agent selected from the group consisting of immunoadhesion molecule, imaging agent, therapeutic agent and cytotoxic agent. Imaging agents that are useful as agent clusters in conjugated binding proteins described herein include, but are not limited to, radiolabel, enzyme, fluorescent label, luminescent label, bioluminescent label, magnetic label, and biotin. In one modality, a radiolabel is selected from the group consisting of: 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho and 153Sm.
[044] In another embodiment, a conjugated binding protein comprises an agent that is a therapeutic or cytotoxic agent selected from the group consisting of: anti-metabolite, alkylating agent, antibiotic, growth factor, cytokine, anti-angiogenic agent, agent anti-mitotic, anthracycline, toxin and apoptotic agent.
[045] In one embodiment, a binding protein, a constructed binding protein, or a conjugated binding protein described herein has a human glycosylation pattern.
The binding proteins, constructed binding proteins and conjugated binding proteins described herein may exist in the form of soluble proteins or in the form of crystals. In one embodiment, these crystals are carrier-free controlled-release pharmaceutical crystals. In another embodiment, a crystal form of a binding protein, constructed binding protein, or conjugated binding protein described herein has a longer in vivo half-life than its soluble version. In another embodiment, a crystal of a binding protein, constructed binding protein or conjugated binding protein described herein retains the biological activity of its soluble version.
[047] Compositions of the invention include a composition for the release of a binding protein, constructed binding protein or conjugated binding protein described herein in crystallized form, comprising: (a) a formulation, wherein said formulation comprises a protein of binding, constructed binding protein or conjugated binding protein described herein in crystallized form and an ingredient; and (b) at least one polymeric carrier.
[048] Polymeric carriers useful in compositions of the invention include, among others, one or more from the group consisting of: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutyrate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly (hydroxypropyl) methacrylamide, poly [(organo) phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof.
[049] In another aspect, an ingredient of a composition of the invention is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.
[050] The invention also provides pharmaceutical compositions containing a binding protein, a constructed binding protein or conjugated binding protein described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions of the invention can further comprise at least one additional agent. In one embodiment, this additional agent includes, but is not limited to, therapeutic agent, imaging agent, cytotoxic agent, angiogenesis inhibitors; kinase inhibitors; blockers of costimulation molecules; adhesion molecule blockers; anti-cytokine antibody or functional fragment thereof; methotrexate; cyclosporine; rapamycin; FK506; detectable marker or reporter; TNF antagonist; antirheumatic; muscle relaxant, narcotic, non-steroidal anti-inflammatory drug (NSAID), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, antimicrobial, antipsoriatic, corticosteroid, anabolic steroid, erythropoietin, immunization, immunoglobulin, immunosuppressant, growth hormone, drug hormone replacement, radiopharmaceutical, antidepressant, antipsychotic, stimulant, asthma medication, beta agonist, inhaled steroid, epinephrine or analogue, cytokine and cytokine antagonist.
[051] In one embodiment, a pharmaceutical composition of the invention comprises a pharmaceutically acceptable carrier, wherein the carrier also serves as an adjuvant to enhance the absorption or dispersion of the binding protein, constructed binding protein or the conjugated binding protein in the composition . An exemplary adjuvant is hyaluronidase.
[052] In another embodiment, a pharmaceutical composition further comprises at least one additional therapeutic agent to treat a disorder in which IL-1β activity is detrimental.
[053] In one embodiment, the invention provides isolated nucleic acids that encode one or more amino acid sequences of a binding protein described herein. Such nucleic acids can be inserted into a vector to perform various genetic analyzes or to express, characterize or improve one or more properties of a binding protein described herein. A vector may comprise one or more nucleic acid molecules that encode one or more amino acid sequences of a binding protein described herein, wherein the single or more nucleic acid molecules are operably linked to appropriate transcriptional and/or translational sequences. that allow the expression of the binding protein in a particular host cell carrying the vector. Examples of vectors for cloning or expressing nucleic acids that encode binding protein amino acid sequences described herein include, but are not limited to, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.
[054] The invention also provides a host cell containing a vector that comprises a nucleic acid encoding one or more amino acid sequences of a binding protein described herein. Host cells useful in the invention can be prokaryotic or eukaryotic. An exemplary prokaryotic host cell is Escherichia coli. Eukaryotic cells useful as host cells in the invention include protist cells, animal cells, plant cells or fungal cells. An example of a fungal cell is a yeast cell, including Saccharomyces cerevisiae. An example of an animal cell useful as a host cell according to the invention includes, but is not limited to, mammalian cell, bird cell and insect cell. Preferred mammalian cells include CHO and COS cells. An insect cell useful as a host cell in accordance with the invention is an Sf9 insect cell.
[055] In another aspect, the invention provides a method for producing a binding protein described herein, comprising culturing a host cell containing a vector encoding the binding protein in culture medium under conditions sufficient to produce the binding protein capable of bind to IL-1a and/or IL-1β. The protein so produced can be isolated and used in the various compositions and methods described herein.
[056] In another embodiment, the invention provides a method for reducing the activity of human IL-1, comprising contacting IL-1 with a binding protein described herein, such that the activity of human IL-1 is reduced .
[057] Another embodiment of the invention provides a method of treating an individual with a disorder by administering to the individual a binding protein described herein such that treatment is achieved.
[058] In another embodiment, a binding protein described herein is useful to treat a disorder selected from the group consisting of: diabetes; uveitis; neuropathic pain; osteoarthritic pain; inflammatory pain; rheumatoid arthritis; osteoarthritis; juvenile chronic arthritis; septic arthritis; Lyme arthritis; psoriatic arthritis; reactive arthritis; spondyloarthropathy; systemic lupus erythematosus (SLE); Crohn's disease; ulcerative colitis; inflammatory bowel disease; autoimmune diabetes; insulin dependent diabetes mellitus; thyroiditis; asthma; allergic diseases; psoriasis; dermatitis; scleroderma; graft versus host disease; transplant organ rejection; acute immune disease associated with organ transplantation; chronic immune disease associated with organ transplantation; sarcoidosis; atherosclerosis; disseminated intravascular coagulation (DIC); Kawasaki disease; Grave's disease; nephrotic syndrome; chronic fatigue syndrome; Wegener's granulomatosis; Henoch-Schoenlein purple; microscopic renal vasculitis; active chronic hepatitis; autoimmune uveitis; septic shock; toxic shock syndrome; septicemic syndrome; cachexia; infectious diseases; parasitic diseases; acute transverse myelitis; Huntington's chorea; Parkinson's disease; Alzheimer's disease; stroke; primary biliary cirrhosis; hemolytic anemia; malignancies; cardiac insufficiency; myocardial infarction; Addison's disease; type I sporadic polyglandular deficiency; type II polyglandular deficiency (Schmidt's syndrome); acute respiratory distress syndrome (ARDS); alopecia; alopecia areata; seronegative arthropathy; arthropathy; Reiter's disease; psoriatic arthropathy; arthropathy in ulcerative colitis; enteropathic synovitis; arthropathy associated with chlamydia infection; Yersinia and Salmonella; spondyloarthropathy; atheromatous disease/arteriosclerosis; atopic allergy; autoimmune bullous disease; pemphigus vulgaris; pemphigus foliaceus; pemphigoid; linear IgA disease; autoimmune hemolytic anemia; Coombs positive hemolytic anemia; acquired pernicious anemia; juvenile pernicious anemia; myalgic encephalitis/Royal Free disease; chronic mucocutaneous candidiasis; giant cell arteritis (GCA); primary sclerosing hepatitis; autoimmune cryptogenic hepatitis; acquired immunodeficiency syndrome (AIDS); diseases related to acquired immunodeficiency; Hepatitis B; hepatitis C; common variable immunodeficiency (common variable hypogammaglobulinemia); dilated cardiomyopathy; female infertility; ovarian failure; premature ovarian failure; fibrotic lung disease; cryptogenic fibrosing alveolitis; post-inflammatory interstitial lung disease; interstitial pneumonitis; interstitial lung disease associated with connective tissue disease; pulmonary disease associated with mixed connective tissue disease; interstitial lung disease associated with systemic sclerosis; interstitial lung disease associated with rheumatoid arthritis; pulmonary disease associated with systemic lupus erythematosus; lung disease associated with dermatomyositis/polymyositis; lung disease associated with Sjogren's syndrome; lung disease associated with ankylosing spondylitis; diffuse pulmonary vasculitis; pulmonary disease associated with hemosiderosis; drug-induced interstitial lung disease; fibrosis; radiation fibrosis; bronchiolitis obliterans; chronic eosinophilic pneumonia; lymphocytic infiltrative lung disease; post-infectious interstitial lung disease; gouty arthritis; autoimmune hepatitis; type 1 autoimmune hepatitis (classic or lupoid autoimmune hepatitis); type 2 autoimmune hepatitis (hepatitis characterized by the presence of anti-LKM antibodies); autoimmunity-mediated hypoglycemia; type B insulin resistance associated with acanthosis nigricans; hypoparathyroidism; osteoarthritis; primary sclerosing cholangitis; type 1 psoriasis; type 2 psoriasis; idiopathic leukopenia; autoimmune neutropenia; kidney disease NOS; glomerulonephritis; microscopic renal vasculitis; Lyme disease; discoid lupus erythematosus; idiopathic male infertility; male infertility associated with nitric oxide; autoimmunity against sperm; multiple sclerosis (all subtypes, including primary progressive, secondary progressive, relapsing-remitting); sympathetic ophthalmia; pulmonary hypertension secondary to connective tissue disease; Goodpasture's syndrome; pulmonary manifestation of polyarteritis nodosa; acute rheumatic fever; rheumatoid spondylitis; Still's disease; systemic sclerosis; Sjorgren's syndrome; Takayasu's disease/arteritis; autoimmune thrombocytopenia (AITP); idiopathic thrombocytopenia; autoimmune thyroid disease; hyperthyroidism; autoimmune hypothyroidism with goiter (Hashimoto's disease); autoimmune atrophic hypothyroidism; primary myxedema; phacogenic uveitis; primary vasculitis; vitiligo; acute liver disease; chronic liver disease; alcoholic cirrhosis; alcohol-induced liver damage; cholestasis; idiosyncratic liver disease; drug-induced hepatitis; non-alcoholic steatohepatitis; allergy; group B Streptococci (GBS) infection; mental disorders (eg depression and schizophrenia); Type Th2 and Type Th1 lymphocyte-mediated diseases; acute and chronic pain (different forms of pain); cancer (such as lung, breast, stomach, bladder, colon, pancreas, ovary, prostate and rectal cancer); hematopoietic malignancies; leukemia; lymphoma; abetalipoproteinemia; acrocyanosis; acute and chronic parasitic or infectious processes; acute leukemia; acute lymphoblastic leukemia (ALL); ALL of T cells ALL; ALL FAB; acute myeloid leukemia (AML); acute or chronic bacterial infection; acute pancreatitis; acute renal failure; adenocarcinomas; atrial ectopic beats; AIDS dementia complex; alcohol-induced hepatitis; allergic conjunctivitis; allergic contact dermatitis; allergic rhinitis; allograft rejection; alpha-1 antitrypsin deficiency; amyotrophic lateral sclerosis; anemia; angina pectoris; anterior horn cell degeneration; anti-CD3 therapy; antiphospholipid syndrome; antireceptor hypersensitivity reactions; aortic and peripheral aneurysms; aortic dissection; arterial hypertension; arteriosclerosis; arteriovenous fistula; ataxia; atrial fibrillation (sustained or paroxysmal); atrial flutter; atrioventricular block; B-cell lymphoma; bone graft rejection; bone marrow transplant rejection (BMT); bundle branch blockage; Burkitt's lymphoma; burns; cardiac arrhythmias; cardiac stun syndrome; cardiac tumors; cardiomyopathy; inflammatory response in cardiopulmonary bypass surgery; cartilage transplant rejection; cortical cerebellar degenerations; cerebellar disorders; chaotic or multifocal atrial tachycardia; disorders associated with chemotherapy; chronic myelocytic leukemia (CML); chronic alcoholism; chronic inflammatory pathologies; chronic lymphocytic leukemia (CLL); chronic obstructive pulmonary disease (COPD); chronic salicylate poisoning; colorectal carcinoma; Congestive heart failure; conjunctivitis; contact dermatitis; pulmonale color; coronary artery disease; Creutzfeldt-Jakob disease; sepsis with negative culture; cystic fibrosis; disorders associated with cytokine therapy; boxer's dementia; demyelinating diseases; dengue hemorrhagic fever; dermatitis; dermatological conditions; diabetes mellitus; diabetic atherosclerotic disease; diffuse Lewy body disease; dilated congestive cardiomyopathy; basal ganglia disorders; Down syndrome in old age; movement disorders induced by drugs that block dopamine receptors in the central nervous system; drug sensitivity; eczema; encephalomyelitis; endocarditis; endocrinopathy; epiglottitis; Epstein-Barr virus infection; erythromelalgia; extrapyramidal and cerebellar disorders; familial hemophagocytic lymphohistiocytosis; fetal thymus implant rejection; Friedreich's ataxia; functional disorders of the peripheral arteries; fungal sepsis; gas gangrene; gastric ulcer; glomerular nephritis; rejection of grafts from any organ or tissue; gram negative sepsis; gram positive sepsis; granulomas due to intracellular organisms; hairy cell leukemia; Hallervorden-Spatz disease; Hashimoto's thyroiditis; hay fever; heart transplant rejection; hemochromatosis; hemodialysis; hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura; bleeding; hepatitis A; His bundle arrhythmias; HIV infection/HIV neuropathy; Hodgkin's disease; hyperkinetic movement disorders; hypersensitivity reactions; hypersensitivity pneumonitis; hypertension; hypokinetic movement disorders; assessment of the hypothalamic-pituitary-adrenal axis; idiopathic Addison's disease; idiopathic pulmonary fibrosis (IPF); antibody-mediated cytotoxicity; asthenia; infantile spinal muscular atrophy; inflammation of the aorta; influenza A; exposure to ionizing radiation; iridocyclitis/uveitis/optic neuritis; ischemia-reperfusion injury; ischemic stroke; juvenile rheumatoid arthritis; juvenile spinal muscular atrophy; Kaposi's sarcoma; kidney transplant rejection; legionellosis; leishmaniasis; leprosy; lesions of the corticospinal system; lipedema; liver transplant rejection; lymphedema; malaria; malignant lymphoma; malignant histiocytosis; malignant melanoma; meningitis; meningococemia; migraine-like headache associated with metabolic syndrome; idiopathic migraine-type headache; mitochondrial multisystem disease; mixed connective tissue disease; monoclonal gammopathy; multiple myeloma; degenerations of multiple systems (Menzel; Dejerine-Thomas; Shy-Drager; and Machado-Joseph); myasthenia gravis; Mycobacterium avium intracellulare infection; Mycobacterium tuberculosis infection; myelodysplastic syndrome; myocardial infarction; myocardial ischemic disorders; nasopharyngeal carcinoma; neonatal chronic lung disease; nephritis; nephrosis; neurodegenerative diseases; neurogenic muscle atrophies I; neutropenic fever; non-Hodgkin's lymphoma; occlusion of the abdominal aorta and its branches; occlusive arterial disorders; OKT3® therapy; orchitis/epididymitis; orchitis/vasectomy reversal procedures; organomegaly; osteoporosis; rejection of pancreas transplantation; pancreatic carcinoma; paraneoplastic syndrome/hypercalcemia of malignancy; rejection of parathyroid transplantation; pelvic inflammatory disease; perennial rhinitis; pericardial disease; peripheral atherosclerotic disease; peripheral vascular disorders; peritonitis; pernicious anemia; Pneumocystis carinii pneumonia; pneumonia; POEMS syndrome (syndrome characterized by polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes); post-perfusion syndrome; post-bomb syndrome; post-MI cardiotomy syndrome; pre eclampsia; progressive supranuclear palsy; primary pulmonary hypertension; radiotherapy; Raynaud's phenomenon; Raynaud's disease; Refsum's disease; regular tachycardia with narrow QRS; renovascular hypertension; reperfusion injury; restrictive cardiomyopathy; sarcomas; senile chorea; senile dementia of the Lewy bodies type; seronegative arthropathies; shock; sickle cell anemia; skin allograft rejection; skin disorder syndrome; small bowel transplant rejection; solid tumors; specific arrhythmias; spinal ataxia; spinocerebellar degenerations; streptococcal myositis; structural lesions of the cerebellum; subacute sclerosing panencephalitis; syncope; syphilis of the cardiovascular system; systemic anaphylaxis; systemic inflammatory response syndrome; systemic onset juvenile rheumatoid arthritis; telangiectasia; thromboangiitis obliterans; thrombocytopenia; toxicity; transplants; trauma/hemorrhage; type III hypersensitivity reactions; type IV hypersensitivity; unstable angina; uremia; urosepsis; heart valve diseases; varicose veins; venous diseases; venous thrombosis; ventricular fibrillation; virus and fungal infections; viral encephalitis/aseptic meningitis; virus-associated hemophagocytic syndrome; Wernicke-Korsakoff syndrome; Wilson's disease; rejection of xenografts from any organ or tissue; acute coronary syndromes; idiopathic acute polyneuritis; acute inflammatory demyelinating polyradiculoneuropathy; acute ischemia; Adult Still's disease; alopecia areata; anaphylaxis; anti-phospholipid antibody syndrome; aplastic anemia; arteriosclerosis; atopic eczema; atopic dermatitis; autoimmune dermatitis; autoimmune disorder associated with Streptococcus infection; autoimmune enteropathy; autoimmune hearing loss; autoimmune lymphoproliferative syndrome (ALPS); autoimmune myocarditis; autoimmune premature ovarian failure; blepharitis; bronchiectasis; bullous pemphigoid; cardiovascular disease; catastrophic antiphospholipid syndrome; celiac disease; cicatricial pemphigoid; clinically isolated syndrome (CIS) with risk for multiple sclerosis; conjunctivitis; childhood-onset psychiatric disorder; dacryocystitis; dermatomyositis; diabetic retinopathy; herniated disc; disc prolapse; drug-induced immune hemolytic anemia; endocarditis; endometriosis; endophthalmitis; episcleritis; erythema multiforme; erythema multiforme major; gestational pemphigoid; Guillan-Barré syndrome (GBS); hay fever; Hughes Syndrome; idiopathic Parkinson's disease; idiopathic interstitial pneumonia; IgE-mediated allergy; immune hemolytic anemia; inclusion body myositis; infectious eye inflammatory disease; inflammatory demyelinating disease; inflammatory heart disease; inflammatory kidney disease; iritis; keratitis; keratoconjunctivitis sicca; Kussmaul's disease or Kussmaul-Meier's disease; Landry's palsy; Langerhans cell histiocytosis; reticular livedo; macular degeneration; microscopic polyangiitis; Morbus Bechterev; motor neuron diseases; mucous membrane pemphigoid; multiple organ failure; myasthenia gravis; myelodysplastic syndrome; myocarditis; nerve root diseases; non-A non-B hepatitis; optic neuritis; osteolysis; pauciarticular JRA; peripheral arterial occlusive disease (PAOD); peripheral vascular disease (PVD); peripheral arterial disease (PAD); phlebitis; polyarteritis nodosa (or periarteritis nodosa); polychondritis; polymyalgia rheumatica; poliosis; polyarticular JRA; polyendocrine deficiency syndrome; polymyositis; polymyalgia rheumatica (PMR); post-pump syndrome; Primary Parkinsonism; Secondary Parkinsonism; prostatitis; pure red series aplasia; primary adrenal insufficiency; recurrent neuromyelitis optica; restenosis; rheumatic heart disease; SAPHO (synovitis, acne, pustulosis, hyperostosis and osteitis); secondary amyloidosis; shock lung; scleritis; sciatica pain; secondary adrenal insufficiency; connective tissue disease associated with silicone; Sneddon-Wilkinson dermatosis; ankylosing spondylitis; Stevens-Johnson syndrome (SJS); systemic inflammatory response syndrome; temporal arteritis; toxoplasmic retinitis; toxic epidermal necrolysis; transverse myelitis; TRAPS (Tumor Necrosis Factor Receptor Type 1 Associated Periodic Syndrome (TNFR)); type B insulin resistance associated with acanthosis nigricans; type 1 allergic reaction; type II diabetes; urticaria; usual interstitial pneumonia (UIP); vernal conjunctivitis; viral retinitis; Vogt-Koyanagi-Harada syndrome (VKH syndrome); wet macular degeneration; wound healing; arthropathy associated with infection by Yersinia and Salmonella.
[059] In a further embodiment of a method of treatment described herein, the step of administering to the subject a binding protein or constructed binding protein or conjugated binding protein described herein is at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intra-abdominal, intracapsular, intracartilaginous, intracavitary, intracellular, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intrapleural, intrapelvic, intrapericardial, intrapropular, intrapropular intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal and transdermal.
[060] Another aspect of the invention is a method for treating a patient suffering from a disorder in which IL-1 is detrimental, comprising the step of administering a binding protein, constructed binding protein or conjugated binding protein described herein above, simultaneously with or following administration of a second agent, wherein the second agent is selected from the group consisting of inhalable steroids; beta-agonists; short-acting or long-acting beta-agonists; leukotriene antagonists or leukotriene receptors; ADVAIR; IgE inhibitors; anti-IgE antibodies; XOLAIR; phosphodiesterase inhibitors; PDE4 inhibitors; xanthines; anticholinergic drugs; mast cell stabilizing agents; Chromoline; IL-4 inhibitors; IL-5 inhibitors; eotaxin/CCR3 inhibitors; antagonists of histamine or its receptors including H1, H2, H3 and H4; antagonists of prostaglandin D or its DP1 and CRTH2 receptors; TNF antagonists; soluble fragment of a TNF receptor; ENBREL®; enzymatic TNF antagonists; TNF-converting enzyme (TACE) inhibitors; muscarinic receptor antagonists; TGF-beta antagonists; interferon gamma; perfenidone; chemotherapeutic agents, methotrexate; leflunomide; sirolimus (rapamycin) or its analogue, CCI-779; COX2 or cPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors; TPL-2, MK-2 and NFkB inhibitors; budesonide; epidermal growth factor; corticosteroids; cyclosporine; sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1β antibodies; anti-IL-6 antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; antibodies or agonists of TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, EMAP-II, GM-CSF, FGF or PDGF; antibodies from CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands; FK506; rapamycin; mycophenolate mofetil; ibuprofen; prednisolone; phosphodiesterase inhibitors; adenosine agonists; antithrombotic agents; complement inhibitors; adrenergic agents; IRAK, NIK, IKK, p38 or MAP kinase inhibitors; IL-1β converting enzyme inhibitors; TNF-α converting enzyme inhibitors; T cell signaling inhibitors; metalloproteinase inhibitors; 6-mercaptopurines; angiotensin converting enzyme inhibitors; soluble cytokine receptors; Soluble p55 TNF receptor; soluble p75 TNF receptor; sIL-1RI; sIL-1RII; sIL-6R; anti-inflammatory cytokines; IL-4; IL-10; IL-11; and TGF-β.
[061] Another aspect of the invention provides at least one anti-IL-1 idiotype antibody against at least one IL-1 binding protein described herein. The anti-idiotype antibody includes any protein or peptide that contains a molecule comprising at least part of an immunoglobulin molecule, such as, among others, at least one heavy or light chain CDR or linker-binding part thereof, a variable region heavy chain or light chain, a heavy chain or light chain constant region, a framework region or any part thereof, which can be incorporated into a binding protein of the invention. Detailed description of the invention
[062] This invention pertains to IL-1β binding proteins, including, among others, anti-IL-1β antibodies or their antigen-binding parts, which bind to IL-1β and multivalent, multispecific binding proteins, such as as DVD-Ig™ which binds to IL-1β and another target. Various aspects of the invention relate to antibodies and antibody fragments, DVD-Ig binding proteins and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for producing such IL-binding proteins. 1β, including antibodies, DVD-Ig binding proteins and fragments thereof. Methods of using the IL-1/3 binding proteins of the invention to detect human IL-1/3; to inhibit human IL-1β, in vitro or in vivo; and to regulate gene expression are also encompassed by the invention.
[063] The invention also encompasses any binding protein or antibody capable of competing with the IL-1β binding protein described in this patent application.
[064] Unless otherwise defined in this specification, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, the definitions given here take precedence over any dictionary or extrinsic definition. Additionally, unless otherwise required by the context, singular terms will include pluralities, and plural terms will include the singular. In this patent application, the use of “or” means “and/or” unless otherwise stated. In addition, the use of the term "including" as well as other forms such as "includes" and "included" does not constitute a limitation. Additionally, terms such as "element" or "component" encompass both elements and components containing a unit and elements and components that contain more than one subunit unless specifically stated otherwise.
[065] In general, the nomenclatures and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization are those well known and commonly used in the prior art . The methods and techniques of the present invention are generally carried out in accordance with conventional methods well known in the art and as described in various general and more specific references which are cited and discussed throughout this specification, unless otherwise indicated. other way. Enzymatic reactions and purification techniques are carried out in accordance with the manufacturer's specifications, as commonly done in the prior art or as described in this patent application. The relatively used nomenclatures and laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry in this descriptive report are those well known and commonly used in the state of the art. Standard techniques are used for chemical syntheses, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient care.
[066] In order that the present invention can be understood more quickly, selected terms are defined below.
[067]The term "polypeptide" refers to any polymeric chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term "polypeptide" encompasses native or artificial proteins, protein fragments, and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric. The term "polypeptide" encompasses fragments and variants (including fragments of variants) thereof, unless contradicted by context. With respect to an antigenic polypeptide, a polypeptide fragment optionally contains at least one contiguous or non-linear epitope of the polypeptide. The precise limits of the at least single epitope fragment can be confirmed by means of common expertise in the prior art. The fragment comprises at least approximately 5 contiguous amino acids, such as at least approximately 10 contiguous amino acids, at least approximately 15 contiguous amino acids, or at least approximately 20 contiguous amino acids. A polypeptide variant is as described in this patent application.
[068]The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, by virtue of its origin or source of derivation, is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins of the same species; is expressed by a cell of a different species; or does not occur in nature. Consequently, a polypeptide, which is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates, will be “isolated” from its naturally associated components. A protein can also be made substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
[069] The term "recovery" refers to the process of rendering a chemical species, such as a polypeptide, substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
[070]The term "human IL-1α" (abbreviated in this specification as hIL-1α or IL-1α) includes a pleiotropic cytokine involved in various immune responses, inflammatory processes, and hematopoiesis. For example, IL-1α includes the human cytokine produced by active macrophages; stimulates thymocyte proliferation by inducing IL-2 release, B cell maturation and proliferation, and fibroblast growth factor activity. The term human IL-1α is intended to include IL- Recombinant human 1α (rh IL-1α) which can be prepared by standard recombinant expression methods.
[071]The term "human IL-1β" (abbreviated in this specification as hIL-1β or IL-1β) includes a pleiotropic cytokine involved in various immune responses, inflammatory processes, and hematopoiesis. The term human IL-1β includes Recombinant human IL-1β (rh IL-1β) which can be prepared by standard recombinant expression methods.
[072]The amino acid sequences of human IL-1α and IL-1β are shown in Table 1. Table 1.

The term "biological activity" refers to all inherent biological properties of the IL-1 cytokine, for example, IL-1α and/or IL-1β. The biological properties of IL-1α and IL-1β include, among others, binding to an IL-1 receptor.
[073] The terms "specific binding" "specifically binding", in reference to the interaction of an antibody, protein or peptide with a second chemical species, means that the interaction depends on the presence of a certain structure (eg, an antigenic determinant or epitope) in the chemical species; for example, an antibody generally recognizes and binds to a specific protein structure rather than to proteins. If an antibody is specific for the "A" epitope, the presence of a molecule containing the A (or free, unlabeled A) epitope in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to antibody.
[074] The term "antibody" refers broadly to any immunoglobulin (Ig) molecule, composed of four polypeptide chains, two heavy (H) chains and two light chains or any fragment, mutant, variant or functional derivative thereof , which retains the essential epitope-binding characteristics of an Ig molecule. Such mutant, variant, or antibody-derived formats are known in the art. Non-limiting modalities thereof are discussed below.
[075]In a complete antibody, each heavy chain is composed of a heavy chain variable region (abbreviated in this specification as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is made up of three domains: CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated in this specification as LCVR or VL) and a light chain constant region. The light chain constant region consists of a domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with regions that are more conserved, called framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgG 1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.
[076]The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which can be generated by papain digestion of an intact antibody. The Fc region can be native sequence Fc region or variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally includes a CH4 domain. Substitution of amino acid residues in the Fc portion to alter antibody effector function is known in the prior art (Winter et al., U.S. Patent Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions, eg, cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and the half-life/clearance rate of antibody and antigen-antibody complexes. In some cases, these effector functions are desirable for a therapeutic antibody, but in other cases they might be unnecessary or even harmful, depending on the therapeutic goals. Certain human IgG isotypes, especially IgG1 and IgG3, mediate ADCC and CDC by binding to FcRs and complement C1q, respectively. Neonatal Fc receptors (FcRn) represent the critical components that determine the circulating half-life of antibodies. In yet another embodiment at least one amino acid residue is substituted in the antibody constant region, e.g., the antibody Fc region, such that the antibody effector functions are altered. Dimerization of two identical heavy chains of an immunoglobulin is mediated by dimerization of the CH3 domains and is stabilized by disulfide bonds within the hinge region (Huber et al., Nature, 264: 415-420 (1976); Thies et al. al., J. Mol. Biol., 293: 67-79 (1999)). Mutation of cysteine residues within the hinge regions to prevent heavy chain-heavy chain disulfide bonds from forming will destabilize the dimerization of CH3 domains. Residues responsible for the dimerization of CH3 have been identified (Dall'Acqua et al., Biochemistry, 37: 9266-9273 (1998)). Therefore, it is possible to generate half-monovalent Ig. Interestingly, such monovalent half-Ig molecules have been found in nature for IgG and IgA subclasses ( Seligmann et al., Ann. Immunol., 129 C: 855-870 (1978); Biewenga et al., Clin. Exp. Immunol. , 51: 395-400 (1983)). The stoichiometry of FcRn:Ig Fc region has been determined to be 2:1 (West et al., Biochemistry, 39: 9698-9708 (2000)) and half Fc is sufficient to mediate binding to FcRn (Kim et al., Eur. J. Immunol., 24: 542-548 (1994)). Mutations that affect the dimerization of the CH3 domain may not exert a major adverse effect on its binding to FcRn, as the important residues for CH3 dimerization are located at the internal interface of the b structure of CH3 b, whereas the region responsible for the binding FcRn is located at the external interface of the CH2-CH3 domains. However, the half-Ig molecule may have some advantage in terms of tissue penetration due to its smaller size than a regular antibody. In one embodiment, at least one amino acid residue is substituted in the constant region of the binding protein of the invention, e.g., in the Fc region, such that heavy chain dimerization is affected, resulting in half Ig molecules with DVD. The anti-inflammatory activity of IgG is completely dependent on the sialylation of the N-linked glycan of the Fc fragment of IgG. Precisely necessary requirements of the glycan for anti-inflammatory activity have been determined such that an appropriate IgG1 Fc fragment can be created, thereby generating a fully recombinant sialylated IgG1 Fc with greatly enhanced potency (Anthony et al., Science , 320:373376 (2008)).
[077]The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, hIL-1β). The antigen-binding function of an antibody can be performed by fragments of a complete antibody. Such antibody modalities can also have bispecific, dual specific, or multispecific formats; specifically, binding to two or more different antigens (eg, hIL -1β and a different antigenic molecule such as hIL-1β and hIL-1a.) Examples of binding fragments encompassed by the term "antigen binding portion" of an antibody include (i) Fab fragment, monovalent fragment consisting of the VL domains , VH, CL and CH1; (ii) F(ab')2 fragment, bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment, consisting of the VH and CH1 domains; (iv) fragment Fv, constituted comprised of the VL and VH domains of a single arm of an antibody, (v) dAb fragment ( Ward et al., Nature, 341: 544-546 (1989); PCT Publication No. WO 90/05144), which comprises a single variable domain; and (vi) isolated complementarity determining region (CDR). Furthermore, although encoded by distinct genes, the two domains of the Fv fragment, VL and VH, can be joined, using recombination methods, by a synthetic linker that allows them to be produced as a single protein chain in which the VL regions and VH pair to form monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad Sci. USA, 85:5879-5883 (1988)). Such single chain antibodies are also intended to be encompassed by the term "antigen binding portion" of an antibody. Other forms of single chain antibodies such as diabodies are covered as well. Diabodies are bivalent bispecific antibodies, in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, whereby the pairing of domains with complementarity domains from another strand and two antigen-binding sites created (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993); Poljak, RJ, Structure, 2: 1121-1123 (1994)). Such antibody binding moieties are known in the art (Kontermann and Dübel eds., Antibody Engineering (Springer-Verlag, New York, 2001), p. 790 (ISBN 3-540-41354-5)). Additionally, single chain antibodies also include "linear antibodies", comprising a pair of Fv segments (VH-CH1-VH-CH1) in tandem, which, together with complementary light chain polypeptides, form a pair of binding regions to antigens (Zapata et al., Protein Eng., 8(10): 1057-1062 (1995; and US Patent No. 5,641,870)).
[078]An immunoglobulin constant domain (c) refers to either a heavy (CH) or a light (CL) chain constant domain. The amino acid sequences of the constant domain of heavy chain and light chain of murine and human IgG are known in the art.
[079] The term "constructed IL-1β binding protein" (or "constructed binding protein") refers to a polypeptide comprising one or more of the antigen binding portions of the invention coupled to a linker or a immunoglobulin constant domain. A "linker polypeptide" comprises two or more amino acid residues joined by peptide bonds and is used to link one or more antigen binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); Poljak, RJ, Structure, 2:1121- 1123 (1994)). An immunoglobulin constant domain refers to a heavy or light chain constant domain. The amino acid sequences of the heavy chain and light chain constant domain of human IgG are known in the art and are represented in Table 2. Table 2. Sequence of the heavy chain constant domain and the light chain constant domain of human IgG



[080] Still further, an IL-1β binding protein, such as an antibody or its antigen-binding portion, may be part of a larger immunoadhesion molecule, formed by covalent or non-covalent association of the antibody or portion of antigen binding with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to produce a tetrameric scFv molecule (Kipriyanov et al., Human Antibod. Hybridomas, 6:93-101 (1995)) and the use of a cysteine residue, a tag peptide and a C-terminal polyhistidine tag to produce bivalent and biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol., 31: 1047-1058 (1994)). Antibody parts, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies, using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Furthermore, antibodies, their antigen-binding portions and immunoadhesion molecules can be obtained by standard recombinant DNA techniques.
[081]"Isolated antibody" in this report refers to an antibody substantially free of other antibodies with different antigenic specificities (eg, an isolated antibody that specifically binds to hIL-1β is substantially free of antibodies that specifically bind to antigens other than hIL-1β). An isolated antibody that specifically binds to hIL-1/3 may, however, be cross-reactive to other antigens, such as IL-1/3 molecules from other species. Additionally, an isolated antibody may be substantially free of other cellular material and/or chemicals.
[082] The term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minimal amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, unlike polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), every mAb is directed against a single determinant on the antigen. The "monoclonal" modifier should not be interpreted as requiring production of the antibody by any particular method.
[083] The term "human antibody" includes antibodies containing variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, randomly introduced mutations or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in CDRs and specifically in CDR3. However, the term "human antibody" does not include antibodies in which CDR sequences are derived from the germline of other mammalian species, such as mice, have been grafted onto human scaffold sequences.
[084]The term "recombinant human antibody" includes all human antibodies that are prepared, expressed, raised or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in more detail in Section II C, below), antibodies isolated from a combinatorial library of recombinant human antibodies ( Hoogenboom, HR, Trends Biotechnol., 15: 6270 (1997); Azzazy and Highsmith, Clin. Biochem., 35: 425-445 (2002) ; Gavilondo and Larrick, BioTechniques, 29: 128-145 (2000); Hoogenboom and Chames, Immunol. Today, 21: 371-378 (2000)), antibodies isolated from an animal (eg, mouse) that is transgenic for genes of human immunoglobulins (see, for example, Taylor et al., Nucl. Acids Res., 20: 6287-6295 (1992); Kellermann and Green, Curr. Opin. Biotechnol., 13: 593-597 (2002); Little et al., Immunol. Today, 21: 364-370 (2000)); or antibodies prepared, expressed, raised or isolated by any other means that involves splicing (intron removal) of human immunoglobulin gene sequences to other DNA sequences. These recombinant human antibodies have variable and constant regions derived from human immunoglobulin germline sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of recombinant antibodies are sequences which, although derived from and related to the VH and VL germline sequences, may not naturally exist within the germline repertoire of human antibodies in vivo.
[085] The term "chimeric antibody" refers to antibodies comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies with murine heavy and light chain variable regions attached to human constant regions.
[086] The term "CDR-grafted antibody" refers to antibodies which comprise heavy and light chain variable region sequences of a species, but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced by CDR sequences from another species, such as antibodies having murine heavy and light chain variable regions in which one or more one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.
[087]The term "CDR" refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the heavy and light chain variable regions, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term "CDR set" in this specification refers to a group of three CDRs that occur in a single variable region capable of binding antigen. The exact limits of these CDRs were defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987) and (1991)) not only provides an unambiguous system for residue numbering applicable to any region variable of an antibody, but also allows to precisely define the residues that delimit the three CDRs. These CDRs can be called CDRs of Kabat. Chothia et al. (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987) ); and Chothia et al., Nature, 342: 877-883 (1989)) found that certain subportions within the Kabat CDRs adopt nearly identical conformations of the peptide backbone, despite having great diversity at the sequence level of These subportions have been named L1, L2 and L3 or H1, H2 and H3, where the "L" and "H" designate the regions of the light chain and the heavy chain, respectively. These regions may be called CDRs of Chothia, whose limits overlap with the CDR s of Kabat. Other limits that define CDRs superimposed on the CDRs of Kabat CDRs were described by Padlan et al. (FASEB J., 9: 133-139 (1995)) and MacCallum et al. (J. Mol. Biol., 262(5): 732-745 (1996)). Still other definitions of CDR limits may not strictly follow one of the above systems, but will nevertheless overlap with Kabat's CDRs, although these may be shortened or extended in light of prediction or experimental findings that certain residues or groups of residues or even entire CDRs do not have a significant impact on antigen binding. The methods used in the present invention can use CDRs defined according to any of these systems, although exemplary embodiments make use of CDRs defined according to Kabat or Chothia.
[088]The terms "Kabat numbering", "Kabat definitions" and "Kabat marking" are used interchangeably in this descriptive report. These terms, which are recognized in the art, refer to a numbering system of amino acid residues that are more variable (i.e. hypervariable) than other amino acid residues in the variable regions of the heavy and light chain. of an antibody or antigen-binding portion thereof ( Kabat et al., Ann. NY Acad. Sci., 190: 382-391 (1971); and Kabat et al., Sequences of Proteins of Immunological Interest, fifth edition, United States Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and from amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and from amino acid positions 89 to 97 for CDR3.
[089]The growth and analysis of extensive public databases of amino acid sequences of heavy and light chain variable regions over the past twenty years have led to an understanding of the typical boundaries between framework regions (FR) and CDR sequences within of variable region sequences, and enabled people skilled in this technique to accurately determine the CDRs according to the Kabat numbering, the Chothia numbering or with other systems. See, for example, Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains", chapter 31, In Antibody Engineering, (Kontermann and Dübel, eds.) (Springer-Verlag, Berlin, 2001), especially pages 432-433 . A useful method for determining the amino acid sequences of Kabat CDRs CDRs within the amino acid sequences of heavy chain variable (VH) and light chain variable (VL) regions is provided below:
[090]To identify an amino acid sequence of CDR-L1: Start at approximately 24 amino acid residues from the amino end of the VL region; The residue before the CDR-L1 sequence is always cysteine (C); The residue after the CDR-L1 sequence is always a tryptophan (W) residue, typically Trp-Tyr-Gln (WYQ), but also Trp-Leu-Gln (WLQ), Trp-Phe-Gln (WFQ) and Trp -Tyr-Leu (WYL); The length is typically 10 to 17 amino acid residues. To identify an amino acid sequence of CDR-L2: Always start at 16 residues after the end of CDR-L1; Residues before the CDR-L2 sequence are generally Ile-Tyr (I-Y), but also Val-Tyr (V-Y), Ile-Lys (I-K) and Ile-Phe (I-F);
[091]The extension is always 7 amino acid residues.
[092]To identify an amino acid sequence of CDR-L3: Always start at 33 amino acids after the end of CDR-L2; The residue before the CDR-L3 amino acid sequence is always a cysteine (C); Residues after the CDR-L3 sequence are always Phe-Gly-X-Gly (F-G-X-G) (SEQ ID NO:11), where X is any amino acid;
[093]The length is typically 7 to 11 amino acid residues.
[094]To identify an amino acid sequence of CDR-H1: Start at approximately 31 amino acid residues from the amino terminus of the VH region and always 9 residues after a cysteine (C); Residues before the CDR-H1 sequence are always Cys-X-X-X-X-X-X-X-X (SEQ ID NO:12), where X is any amino acid; The residue after the CDR-H1 sequence is always a Trp (W), typically Trp-Val (W-V), but also Trp-Ile (W-I) and Trp-Ala (W-A);
[095]The length is typically 5 to 7 amino acid residues.
[096]To identify an amino acid sequence of CDR-H2: Always start at 15 amino acid residues after the end of CDR-H1; Residues before the CDR-H2 sequence are typically Leu-Glu-Trp-Ile-Gly (L-E-W-I-G) (SEQ ID NO:23), but other variations as well; Residues after the CDR-H2 sequence are Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala (K/RL/I/V/F/T/AT/S/ I/A);
[097]The length is typically 16 to 19 amino acid residues.
[098]To identify a CDR-H3 amino acid sequence: Always starts at 33 amino acid residues after the end of CDR-H2 and always 3 after a cysteine (C)' Residues before the CDR-H3 sequence are always Cys-XX (CXX), where X is any amino acid, typically Cys-Ala-Arg (CAR); Residues after the CDR-H3 sequence are always Trp-Gly-X-Gly (W-G-X-G) (SEQ ID NO:24), where X is any amino acid;
[099]The range is typically from 3 to 25 amino acid residues.
[0100]In this specification, the term "canonical" residue refers to a residue in a CDR or framework that defines a certain canonical CDR structure as defined by Chothia et al. (J. Mol. Biol., 196: 901-917 (1987)); and Chothia et al. (J. Mol. Biol., 227: 799-817 (1992)), both of which are incorporated herein by reference in this patent application). According to Chothia et al., critical parts of the CDRs of many antibodies have nearly identical main-chain peptide conformations despite great diversity at the amino acid sequence level. Every canonical structure primarily specifies a set of twist angles from the peptide backbone to a contiguous segment of amino acid residues that form a loop.
[0101] "Affinity-matured" antibody is an antibody with one or more changes in one or more of its CDRs that result in an improvement in the antibody's affinity for a target antigen, when compared to a precursor antibody that does not have the(s) change(s). Exemplary antibodies with matured affinity will show affinities in the nanomolar or even picomolar order for the target antigen. A variety of procedures for producing affinity matured antibodies are known in the art. For example, Marks et al., BioTechnology, 10: 779-783 (1992) describe affinity maturation by shuffling the VH and VL domain. Random mutagenesis of CDR and/or scaffold residues is described by Barbas et al., Proc. Nat. Sci. USA, 91: 3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton et al., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol., 154(7): 3310-3319 (1995); Hawkins et al., J. Mol. Biol., 226: 889-896 (1992). Selective mutation at positions for selective mutagenesis and at contact or hypermutation positions with an amino acid residue exhibiting enhanced activity is described in U.S. Patent No. 6,914,128 B1.
[0102]The term "multivalent binding protein" indicates a binding protein comprising two or more antigen binding sites. A multivalent binding protein is preferably constructed to have three or more antigen binding sites and is generally not a naturally occurring antibody. The term "multispecific binding protein" refers to a binding protein capable of binding to two or more related or unrelated targets. The "double variable domain" ("DVD") binding proteins of the invention comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. DVDs can be monospecific, that is, capable of binding to one antigen, or multispecific, that is, capable of binding to two or more antigens. A DVD binding protein, comprising two DVD heavy chain polypeptides and two DVD light chain polypeptides, is termed "DVD immunoglobulin" or "DVD-Ig". Each half of a DVD-Ig comprises a heavy chain polypeptide with DVD and a light chain polypeptide with DVD and two or more antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain, totaling six CDRs involved in antigen binding per antigen binding site.
[0103] A description of the design, expression and characterization of DVD-Ig molecules is provided in PCT Publication No. WO 2007/024715; U.S. Patent No. 7,612,181; and in Wu et al., Nature Biotechnol., 25: 1290-1297 (2007). A preferred example of such DVD-Ig molecules comprises a heavy chain having the structural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a heavy chain first variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker with the proviso that it is not CH1, X2 is an Fc region and n is 0 or 1, but preferably 1; and a light chain having the structural formula VD1-(X1)n-VD2-C-(X2)n, where VD1 is a light chain first variable domain, VD2 is a light chain second variable domain, C is a light chain constant domain, X1 is a linker with the proviso that it is not CH1 and X2 does not comprise an Fc region; and n is 0 or 1, but preferably 1. Such DVD-Ig may comprise two such heavy chains and two such light chains, each chain comprising variable domains linked in tandem without one without a constant region intervening between the variable regions, wherein one heavy and one light chain associate to form functional antigen-binding sites in tandem, and one heavy and light chain pair can associate with another heavy and light chain pair to form a tetrameric binding protein with four functional sites of antigen binding. In another example, a DVD-Ig molecule can comprise heavy and light chains, each comprising three variable domains (VD1, VD2, VD3) linked in tandem without a constant region intervening between the variable domains, wherein a pair of chains, heavy and light, can associate to form three antigen-binding sites and wherein a pair of heavy and light chains can associate to form a tetrameric binding protein with six antigen-binding sites.
[0104]A DVD-Ig binding protein can bind to one or more epitopes of IL-1β. A DVD-Ig binding protein can also bind an IL-1β epitope and a second target antigen epitope other than an IL-1β polypeptide.
[0105] The term "bispecific antibody" in this specification refers to complete antibodies that are generated by quadroma technology (see Milstein and Cuello, Nature, 305: 537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314: 628-631 (1985)) or by the knob-into-hole approach or the like that introduce mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci USA, 90(14):6444-6448 (1993)), resulting in multiple different species of immunoglobulins of which only one is the functional bispecific antibody. The molecular function allows a bispecific antibody to bind to an antigen (or epitope) on one of its two binding arms (a HC/LC pair) and to bind to a different antigen (or epitope) on its second arm ( a different pair of HC/LC). According to this definition, a bispecific antibody has two distinct binding arms (both in terms of specificity and sequence of CDRs) and is monovalent for each antigen to which it binds.
[0106] The term "dual-specific antibody" in this specification refers to complete antibodies that are capable of binding to two different antigens (or epitopes) on each of their binding arms (a pair of HC/ LC) (see PCT Publication No. WO 02/02773). Thus, a dual-specific binding protein has two identical antigen-binding arms, with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.
[0107]"Functional antigen-binding site" of a binding protein is one capable of binding to a target antigen. The antigen-binding affinity of the antigen-binding site is not necessarily as strong as that of the precursor antibody from which the antigen-binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of known methods for assessing antibody binding to an antigen. Furthermore, the antigen-binding affinity of each of the antigen-binding sites of a multivalent antibody in this patent application need not be the same in quantitative terms.
[0108]The term "cytokine" is a generic term for proteins that are released by one cell population and act on another cell population as intracellular mediators. Examples of these cytokines are lymphokines, monokines and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyrostimulating hormone (TSH) and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor such as tumor necrosis factor-alpha (TNF-α) and tumor necrosis factor-beta (TNF-β); Müller-inhibiting substance; mouse gonodatrophin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); neural growth factors such as NGF-alpha (NGF-a); platelet growth factor; placental growth factor; transforming growth factors (TGFs) such as TGF-alpha (TGF-α) and TGF-beta (TGF-β); insulin-like growth factor 1 and 11; erythropoietin (EPO); bone induction factors; interferons such as interferon-alpha (IFN-α), interferon-beta (IFN-β) and interferon-gamma (IFN-y); colony stimulating factors (CSFs) such as macrophage CSF (M-CSF); granulocyte and macrophage CSF (GM-CSF); and granulocyte CSF (G-CSF); interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-33; and other polypeptide factors including LIF and the kit linker (KL). In this descriptive report, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
[0109]In this specification, the terms "donor" and "donor antibody" refer to an antibody that provides one or more CDRs. In an exemplary embodiment, the donor antibody is an antibody of a species other than the antibody from which the framework regions are obtained or derived. In the context of a humanized antibody, the term "donor antibody" refers to a non-human antibody that provides one or more CDRs.
[0110]In this descriptive report, the term "framework" or "framework sequence" refers to the other sequences of a variable region minus the CDRs. Since the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2 and -L3 light chain and CDR-H1, -H2 and -H3 heavy chain) also divide the light chain and heavy chain framework regions into four subregions (FR1 , FR2, FR3 and FR4) on each chain, where the CDR1 is positioned between FR1 and FR2, the CDR2 between FR2 and FR3 and the CDR3 between FR3 and FR4. If the particular subregions are not specified as FR1, FR2, FR3 or FR4, a framework region, as designated by others, represents that of the FRs combined within the variable region of a single naturally occurring immunoglobulin chain. In this descriptive report, FR represents one of the four subregions and FRs represent two or more of the four subregions that make up a framework region.
[0111] In this specification, the terms "acceptor" and "acceptor antibody" refer to the antibody that provides or the nucleic acid sequence that encodes at least 80%, at least 85%, at least 90%, at least 95 %, at least 98% or 100% of the amino acid sequences of one or more of the framework regions. In some embodiments, the term "acceptor" refers to the antibody that provides the amino acid or nucleic acid sequence that encodes the constant region(s). In yet another embodiment, the term "acceptor" refers to the antibody that provides the amino acid sequence or the nucleic acid sequence that encodes one or more of the framework regions and the constant region(s). In a specific embodiment, the term "acceptor" refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, preferably, at least 85%, at least 90%, at least 95% , at least 98% or 100% of the amino acid sequences of one or more of the framework regions. According to this embodiment, an acceptor may contain at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid residues that do not occur at one or more specific positions of a human antibody. An acceptor region and/or acceptor constant region(s) can be, for example, derived or obtained from an antibody germline gene, a mature antibody gene, a functional antibody (for example , antibodies well known in the art, antibodies under development or commercially available antibodies).
[0112] Human heavy chain and light chain acceptor sequences are known in the state of the art. In one embodiment of the invention, human heavy and light chain acceptor sequences are selected from the V-base (http://vbase.mrc-cpe.cam.ac.uk/) or IMGT® listed sequences , the international information system ImMunoGeneTics system® (http://imgt.cines.fr/textes/IMGTrepertoire/LocusGenes/). In another embodiment of the invention, human heavy and light chain acceptor sequences are selected from the sequences described in Table 3 and Table 4. Table 3. Heavy chain acceptor sequences











[0113] In this specification, the term "antibody germline gene" or "gene fragment" refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone a maturation process leading to genetic rearrangement and mutation for the expression of a particular immunoglobulin (See, for example, Shapiro et al., Crit. Rev. Immunol., 22(3): 183-200 (2002); Marchalonis et al., Adv. Exp. Med. Biol., 484 : 13-30 (2001)). One of the advantages offered by various embodiments of the present invention derives from the recognition that germline antibody genes are likely than mature antibody genes to conserve essential structures of amino acid sequences characteristic of individuals in the species, and therefore less likely to be recognized as coming from a foreign source when used therapeutically in that species.
[0114] In this specification, the term "fundamental" residues refers to certain residues within the variable region that exert more impact on the specificity and/or binding affinity of an antibody, specifically a humanized antibody. A fundamental residue includes, but is not limited to, one or more of the following: a residue adjacent to a CDR, a potential glycosylation site (may be N or O-glycosylation site), a rare residue, a residue capable of interacting with antigen , a residue capable of interacting with a CDR, a canonical residue, a contact residue between heavy chain variable region and light chain variable region, a residue within the Vernier zone, and a residue in the region that overlaps a region between a heavy-chain variable CDR1, according to Chothia's definition, and the first heavy-chain scaffolding according to Kabat's definition.
[0115] The term "humanized antibody" refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (eg mouse), but in which at least part of the sequence of VH and/ or VL has been altered to be more “human-like”, that is, more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are inserted into non-human VH and VL sequences to replace the corresponding non-human sequences of the CDRs. Additionally, "humanized antibody" is an antibody or variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework region (FR) substantially containing the amino acid sequence of a human antibody and a region complementarity determinant (CDR) substantially having the amino acid sequence of a non-human antibody. In this specification, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 98 % or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one and typically two variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In one embodiment, a humanized antibody also comprises at least part of a constant region (Fc) of an immunoglobulin, typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody may also include the heavy chain CH1, hinge, CH2, CH3 and CH4 regions. In some embodiments, a humanized antibody contains only one humanized light chain. In some embodiments, a humanized antibody contains only one humanized heavy chain. In specific embodiments, a humanized antibody contains only a humanized variable domain of a humanized light chain and/or heavy chain.
[0116] A humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE and any isotope including, but not limited to, IgG1, IgG2, IgG3 and IgG4. The humanized antibody can comprise sequences from more than one class or isotype, and specific constant domains can be selected to enhance the desired effector functions using techniques well known in the art.
[0117] The framework regions and CDRs of a humanized antibody do not need to precisely match the precursor sequences, for example, the CDR of the donor antibody or the consensus framework can undergo mutagenesis by substitution, insertion and/or deletion of at least one residue of amino acid such that the residue of the CDR or framework at that site does not match that of the donor antibody or the consensus framework. In an exemplary modality, such mutations, however, will not be extensive. In general, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those in the FR and CDR precursor sequences. In this descriptive report, the term "consensus framework" refers to the framework region in the immunoglobulin consensus sequence. In this specification, the term "immunoglobulin consensus sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in an immunoglobulin family of related sequences (see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In an immunoglobulin family, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur with equal frequency, either one can be included in the consensus sequence.
[0118] With respect to the construction of DVD-Ig or other binding protein molecules, a "linker" is used to indicate a single amino acid or a polypeptide ("linker polypeptide") comprising two or more amino acid residues joined by peptide bonds and used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); Poljak, RJ, Structure, 2:1121- 1123 (1994)). Exemplary linkers include, but are not limited to, GGGGSG (SEQ ID NO:26), GGSGG (SEQ ID NO:27), GGGGSGGGGS (SEQ ID NO:28), GGSGGGGSG (SEQ ID NO:223), GGSGGGGSGS (SEQ ID NO:29 ), GGSGGGGSGGGGS (SEQ ID NO:30), GGGGSGGGGSGGGG (SEQ ID NO:31), GGGGSGGGGSGGGGS (SEQ ID NO:32), ASTKGP (SEQ ID NO:33), ASTKGPSVFPLAP (SEQ ID NO:34), TVAAP (SEQ ID NO:35), RTVAAP (SEQ ID NO:224),TVAAPSVFIFPP (SEQ ID NO:36), RTVAAPSVFIFPP (SEQ ID NO:225), AKTTPKLEEGEFSEAR (SEQ ID NO:37), AKTTPKLEEGEFSEARV (SEQ ID NO:38) , AKTTPKLGG (SEQ ID NO:39), SAKTTPKLGG (SEQ ID NO:40), SAKTTP (SEQ ID NO:41), RADAAP (SEQ ID NO:42), RADAAPTVS (SEQ ID NO:43), RADAAAAGGPGS (SEQ ID NO:44), RADAAAAGGGGSGGGGGSGGGGSGGGGS (SEQ ID NO:45), SAKTTPKLEEGEFSEARV (SEQ ID NO:46), ADAAP (SEQ ID NO:47), ADAAPTVSIFPP (SEQ ID NO:48), QPKAAP (SEQ ID NO:49), QPKAAPSVTLFPP (SEQ ID NO:50), AKTTPP (SEQ ID NO:51), AKTTPPSVTPLAP (SEQ ID NO:52), AKTTAP (SEQ ID NO:53), AKTTAPSVYPLAP (SEQ ID NO:54), GENKVEYAPALMALS (SEQ ID NO:53) :55), GPAKELTPLKEAKVS (SEQ ID NO:56) and GHEA AAVMQVQYPAS (SEQ ID NO:57).
[0119]In this specification, "Vernier" zone refers to a subset of framework residues that can adjust the structure of CDRs and tune the fit to antigen, as described by Foote and Winter, J. Mol. Biol., 224:487-499 (1992), the contents of which are incorporated herein by reference in this patent application). Residues from the Vernier's Zone form an underlying layer of CDRs and can affect the structure of CDRs and antibody affinity.
[0120]In this specification, the term "neutralizing" refers to the neutralization of the biological activity of an antigen (eg, the IL-1β cytokine) when a binding protein specifically binds to the antigen. Preferably, a neutralizing binding protein described herein binds to hIL-1β resulting in inhibition of a biological activity of hIL-1β. Preferably, the neutralizing binding protein binds hIL-1β and reduces a biological activity of hIL-1β by at least approximately 20%, 40%, 60%, 80%, 85% or more. Inhibition of a biological activity of hIL-1β by a neutralizing binding protein can be evaluated by measuring one or more indicators of the biological activity of hIL-1β, well known in the art. For example, the inhibition of human IL-6 secretion by IL-1β induction in HS27 cells.
[0121]The term "activity" includes activities such as the binding specificity/affinity of an antibody for an antigen, eg an anti-h IL-1β antibody that binds to an IL-1β antigen and/or potency neutralizing an antibody, for example, an anti-IL-1β antibody whose binding to h IL-1β inhibits the biological activity of h IL-1β, eg inhibition of human IL-6 secretion by inducing IL-1β in HS27 cells.
[0122] The term "epitope" includes any polypeptide determinant capable of specific binding to an immunoglobulin or T cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains , phosphoryl or sulfonyl, and, in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when the latter preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. Antibodies are said to "bind to the same epitope" if the antibodies cross-compete (one prevents the binding or modulating effect of the other). Additionally, structural definitions of epitopes (overlays, similar, identical) are informative, but functional definitions are often more important as they cover structural (binding) and functional (modulation, competition) parameters.
[0123] The term "surface plasmon resonance" in this descriptive report refers to any optical phenomenon that enables the analysis of bispecific interactions in real time by changes in protein concentrations detected within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey). For more detailed descriptions, see Jonsson et al., Ann. Biol. Clin., 51:1926 (1993); Jonsson et al., BioTechniques, 11: 620-627 (1991); Johnsson et al., J. Mol. Recognit., 8: 125-131 (1995); and Johnsson et al., Anal. Biochem., 198: 268-277 (1991).
[0124]The term "Kon" (also "Kon", "kon") in this specification is intended to refer to the constant for the rate of association of a binding protein (eg antibody) with an antigen to form an association complex, e.g. antibody/antigen complex, as known in the prior art. "Kon" is also known by the terms "membership fee constant" or "ka" as used interchangeably in this specification. This value indicates the binding rate of an antibody to its target antigen or the rate of complex formation between antibody and antigen as shown by the equation below:

[0125]The term "Koff" (also "Koff", "koff") in this specification is intended to refer to the dissociation rate constant or "rate to dissociation constant" of a binding protein (by antibody) of an association complex (eg antibody/antigen complex) as known in the prior art. This value indicates the dissociation rate of an antibody from its target antigen or the separation of the Ab-Ag complex over time into free antibody and antigen, as shown by the equation below:

[0126] The term "KD" (also "Kd"), in this descriptive report, is intended to refer to the "equilibrium dissociation constant" and refers to the value obtained in titration measured in equilibrium state or by dividing the constant to the off-rate (Koff) by the constant for the on-rate (Kon). The on-rate constant (Kon), the off-rate constant (Koff), and the equilibrium dissociation constant (K) are used to represent the binding affinity of an antibody for an antigen. Methods for determining on-rate and off-rate constants are well known in the art. Fluorescence-based techniques offer high sensitivity and allow you to examine samples in physiological equilibrium buffers. Other experimental approaches and instruments such as the BIAcore® assay (biomolecular interaction analysis) can be used (eg the instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay), available from Sapidyne Instruments (Boise, Idaho) may also be used.
[0127] The terms "marker" and "detectable marker" mean a group coupled to a specific binding partner, such as antibody or analyte, for example, to make the reaction between members of a specific binding pair, such as antibody and analyte, detectable. The specific binding partner, e.g. antibody or analyte, so labeled is referred to as "detectably labeled". Therefore, the term "labeled binding partner" in this specification refers to a protein with an incorporated label that enables identification of the binding protein. In one embodiment, the marker is a detectable marker that can produce a detectable signal by visual or instrumental means, for example, incorporation of a radiolabeled amino acid or coupling to a polypeptide of biotinylated groups that can be detected by avidin or labeled streptavidin ( for example, streptavidin containing a fluorescent label or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (for example, 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho or 153Sm), chromogens, fluorescent labels (for example , FITC, rhodamine, lanthanide phosphors), enzyme markers (eg, horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinylated groups, predetermined polypeptide epitopes recognized by a secondary reporter (eg, leucine zipper pair sequences , binding sites for secondary antibodies, metal binding domains, epitope tags) and magnetic agents (eg gadolinium chelates). Representative examples of commonly used markers for immunoassays include light-producing clusters, eg, acridinium compounds, and fluorescence-producing clusters, eg, fluorescein. Other markers are described in this patent application. In this respect, the cluster itself may not be detectable, but it may become detectable upon reaction with yet another cluster. The use of the term "detectably tagged" is intended to encompass the last type of detectable tag.
[0128]The term "conjugate IL-1β binding protein" refers to an IL-1β binding protein described herein linked to a second chemical group, such as a therapeutic or cytotoxic agent. The term "agent" is used in this specification to indicate chemical compound, mixture of chemical compounds, biological macromolecule or extract made from biological materials. Preferably, therapeutic or cytotoxic agents include, but are not limited to, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy-anthracin mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogues or homologues thereof. When employed in the context of an immunoassay, an IL-1β binding protein conjugate can be a detectably labeled antibody, which is used as the detection antibody.
[0129] The terms "crystal" and "crystallized" in this specification refer to a binding protein (eg, antibody) or its antigen-binding portion, existing in crystal form. Crystals represent a solid-state form of matter that is distinct from other forms, such as the amorphous solid state or the liquid crystalline state. Crystals are composed of regular, repeating, three-dimensional arrays of atoms, ions, molecules (eg, proteins such as antibodies) or molecular assemblies (eg, antigen/antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well understood in the field. The fundamental unit or building block, which is repeated in a crystal is called the asymmetric unit. The repetition of the asymmetric unit in an arrangement that conforms to a certain well-defined crystallographic symmetry provides the “unit cell” of the crystal. Repetition of the unit cell by regular translations in all three dimensions provides the crystal. See Giegé et al., chapter 1, In Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd edition, (Ducruix and Giegé, eds.) (Oxford University Press, New York, 1999) p. 1-16.
[0130]The term "polynucleotide" means a polymeric form of two or more nucleotides, either ribonucleotides or deoxynucleotides, or a modified form of any type of nucleotide. The term includes single-stranded and double-stranded forms of DNA.
[0131] The term "isolated polynucleotide" shall mean a polynucleotide (for example, of genomic DNA, cDNA or synthetic origin or some combination thereof) which, by virtue of its origin, the "isolated polynucleotide" is not associated with all or with part of a polynucleotide with which the "isolated polynucleotide" is found in nature, is operably linked to a polynucleotide to which it is not linked in nature, or does not occur in nature as part of a larger sequence.
[0132]The term "vector" in this specification is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a double-stranded, circular loop DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby replicate throughout the host genome. Additionally, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to in this specification as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In this descriptive report, "plasmid" and "vector" can be used interchangeably, as plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, as viral vectors (e.g., retroviruses, adenoviruses and adeno-associated viruses with defective replication), which perform equivalent functions.
[0133]The term "operationally linked" refers to a juxtaposition, in which the described components are in a relationship that allows them to act in their intended way. A control sequence "operably linked" to a coding sequence is joined in such a way that expression of the coding sequence is carried out under conditions compatible with the control sequences. "Operably linked" sequences include both expression control sequences, contiguous to the gene of interest, and expression control sequences that act in trans or at a distance to control the gene of interest. The term "expression control sequence" in this specification refers to polynucleotide sequences that are necessary to effect expression and processing of coding sequences to which they are joined. Expression control sequences include appropriate transcription initiation and termination, promoter and enhancer sequences; efficient signaling for RNA processing, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and, where desired, sequences that enhance protein secretion. The nature of these control sequences differs depending on the host organism; in prokaryotes, these control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, these control sequences generally include promoters and transcription termination sequence. The term "control sequences" is intended to include components whose presence is essential for expression and processing, and may also include components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
[0134]"Transformation", as defined in this specification, refers to any process by which exogenous DNA enters a host cell. The transformation can take place under natural or artificial conditions, using various methods well known in the art. Transformation can be based on any known method for inserting foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the transforming host cell and may include, but are not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication either on an autonomously replicating plasmid or as part of the host chromosome. In addition to these, they include cells that transiently express the inserted DNA or RNA for limited periods of time.
[0135]The term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which exogenous DNA has been introduced. In one embodiment, the host cell comprises two or more (e.g., multiple) nucleic acids that encode antibodies, such as the host cells described in U.S. Patent No. 7,262,028, for example. These terms are intended to refer not only to the particular object cell, but also to the progeny of that cell. Since certain modifications can occur in succeeding generations as a result of mutation or environmental influences, such progeny may not actually be identical to the parent cell, but still fall within the scope of the term "host cell" in this specification. In one embodiment, host cells include prokaryotic cells and eukaryotic cells selected from any of the Realms of life. In another embodiment, eukaryotic cells include protist, fungal, plant and animal cells. In another embodiment, host cells include, but are not limited to, the prokaryotic cell line of Escherichia coli; the mammalian cell lines CHO, HEK 293, COS, NS0, SP2 and PER.C6; the Sf9 insect cell line; and cells of the fungus Saccharomyces cerevisiae.
[0136]Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques may be carried out according to the manufacturer's specifications or as commonly performed in the prior art or as described herein. The foregoing techniques and procedures can generally be carried out in accordance with conventional methods well known in the art and as described in various general and more specific references which are cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
[0137] "Transgenic organism", as known in the prior art, refers to an organism with cells that contain a transgene, in which the transgene introduced into the organism (or into an ancestor of the organism) expresses a polypeptide that is not expressed naturally in the body. "Transgene" is constructed DNA that is stable and operationally integrated into the genome of a cell from which a transgenic organism develops, directing the expression of a gene product encoded in one or more cell types or tissues of the transgenic organism.
[0138]The terms "regular" and "modular" are used interchangeably and, in this specification, refer to a modification or change in the activity of a molecule of interest (eg, the biological activity of hIL-1β). Modulation can be an increase or decrease in the magnitude of a certain activity or function of the molecule of interest. Exemplary activities and functions of a molecule include, among others, binding characteristics, enzymatic activity, activation of cell receptors, and signal transduction.
[0139]Correspondingly, the term "modulator," in this specification, is a compound capable of modifying or altering an activity or function of a molecule of interest (eg, the biological activity of hIL-1β). For example, a modulator can cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. In certain embodiments, a modulator is an inhibitor, which decreases the magnitude of at least one activity or function of a molecule. Exemplary inhibitors include, among others, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described, for example, in PCT Publication No. WO 01/83525.
[0140] The term "agonist" in this specification refers to a modulator that, when brought into contact with a molecule of interest, causes an increase in the magnitude of a certain activity or function of the molecule, when compared to the magnitude of the activity or function observed in the absence of the agonist. Agonists of particular interest may include, among others, IL-1β polypeptides, nucleic acids, carbohydrates, or any other molecule that binds to hIL-1β.
[0141] The terms "antagonist" and "inhibitor" in this specification refer to a modulator that, when brought into contact with a molecule of interest, causes a decrease in the magnitude of a certain activity or function of the molecule, when compared to magnitude of activity or function observed in the absence of the antagonist. Antagonists of particular interest include those that block or modulate the biological or immunological activity of human IL-1β. Antagonists and inhibitors of human IL-1/3 can include, but are not limited to, proteins, nucleic acids, carbohydrates, or any other molecules that bind to human IL-1/3.
[0142]In this descriptive report, the term "effective amount" refers to the amount of a therapy that is sufficient to reduce or ameliorate the severity and/or duration of a disorder or one or more of its symptoms; prevent the advancement of a disorder; cause the regression of a disorder; prevent the recurrence, development, appearance or progression of one or more symptoms associated with a disorder; detect a disorder; or enhance or ameliorate the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent).
[0143] "Patient" and "individual" may be used interchangeably in this specification to refer to an animal, such as a mammal, including primate (eg, human, monkey, and chimpanzee), non-primate (eg, cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, whale), bird (eg duck or goose) and shark. Preferably, a patient or individual is a human being, such as one being treated or evaluated for a disease, disorder or condition, one at risk for a disease, disorder or condition, one suffering from a disease, disorder or condition and/ or one being treated for an illness, disorder or condition.
[0144]The term "sample" in this descriptive report is used in its broadest sense. "Biological sample" in this descriptive report includes, among others, any quantity of a substance from a living being or formerly a living being. Such living beings include, but are not limited to, humans, non-human primates, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissue, bone marrow, lymph nodes, and spleen.
[0145] "Component", "components" and "at least one component" generally refer to capture antibody, detection antibody or conjugate, control, calibrator, calibrator series, sensitivity panel, container, buffer, diluent, salt, enzyme, cofactor for an enzyme, detection reagent, pretreatment reagent/solution, substrate (eg in solution), stopping solution and the like that can be included in a kit for assaying a test sample , such as a patient's urine, serum or plasma sample, according to the methods described herein and other methods known in the art. Therefore, in the context of the present invention, "at least one component", "component" and "components" may include a polypeptide or other analyte as above, such as a composition comprising an analyte such as a polypeptide, which is optionally immobilized on a solid support, such as by binding to an anti-analyte antibody (e.g., anti-polypeptide). Some components may be in solution or lyophilized for reconstitution and use in an assay.
[0146]"Control" refers to a composition known to not contain the analyte ("negative control") or to contain the analyte ("positive control"). A positive control can comprise a known concentration of the analyte. "Control", "positive control" and "calibrator" may be used interchangeably in this specification to refer to a composition comprising a known concentration of analyte. A "positive control" can be used to establish assay performance characteristics and is a useful indicator of the integrity of reagents (eg, analytes).
[0147] "Predetermined cutoff" and "predetermined level" refer generally to the cutoff value of an assay that is used to evaluate diagnostic/prognostic/therapeutic efficacy results by comparing the assay results against the limit cutoff/default level, where the predetermined cutoff/level threshold has already been linked or associated with various clinical parameters (eg disease severity, progression/non-progression/improvement, etc.). Although the present invention can provide exemplary predetermined levels, it is well known that cut-off values can vary depending on the nature of the immunoassay (e.g., antibodies employed, etc.). It is still well within the ability of any person skilled in the art to adapt the present invention to other immunoassays to obtain immunoassay-specific cutoff values for the other immunoassays based on the present invention. While the precise value of the predetermined cutoff/level may vary between tests, the correlations described in this patent application (if any) should generally apply.
[0148] "Pretreatment reagent", for example, a lysis, precipitation and/or solubilization reagent, as used in a diagnostic assay, as described herein, is one that lyses any cells and/or that solubilizes any analyte that is/are present in a sample under test. Pretreatment is not required for all samples, as described in more detail below. Among other effects, solubilizing the analyte (eg, polypeptide of interest) may entail releasing the analyte from any endogenous binding proteins present in the sample. A pretreatment reagent can be homogeneous (not requiring a separation step) or heterogeneous (requiring a separation step). The use of a heterogeneous pretreatment reagent allows you to remove any analyte-binding proteins that have precipitated out of the test sample before proceeding to the next step of the assay.
[0149] "Quality control reagents", in the context of immunoassays and kits described herein, include, among others, calibrators, controls and sensitivity panels. A "calibrator" or "standard" is typically used (e.g., one or more, such as a plurality) to establish calibration (standard) curves for interpolating the concentration of an analyte, such as antibody or analyte. Alternatively, a single calibrator, which is close to a predetermined positive/negative cutoff limit, can be used. Multiple calibrators (ie, more than one calibrator or a varying amount of calibrator(s)) can be used together to comprise a “sensitivity panel”.
[0150]"Risk" refers to the possibility or probability that a particular event will occur, either in the present or at some point in the future. "Risk stratification" refers to an array of known clinical risk factors that allows clinicians to classify patients at low, moderate, high, or higher risk of developing a particular disease, disorder, or condition.
[0151] "Specific" and "specificity", in the context of an interaction between members of a specific binding pair (for example, antigen (or its fragment) and antibody (or its antigenically reactive fragment)) refer to selective reactivity of the interaction. The term "specifically binds to" and the like refer to the ability of antibodies (or antigenically reactive fragments thereof) to specifically bind to the analyte (or fragment thereof) and not specifically bind to other entities.
[0152]"Specific binding partner" is a member of a specifically binding pair. A specifically bound pair comprises two different molecules, which specifically bind to each other by chemical or physical means. Therefore, in addition to specific binding pairs, formed by antigen and antibody, of common immunoassays, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme and enzyme inhibitors and the like. Furthermore, specific binding pairs can include members that are analogous to the original members of the specific binding, e.g., analyte analog. Specific binding immunoreactive members include antigens, antigenic fragments and antibodies, including monoclonal and polyclonal antibodies, as well as their complexes, fragments and variants (including fragments of variants), whether isolated or produced by recombination.
[0153]"Variant" in this specification means a polypeptide that differs from a particular polypeptide (eg, IL-1β, BNP, NGAL or HIV polypeptide or anti-polypeptide antibody) in amino acid sequence by addition (eg, insertion ), conservative amino acid deletion or substitution, but which retains the biological activity of the particular polypeptide (for example, a variant IL-1β may compete with the anti-IL-1β antibody for binding to IL-1β). Conservative replacement of an amino acid, i.e. replacement of an amino acid by a different one with similar properties (for example, hydrophilicity and degree and distribution of charged regions) is recognized in the prior art as typically involving minimal change. These minor changes can be identified, in part, by considering the hydropathic amino acid index as understood in the prior art (see, for example, Kyte et al., J. Mol. Biol., 157: 105132 (1982)). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids with hydropathic indices of ± 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins that retain biological function. A consideration of the hydrophilicity of amino acids, in the context of a peptide, allows one to calculate the greatest mean local hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (see, for example, US Patent No. 4,554,101 ). Substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity, as understood in the prior art. In one aspect, substitutions are made with amino acids having hydrophilicity values within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the specific side chain of that amino acid. In line with this observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids and especially the side chains of these amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties. "Variant" can also be used to describe a polypeptide or fragment thereof that has been processed differently, such as by proteolysis, phosphorylation or other post-translational modification, although it still retains its biological activity or antigen reactivity, eg, capacity to bind to IL-1β. The use of "variant" in this descriptive report is intended to cover fragments of a variant unless contradicted by context. I. Antibodies that bind to human IL-1β
[0154] One aspect of the present invention provides isolated murine monoclonal antibodies, or antigen-binding portions thereof, that bind IL-1β with high affinity, low dissociation rate, and high neutralizing capacity. A second aspect of the invention provides chimeric antibodies that bind to IL-1β. A third aspect of the invention provides CDR-grafted antibodies, or antigen-binding portions thereof, which bind to IL-1β. A fourth aspect of the invention provides humanized antibodies, or antigen-binding portions thereof, which bind to IL-1β. A fifth aspect of the invention provides double variable domain immunoglobulin molecules (DVD-Ig™) that bind to IL-1β and another target. Preferably, the antibodies, or portions thereof, are isolated antibodies. Preferably, the antibodies of the invention are neutralizing anti-human IL-1β antibodies. A. Method for producing anti-IL-1β antibodies
[0155] The anti-IL-1β antibodies of the present invention can be produced by any of a few techniques known in the state of the art. 1. Monoclonal antibodies against IL-1β using hybridoma technology
[0156] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant and phage display technologies or a combination of these. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, 1988); Hammerling et al., eds., "Monoclonal Antibodies and T-Cell Hybridomas," in Research Monographs in Immunology, vol. 3 (J.L. Turk, General Editor) (Elsevier, New York, 1981) p. 563-587 (the contents of which are incorporated herein in their entirety by reference in this patent application). The term "monoclonal antibody" in this specification is not restricted to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including eukaryotic, prokaryotic or actual clone and not the method by which it is produced.
[0157] Methods to produce and screen specific anti-IL-1β antibodies using hybridoma technology are routine and well known in the art. In one embodiment, the present invention provides methods for generating monoclonal antibodies, as well as antibodies produced by the method comprising culturing a hybridoma cell that secretes an antibody of the invention, wherein, preferably, the hybridoma is generated by fusion of splenocytes isolated from a mouse, immunized with an antigen of the invention, myeloma cells, and then screening for hybridomas resulting from the fusion by hybridoma clones that secrete an antibody capable of binding to a polypeptide of the invention. Briefly, mice can be immunized with an IL-1β antigen. In an exemplary embodiment, the IL-1β antigen is administered with an adjuvant to stimulate the immune response. These adjuvants include Freund's complete or incomplete adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). These adjuvants can protect the polypeptide against rapid dispersal by sequestering it in a local depot, or they can contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if a polypeptide is being administered, the immunization schedule will involve two or more administrations of the polypeptide, spread over several weeks.
[0158]After immunizing an animal with an IL-1β antigen, antibodies and/or antibody-producing cells can be obtained from the animal. A serum containing anti-IL-1β antibody is obtained from the animal by bleeding or sacrificing the animal. Serum can be used as obtained from the animal, an immunoglobulin fraction can be obtained from the serum, or anti-IL-1β antibodies can be purified from the serum. The serum or immunoglobulins obtained in this way are polyclonal, thus displaying a heterogeneous set of properties.
[0159]Once an immune response is detected, for example, antibodies specific to the IL-1β antigen are detected in the mouse serum, the mouse spleen is harvested and the splenocytes are isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, e.g., cells of the SP20 cell line, available from the American Type Culture Collection (ATCC, Manassas, Virginia). Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then analyzed by methods known in the art for cells that secrete antibodies capable of binding to IL-1β. Ascitic fluid, which usually contains high levels of antibodies, can be generated by immunizing mice with positive clones of the hybridomas.
[0160] In another embodiment, antibody-producing immortalized hybridomas can be prepared from the immunized animal. After immunization, the animal is sacrificed, and the splenic B cells are fused to immortalized myeloma cells as is well known in the art. See, for example, Harlow et al., supra. In an exemplary embodiment, myeloma cells do not secrete immunoglobulin polypeptides (non-secretory cell lineage). After fusion and antibiotic selection, hybridomas are screened using IL-1β or part thereof or a cell that expresses IL-1β. In an exemplary modality, initial screening is performed with an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably an ELISA. An example of screening by ELISA is provided in PCT Publication No. WO 00/37504, incorporated herein by reference in this application.
[0161] Anti-IL-1β antibody-producing hybridomas are selected, cloned, and further screened for desirable characteristics, including robust hybridoma growth, high antibody production, and desirable antibody characteristics, as discussed in more detail below. Hybridomas can be cultivated and expanded in vivo in syngeneic animals, in animals lacking an immune system, for example, nude mice, or in in vitro cell culture. Methods for selecting, cloning and expanding hybridomas are well known to those skilled in the art.
[0162] In an exemplary modality, the hybridomas are from mice, as described above. In another preferred embodiment, the hybridomas are produced in a non-human, non-mouse species, such as from rats, sheep, pigs, goats, cattle or horses. In another embodiment, hybridomas are human hybridomas, in which a non-secretory human myeloma is fused to a human cell that expresses an anti-IL-1β antibody.
[0163] Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, Fab and F(ab')2 fragments of the invention can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the heavy chain CHI domain. 2. Anti-IL-1β monoclonal antibodies using SLAM
[0164] In another aspect of the invention, recombinant antibodies are generated from single lymphocytes, isolated using a procedure designated in the prior art selected lymphocyte method for antibodies (SLAM), as described in U.S. Patent No. 5,627,052; PCT Publication No. WO 92/02551; and Babcook et al., Proc. Natl. Academic Sci. USA, 93: 7843-7848 (1996). In this method, single antibody-secreting cells of interest, eg lymphocytes, derived from any of the immunized animals described in Section 1, are screened using an antigen-specific hemolytic plaque assay, in which the IL-1β antigen, a subunit of IL-1β, or a fragment thereof, is coupled to sheep red blood cells using a linker such as biotin and used to identify single cells that secrete antibodies with specificity for IL-1β. After identification of antibody-secreting cells of interest, heavy and light chain variable region cDNAs (VH and VL) are rescued from the cells by PCR via reverse transcriptase, and these variable regions can then be expressed in the context of constant regions of appropriate immunoglobulins (e.g., human constant regions), in mammalian host cells, such as COS or CHO cells. Host cells transfected with the amplified immunoglobulin sequences, derived from in vivo selected lymphocytes, can then be subjected to further analysis and in vitro selection, for example by panning techniques from the transfected cells to isolated cells expressing antibodies against IL -1β. Amplified immunoglobulin sequences can be manipulated in vitro, for example, by in vitro affinity maturation methods such as those described in PCT Publication No. WO 97/29131 and PCT Publication No. WO 00/56772. 3. Anti-IL-1β monoclonal antibodies using transgenic animals
[0165] In another embodiment of the invention, antibodies are produced by immunizing a non-human animal, comprising some or all of the human immunoglobulin loci, with an IL-1β antigen. In an exemplary modality, the non-human animal is a transgenic mouse XENOMOUSE, a constructed mouse strain that comprises large fragments of the human immunoglobulin locus and is deficient in mouse antibody production. See, for example, Green et al., Nature Genetics, 7: 13-21 (1994) and U.S. Patent Nos. 5,916,771 ; 5,939,598; 5,985,615; 5,998,209; 6,075,181; 6 091 001; 6,114,598 and 6,130,364. See also PCT Publication Nos. WO 91/10741, published July 25, 1991; WO 94/02602, published February 3, 1994; WO 96/34096 and WO 96/33735, both published October 31, 1996; WO 98/16654, published April 23, 1998; WO 98/24893, published June 11, 1998; WO 98/50433, published November 12, 1998; WO 99/45031, published September 10, 1999; WO 99/53049, published October 21, 1999; WO 00/09560, published February 24, 2000; and WO 00/037504, published June 29, 2000. The XENOMOUSE® transgenic mouse produces an adult-like human repertoire of fully human antibodies and generates antigen-specific human Mabs. The XENOMOUSE® transgenic mouse contains approximately 80% of the human antibody repertoire through the introduction of YAC fragments with germline configuration and megabase-sized human heavy chain locus and x human light chain locus. See, Mendez et al., Nature Genetics, 15:146-156 (1997); and Green and Jakobovits, J. Exp. Med., 188: 483-495 (1998), the contents of which are incorporated herein by reference in this application. 4. Anti-IL-1β Monoclonal Antibodies Using Recombinant Antibody Libraries
[0166] In vitro methods can also be used to produce the antibodies of the invention, in which an antibody library is screened to identify an antibody with the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, Ladner et al., U.S. Patent No. 5,223,409; Kang et al., PCT Publication No. WO 92/18619; Dower et al., PCT Publication No. WO 91/17271; Winter et al., PCT Publication No. WO 92/20791; Markland et al., PCT Publication No. WO 92/15679; Breitling et al., PCT Publication No. WO 93/01288; McCafferty et al., PCT Publication No. WO 92/01047; Garrard et al., PCT Publication No. WO 92/09690; Fuchs et al., Bio/Technology, 9: 1369-1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3:81-85 (1992); Huse et al., Science, 246: 1275-1281 (1989); McCafferty et al., Nature, 348: 552-554 (1990); Griffiths et al., EMBO J., 12: 725-734 (1993); Hawkins et al., J. Mol. Biol., 226: 889-896 (1992); Clackson et al., Nature, 352: 624-628 (1991); Gram et al., Proc. Natl. Academic Sci. USA, 89: 3576-3580 (1992); Garrard et al., Bio/Technology, 9:1373-1377 (1991); Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991); and Barbas et al., Proc. Natl. Academic Sci. USA, 88:7978-7982 (1991); U.S. Publication No. 2003/0186374; and PCT Publication No. WO 97/29131, the contents of which are incorporated herein by reference in this application.
[0167]The recombinant antibody library can come from an individual immunized with IL-1/3 or with part of IL-1/3. Alternatively, the recombinant antibody library can be from a naive individual, i.e., who has not been immunized with IL-1/3, such as a human antibody library from a human individual who has not been immunized with human IL-1/3. Antibodies of the invention are selected by screening the recombinant antibody library with the peptide comprising human IL-1/3, thus selecting the antibodies that recognize IL-1/3. Methods to conduct such screening and selection are well known in the art, such as those described in the references cited in the preceding paragraph. To select antibodies of the invention having particular binding affinities for human IL-1β, such as those that dissociate from human IL-1β with a certain constant for the Koff rate, the method known in the prior art surface plasmon resonance technique can be used to select antibodies showing the constant for the desired Koff rate. To select antibodies of the invention having a certain neutralizing activity for human IL-1/3, such as those with a certain IC50, standard methods known in the art for evaluating inhibition of human IL-1/3 activity can be used. In one aspect, the invention pertains to an isolated antibody, or an antigen-binding portion thereof, that binds to human IL-1β. Preferably, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.
[0168] For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, the functional domains of the antibody are displayed on the surface of phage particles, which carry the polynucleotide sequences encoding them. Specifically, this phage can be used to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phages expressing an antigen-binding domain that bind to the antigen of interest can be selected or identified with the antigen, for example, using tagged antigen or antigen bound or captured on a solid surface or microsphere. The phages used in these methods are typically filamentous phages, including the fd and M13 binding domains, expressed from phages with disulfide-stabilized Fab, Fv or antibody domains with Fv, fused by recombination to gene III or gene VIII proteins of the phages. Examples of phage display methods that can be used to produce the antibodies of the present invention include those disclosed in Brinkmann et al., J. Immunol. Methods, 182: 41-50 (1995); Ames et al., J. Immunol. Methods, 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24: 952-958 (1994); Persic et al., Gene, 187: 918 (1997); Burton et al., Adv. Immunol., 57: 191-280 (1994); PCT Publication Nos. WO 90/02809; WO 91/10737; WO 92/01047 (PCT/GB91/01134); WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; the contents of which are incorporated herein in their entirety by reference in this patent application.
[0169] As described in the above references, after phage selection, the antibody coding regions of the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in more detail below. For example, techniques to produce Fab, Fab' and F(ab')2 fragments by recombination can also be employed, using methods known in the art, such as those described in PCT Publication No. WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); and Sawai et al., Am. J. Reprod. Immunol., 34: 26-34 (1995); and Better et al., Science, 240: 1041-1043 (1988), (the contents of which are incorporated herein in their entirety by reference in this patent application). Examples of techniques that can be used to produce single-chain Fvs and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods Enzymol., 203: 46-88 (1991); Shu et al., Proc. Natl. Academic Sci. USA, 90: 7995-7999 (1993); and Skerra et al., Science, 240: 1038-1041 (1988).
[0170] Alternatively to screening recombinant antibody libraries by phage display, other methodologies known in the art to screen large combinatorial libraries can be applied for the identification of antibodies with dual specificity of the invention. One type of alternative expression system is one in which the recombinant antibody library is expressed in RNA-protein fusions, as described in PCT Publication No. WO 98/31700 by Szostak and Roberts; and in Roberts and Szostak, Proc. Natl. Academic Sci. USA, 94: 12297-12302 (1997). In this system, a covalent fusion is created between an mRNA and the peptide or protein it encodes by in vitro translation of synthetic mRNAs carrying puromycin, a peptidyl-acceptor antibiotic at its 3' end. In this way, a specific mRNA can be enriched from a complex mixture of mRNAs (eg a combinatorial library) based on the properties of the peptide or encoded protein, eg antibody or portion thereof, such as antibody binding or part of this to the antigen with dual specificity. Nucleic acid sequences encoding antibodies or parts thereof, recovered from screening these libraries, can be expressed recombinantly as described above (for example, in mammalian host cells) and, in addition, can be further subjected to affinity maturation by additional rounds of screening of mRNA-peptide fusions, in which mutations have been introduced in the originally selected sequence(s), or by others for in vitro affinity maturation of recombinant antibodies, as described above.
[0171] In another approach, the antibodies of the present invention can also be generated using yeast display methods, known in the state of the art. In yeast display methods, genetic methods are used to attach antibody domains to the yeast cell and display them on the yeast surface. Specifically, this yeast can be used to display antigen binding domains expressed from a combinatorial antibody repertoire or library (e.g., human or murine). Examples of yeast display methods that can be used to produce the antibodies of the present invention include those disclosed by Wittrup et al. in U.S. Patent No. 6,699,658, incorporated herein by reference in this patent application. B. Production of recombinant antibodies against IL-1β
[0172] Antibodies of the present invention can be produced by any of a few techniques known in the art. For example, expression from host cells, wherein expression vector(s) encoding the heavy and light chain is (are) transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly employed for introducing exogenous DNA into a prokaryotic or eukaryotic host cell, eg electroporation, calcium phosphate precipitation, DEAE-dextran transfection and the like. While it is possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells, the expression of antibodies in eukaryotic cells is preferable, and most preferable, in mammalian host cells, because these eukaryotic cells (and especially, the cells) are more likely of mammals) clump together and secrete a properly folded and immunologically active antibody than prokaryotic cells.
Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary cells (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77, 77 :4216-4220 (1980), used with selectable marker DHFR, for example, as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982)), myeloma NS0 cells, COS cells and cells SP2. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a sufficient period of time to allow expression of the antibody in the host cells or, more preferably, secretion of the antibody in the culture medium in which the host cells are cultured. Antibodies can be recovered from the culture medium with standard protein purification methods.
[0174] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are included within the scope of the present invention. For example, it may be convenient to transfect a host cell with DNA encoding functional fragments of the light chain and/or heavy chain of an antibody of this invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both the light and heavy chains that is not needed for binding to the antigens of interest. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. Additionally, bifunctional antibodies can be produced, in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than the antigens of interest, by cross-linking one antibody of the invention to a second antibody by standard chemical cross-linking methods.
[0175] In an exemplary system for recombinant expression of an antibody, or its antigen-binding portion, of the invention, a recombinant expression vector, encoding the antibody heavy chain and antibody light chain, is introduced into cells dhfr-CHO by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows selection of CHO cells that have been transfected with the vector using methotrexate/amplification selection. Selected transforming host cells are cultured to allow expression of the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are employed to prepare the recombinant expression vector, to transfect host cells, to select transformants, to culture the host cells, and to recover the antibody from the culture medium. Still further, the invention provides a method for synthesizing a recombinant antibody of the invention, comprising culturing a host cell of the invention in a suitable culture medium until the recombinant antibody of the invention is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium. 1. Chimeric anti-human IL-1β antibodies
[0176] Chimeric antibody is a molecule in which different parts of the antibody are derived from different animal species, such as antibodies with a variable region derived from a murine monoclonal antibody and a constant region from human immunoglobulin. Methods for producing chimeric antibodies are known in the art and discussed in detail in the Examples section. See, for example, Morrison, S.L., Science, 229: 1202-1207 (1985); Oi et al., BioTechniques, 4:214-221 (1986); Gillies et al., J. Immunol. Methods, 125:191202 (1989); U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816,397, the contents of which are incorporated herein in their entirety by reference in this patent application. Additionally, techniques developed for the production of "chimeric antibodies" ( Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984); Neuberger et al., Nature, 312: 604-608 ( 1984); Takeda et al., Nature, 314: 452-454 (1985), the contents of which are hereby incorporated in their entirety by reference in this patent application) by gene splicing of an antigen-specific mouse antibody molecule appropriate together with genes from a human antibody molecule of desired biological activity can be used.
[0177] In one embodiment, the chimeric antibodies of the invention are produced by replacing the constant region of the heavy chain of murine monoclonal anti-human IL-1β antibodies, in Section 1, with a constant region of human IgG1. 2. Anti-IL-1β CDR-grafted antibodies
CDR-grafted antibodies of the invention comprise heavy and light chain variable region sequences of a human antibody, wherein one or more of the VH and/or VL CDR regions are replaced by CDR sequences of the murine antibodies of the invention. A framework sequence from any human antibody can serve as the template for grafting CDRs. However, direct replacement of chains in such a framework often leads to some loss of antigen-binding affinity. The more homologous a human antibody is to the original murine antibody, the less likely it is that murine CDRs combined with the human framework introduce distortions into the CDRs that could reduce affinity. Therefore, it is preferable that the human variable framework that is chosen to replace the murine variable framework, excluding the CDRs, has at least 65% sequence identity with the murine antibody variable region framework. It is more preferable that the human and murine variable regions, excluding the CDRs, have at least 70% sequence identity. It is even more preferable that the human and murine variable regions, excluding the CDRs, have at least 75% sequence identity. Most preferably, the human and murine variable regions, excluding the CDRS, have at least 80% sequence identity. Methods for producing chimeric antibodies are known in the art. See, for example, European Patent No. EP 0 239 400; PCT Publication No. WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089). For veneering or resurfacing of antibodies, see, for example, European Patent Nos. EP 0 592 106 and EP 0 519 596; Padlan, Mol. Immunol., 28 (4/5): 489-498 (1991); Studnicka et al., Protein Eng., 7(6): 805-814 (1994); and Roguska et al., Proc. Natl. Academic Sci. USA, 91: 969-973 (1994)). With regard to antibody chain stacking, see, for example, U.S. Patent No. 5,565,352. 3. Humanized anti-human IL-1β antibodies
[0179] Humanized antibodies are antibody molecules derived from a non-human species antibody that binds to the desired antigen, containing one or more complementarity determining regions (CDRs) from the non-human species antibody and framework regions from an immunoglobulin molecule human. Known human Ig sequences are described, for example, on World Wide Web sites: www.ncbi.nlm.nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/; www.anticorporesource.com/onlinecomp.html; www.public.iastate.edu/.about.pedro/research_tools.html; www.mgen.uni-heidelberg.de/SD/IT/IT.html; www.whfreeman.com/immunology/CH-05/kuby05.htm; www.library.thinkquest.org/12429/Immune/Antibody.html; www.hhmi.org/grants/lectures/1996/vlab/; www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html; www.anticorporesource.com/; mcb.harvard.edu/BioLinks/Immuno-logy.html.www.immunologylink.com/; pathbox.wustl.edu/.about.hcenter/index.-html; www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913.html- ; www.nal.usda.gov/awic/pubs/antibody/; www.m.ehime-u.acjp/.about.yasuhito- /Elisa.html; www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/links.html; www.biotech.ufl.edu/.about.fccl/protocol.html; www.isac-net.org/sites_geo.html; aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html; baser.uci.kun.nl/.about.jraats/linksl.html; www.recab.uni-hd.de/immuno.bme.nwu.edu/; www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html; www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/; www.biochem.ucl.ac.uk/.about.martin/abs/index.html; antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html; www.unizh.ch/.about.honegger/AHOseminar/Slide01.html; www.cryst.bbk.ac.uk/.about.ubcg07s/; www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm; www.path.cam.ac.uk/.about.mrc7/h-umanisation/TAHHP.html; www.ibt.unam.mx/vir/structure/stat_aim.html; www.biosci.missouri.edu/smithgp/index.html; www.cryst.bioc.cam.ac.uk/.abo-ut.fmolina/Web-pages/Pept/spottech.html; www.jerini.de/fr roducts.htm; www.patents.ibm.com/ibm.html.Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health (1983), the contents of which are fully incorporated herein by reference in this application. Such imported sequences can be used to reduce immunogenicity or to reduce, enhance or modify binding, affinity, rate of association, rate of dissociation, avidity, specificity, half-life or any other suitable characteristic, as known in the prior art.
[0180]Framework (FR) residues in the human framework regions can be replaced by the corresponding residue from the CDR donor antibody to preferentially alter antigen binding. These scaffold substitutions are identified by methods well known in the art, for example, modeling the interactions of CDRs and scaffold residues to identify important scaffold residues for antigen binding, and sequence comparison to identify unusual residues in the framework in specific positions. See, for example, Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature, 332: 323-327 (1988), the contents of which are incorporated herein in their entirety by reference in this patent application. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of residues in the functioning of the candidate immunoglobulin sequence, that is, analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus sequence and those imported so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, CDR residues are directly and most substantially involved in influencing antigen binding. Antibodies can be humanized using a variety of techniques known in the art, such as, but not limited to, those described in Jones et al., Nature, 321:522-525 (1986); Verhoeyen et al., Science, 239:1534-1536 (1988); Sims et al., J. Immunol., 151: 2296-2308 (1993); Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987), Carter et al., Proc. Natl. Academic Sci. USA, 89: 4285-4289 (1992); Presta et al., J. Immunol., 151:2623-2632 (1993); Padlan, Mol. Immunol., 28 (4/5): 489-498 (1991); Studnicka et al., Protein Eng., 7(6): 805-814 (1994); Roguska et al., Proc. Natl. Academic Sci. USA, 91: 969-973 (1994); PCT Publication Nos. WO 91/09967; WO 90/14443; WO 90/14424; WO 90/14430; WO 99/06834 (PCT/US98/16280); WO 97/20032 (PCT/US96/18978); WO 92/11272 (PCT/US91/09630); WO 92/03461 (PCT/US91/05939); WO 94/18219 (PCT/US94/01234); WO 92/01047 (PCT/GB91/01134); and WO 93/06213 (PCT/GB92/01755); European Patent Nos. EP 0 592 106; EP 0 519 596 and EP 0 239 400; U.S. Patent Nos. 5,565,332; 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6 180 370; 5,693,762; 5,530,101; 5,585,089; 5,225,539 and 4,816,567, the contents of which are fully incorporated herein by reference in this patent application, including references cited therein. 5. Anti-IL-1β DVD-Ig™ binding proteins
[0181] Additionally, double variable domain immunoglobulin-like binding proteins (DVD-Igs) are provided which bind to one or more epitopes of IL-1β. A DVD-Ig binding protein can also bind to one epitope of IL-1/3 and another of a second target antigen other than an IL-1/3 polypeptide. An exemplary embodiment of such DVD-Ig molecules, which contain a heavy chain, comprises the structural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker with the proviso that it is not CH1, X2 is an Fc region and n is 0 or 1 and preferably 1; and a heavy chain comprising the structural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a light chain first variable domain, VD2 is a light chain second variable domain, C is a domain light chain constant, X1 is a linker with the proviso that it is not CH1 and X2 does not comprise an Fc region; and n is 0 or 1 and preferably 1. Such DVD-Ig may comprise two such heavy chains and two such light chains, each chain comprising variable domains linked in tandem without a constant region intervening between the variable regions, wherein one heavy chain and one light chain associate to form two antigen-binding sites in tandem and a pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with four binding sites. antigens. In another embodiment, a DVD-Ig molecule may comprise heavy and light chain, each comprising three variable domains, e.g., VD1, VD2, VD3, joined in tandem without a constant region intervening between the variable domains, wherein a pair heavy and light chains can associate to form three antigen-binding sites and one pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen-binding sites.
[0182]Each variable domain (VD) on a DVD-Ig can be derived from one or more "precursor" monoclonal antibodies, which bind to one or more desired antigens or epitopes, such as IL-antigens or epitopes 1β and/or IL-1α. 1. Generation of precursor monoclonal antibodies
[0183] The variable domains of DVD-Ig binding protein can be obtained from precursor antibodies, including monoclonal antibodies (mAb), capable of binding to antigens of interest. These antibodies can be natural or be generated by recombinant technology. It is understood that if an antibody that binds to a desired target antigen or epitope is polyclonal, it is still necessary to obtain the variable domains of an antigen-binding site from a single antibody of the polyclonal population, i.e., from a single monoclonal member of the polyclonal population, for use in generating a DVD-Ig. Monoclonal antibodies can be generated by any of a variety of methods known in the art, including those described in this patent application (see Sections A.1.-A.4., above). 8. Criteria for selecting precursor monoclonal antibodies
[0184] One embodiment of the invention pertains to the selection of precursor antibodies with at least one or more desired properties on the DVD-Ig molecule. In one embodiment, the desired property is selected from one or more antibody parameters. In another embodiment, antibody parameters are selected from the group consisting of antigen specificity, antigen affinity, potency, biological function, epitope recognition, stability, solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, cross-reactivity in tissues and orthologous binding to antigens. 81. Affinity for the antigen
[0185]The desired affinity of a therapeutic mAb may depend on the nature of the antigen and the desired therapeutic outcome. In one embodiment, monoclonal antibodies have higher affinities (Kd = 0.01 - 0.50 pM) when they block a cytokine-cytokine receptor-cytokine receptor interaction, as interactions of this type are generally high-affinity (eg, bands <pM - <nM). In such circumstances, the affinity of the mAb for its target must be equal to or better than the affinity of the cytokine (ligand) for its receptor. On the other hand, a mAb with lower affinity (>nM range) could be therapeutically effective, for example, to purge potentially pathogenic proteins in circulation, for example, monoclonal antibodies that bind, sequester and clear circulating species of a target antigen, such as as A-β amyloid. In other circumstances, reducing the affinity of an existing high-affinity mAb by site-directed mutagenesis or using a mAb with lower affinity for its target could be used to avoid potential side effects, eg, a high-affinity mAb may hijack or neutralize its entire intended target, thus depleting/completely eliminating the function(s) of the target protein. In this scenario, a low-affinity mAb can hijack/neutralize a fraction of the target that may be responsible for disease symptoms (pathological or overproduction levels), thereby allowing a fraction of the target to continue to perform its normal physiological function or functions. . Therefore, it may be possible to reduce the Kd to adjust the dose and/or to reduce side effects. The affinity of the precursor mAb could contribute to the proper targeting of cell surface molecules and the desired therapeutic outcome to be achieved. For example, if a target is expressed in cancer cells with high density and normal cells with low density, a lower affinity mAb will bind to a greater number of targets in tumor cells than in normal cells, resulting in elimination of tumor cells via ADCC or CDC and therefore could have therapeutically desirable effects. Thus, selecting a mAb with the desired affinity can be of importance for both soluble and surface-present targets.
[0186]Signaling through a receptor when interacting with its ligand may depend on the affinity of the receptor-ligand interaction. Likewise, it is conceivable that the affinity of a mAb for a surface receptor could determine the nature of intracellular signaling and whether the mAb can deliver an agonistic or antagonistic signal. The affinity-based nature of mAb-mediated signaling can affect its side-effect profile. Therefore, the desired affinity and desired functions of therapeutic monoclonal antibodies need to be carefully determined by in vitro and in vivo experimentation.
[0187] The desired Kd of a binding protein (eg antibody) can be determined experimentally, depending on the desired therapeutic outcome. In one embodiment, precursor antibodies are selected so as to have an affinity (Kd) for a given antigen equal to or better than DVD-Ig's desired affinity for the same antigen. Antigen binding affinity and kinetics are evaluated by the Biacore or similar technique. In one modality, each precursor antibody has a dissociation constant (Kd) against its antigen selected from the group consisting of: at most approximately 10-7 M; at most approximately 10-8 M; at most approximately 10-9 M; at most approximately 10-10 M; at most approximately 10-11 M; at most approximately 10-12 M; and at most 10-13 M. The first precursor antibody from which VD1 is obtained and the second precursor antibody from which VD2 is obtained may have similar or different affinity (KD) for the respective antigen. Each precursor antibody has a constant for the rate of association (Kon) with the antigen selected from the group consisting of: at least approximately 102 M-1s-1; at least approximately 103M-1s-1; at least approximately 104 M-1s-1; at least approximately 105 M-1s-1; and at least approximately 106 M-1s-1 as measured by surface plasmon resonance. The first precursor antibody from which, for example, a VD1 is obtained and the second precursor antibody from which a VD2 is obtained may show similar or different on-rate constant (Kon) for the respective antigen. In one embodiment, each precursor antibody has a constant for the dissociation rate (Koff) of the antigen selected from the group consisting of: at most approximately 10-3s-1; at most approximately 10-4 s-1; at most approximately 10-5 s-1; and at most approximately 10-6 s-1 as measured by surface plasmon resonance. The first precursor antibody from which VD1 is obtained and the second precursor antibody from which VD2 is obtained may have similar or different dissociation rate (Koff) constants for the respective antigen. 82. Power
[0188] The desired affinity/potency of precursor monoclonal antibodies will depend on the desired therapeutic outcome. For example, for receptor-ligand (R-L) interactions, the affinity (kd) is equal to or better than the kd of R-L (pM range). For clearance of circulating pathological proteins, Kd could be in the low nM range, eg clearance of various species of A-β peptide in circulation. Additionally, Kd will also depend on whether the target expresses multiple copies of the same epitope, for example, a mAb directed to a conformational epitope on in Aβ oligomers.
[0189] When VDI and VD2 bind to the same antigen, but different epitopes, DVD-Ig will contain binding sites for the same antigen, thus increasing the avidity and with it the apparent Kd of DVD-Ig. In one embodiment, precursor antibodies with Kd equal to or lower than that desired on DVD-Ig are chosen. Affinity considerations for a precursor mAb may also depend on whether the DVD-Ig contains four or more identical antigen-binding sites (i.e., a DVD-Ig from a single mAb). In this case, the apparent Kd would be greater than that of the mAb due to avidity. Such DVD-Igs can be used to cross-link surface receptors, increase the clearance of pathogenic proteins, etc.
[0190]In another embodiment, precursor antibodies with neutralizing potency for a specific antigen equal to or better than the desired neutralizing potential of DVD-Ig for the same antigen are selected. Neutralizing potency can be assessed by a target-dependent bioassay, in which cells of the appropriate type that produce a measurable signal (i.e., proliferation or cytokine production) in response to target stimulation and target neutralization by the mAb, can reduce the signal in a dose-dependent manner. 83. Biological functions
[0191] Monoclonal antibodies can potentially perform several functions. Some of these functions are listed in Table 5. These functions can be evaluated by in vitro assays (eg cellular and biochemical assays) and in in vivo animal models. Table 5. Some potential applications for therapeutic antibodies


[0192]MAbs with distinct functions described in the examples in this descriptive report and in Table 5 can be selected to achieve the desired therapeutic results. Two or more selected monoclonal precursor antibodies can be used in DVD-Ig format to achieve two distinct functions on a single DVD-Ig molecule. For example, a DVD-Ig can be generated by selecting a precursor mAb that neutralizes the function of a specific cytokine, such as IL-1β, and selecting a precursor mAb that enhances clearance of a pathological protein. Likewise, two precursor mAbs can be selected that recognize two different cell surface receptors, one mAb with agonist function on one receptor and the other mAb with antagonist function on a different receptor. These two selected mAbs, each with a distinct function, can be used to construct a single DVD-Ig molecule that will have the two distinct functions (agonist and antagonist) of the selected monoclonal antibodies in a single molecule. Likewise, two antagonist mAbs to cell surface receptors, each blocking the binding of the respective receptor ligands (e.g., EGF and IGF), can be used in DVD-Ig format. On the other hand, an anti-receptor antagonist mAb (eg, anti-EGFR) and an anti-soluble neutralizing mediator mAb (eg, anti-IGF1/2) can be selected to produce a DVD-Ig. 84. Epitope recognition
[0193]Different regions of proteins can perform different functions. For example, specific regions of a cytokine, such as IL-1β, interact with the cytokine receptor to bring about receptor activation, whereas other regions of the protein may be needed to stabilize the cytokine. In this circumstance, it is possible to select a mAb that specifically binds to the region (regions) that interact with the cytokine receptor and thereby block the cytokine-receptor interaction. In some cases, for example, certain chemokine receptors can be selected which bind to multiple ligands, a mAb that binds to the epitope (region on the chemokine receptor) that interacts with only a single ligand. In other circumstances, monoclonal antibodies may bind to epitopes on a target that are not directly responsible for the protein's physiological functions, but binding a mAb to these regions could interfere with physiological functions (steric hindrance) or alter the protein's conformation such that the protein cannot function (mAb against receptors with multiple ligands that alter the conformation of the receptor in such a way that none of the ligands can bind). Anti-cytokine monoclonal antibodies have also been identified that do not block cytokine binding to its receptor, but block signal transduction (eg, 125-2H, an anti-IL-18 mAb).
[0194]Examples of mAb epitopes and functions include, among others, blocking the Receptor-Link (R-L) interaction (neutralizing mAb that binds to the R interaction site); steric hindrance resulting in decreased or no binding to R. An antibody may bind to the target at a site other than the receptor binding site, but still interfere with receptor binding and target functions by inducing a conformational change and eliminating a function (eg, XOLAIR® omalizumab, Genetech/Novartis), binds to R but blocks signaling (mAb 125-2H).
[0195] In one embodiment, the precursor mAb needs to be targeted to the appropriate epitope for maximum effectiveness. This epitope must be preserved on DVD-Ig. The binding epitope of a mAb can be determined by several approaches, including co-crystallography, limited proteolysis of the mAb-antigen complex, plus peptide mapping by spectrometry (Legros et al., Protein Sci., 9:1002-1010 (2000)) , libraries of phage-displayed peptides (O'Connor et al., J. Immunol. Methods., 299: 21-35 (2005)), as well as by mutagenesis (Wu C. et al., J. Immunol., 170 : 5571-5577 (2003)). 85. Physical-chemical and pharmaceutical properties
[0196] Therapeutic antibody treatment often requires administration of high doses, often several mg/kg (due to low potency on a mass basis as a consequence of a typically large molecular weight). In order to accommodate patient compliance and adequately address chronic disease therapies and outpatient treatment, subcutaneous (s.c.) or intramuscular (i.m.) administration of therapeutic mAbs is desirable. For example, the maximum desirable volume for s.c. administration is ~1.0 mL and therefore concentrations >100 mg/mL are desirable to limit the number of injections per dose. In one embodiment, the therapeutic antibody is administered in one dose. The development of such formulations is restricted, however, by protein-protein interactions (eg, aggregation, which potentially increases the risks of immunogenicity) and by limitations during processing and release (eg, viscosity). Consequently, the large amounts required for clinical efficacy and the associated restrictions on development limit the full exploitation of the potential of antibody-containing formulations and s.c. administration in high dose regimens. It is evident that the physicochemical and pharmaceutical properties of a protein molecule and the protein-containing solution are of major importance, for example stability, solubility and viscosity characteristics. 85.1. Stability
[0197] A "stable" antibody formulation is one in which the contained antibody retains its physical stability and/or chemical stability and/or biological activity upon storage. Stability can be measured at a selected temperature for a selected period of time. In one embodiment, the antibody in the formulation is stable at room temperature (around 30 °C) or at 40 °C for at least 1 month and/or stable at approximately 2 - 8 °C for at least 1 year, for example , for at least 2 years. Additionally, in one embodiment, the formulation is stable after freezing (to, for example, -70°C) and thawing of the formulation, hereinafter referred to as the "freeze/thaw cycle". In another example, a "stable" formulation may be one in which less than approximately 10% and less than approximately 5% of the protein is present in aggregate form in the formulation.
[0198]A DVD-Ig stable in vitro at various temperatures for an extended period of time is desirable. This can be achieved by rapid screening of stable precursor mAbs in vitro at an elevated temperature, eg 40 °C for 2 - 4 weeks, and then assessing stability. During storage at 2 - 8 °C, the protein shows stability for at least 12 months, eg at least 24 months. Stability (% monomeric molecule, intact) can be assessed using various techniques such as cation exchange chromatography, size exclusion chromatography, SDS-PAGE, as well as by bioactivity tests. For a more comprehensive list of analytical techniques that can be employed to analyze covalent and conformational modifications, see Jones, AJS, "Analytical Methods for the Assessment of Protein Formulations and Delivery Systems," Chapter 2, In Formulation and Delivery of Peptides and Proteins , 1st ed., (Cleland and Langer, eds.) (American Chemical Society, Washington, DC, 1994) p. 22-45; and Pearlman and Nguyen, "Analysis of protein drugs", chapter 6, In Peptide and protein drug delivery, 1st ed. [In Advances in Parenteral Sciences, vol. 4] (Lee, V.H., ed.) (Marcel Dekker, Inc., New York, 1991) p. 247-301.
[0199] Heterogeneity and aggregate formation: the stability of the antibody may be such that the formulation may reveal less than approximately 10% and, in one embodiment, less than approximately 5%, in another embodiment, less than approximately 2%, or in another embodiment one embodiment, within the range of 0.5% to 1.5% or less in the antibody GMP material that is present as an aggregate. Size exclusion chromatography is a sensitive, reproducible and very robust method for detecting protein aggregates.
[0200] In addition to low levels of aggregates, the antibody needs to be, in one embodiment, chemically stable. Chemical stability can be determined by ion exchange chromatography (eg cation or anion exchange chromatography), hydrophobic interaction chromatography, or other methods such as isoelectric focusing or capillary electrophoresis. For example, the chemical stability of the antibody may be such that, after storage for at least 12 months at 2 - 8°C, the peak representing unmodified antibody in cation exchange chromatography may increase by a maximum of 20% in one modality, a maximum of 10% or, in another modality, a maximum of 5%, when compared to the antibody solution prior to storage testing.
[0201] In one embodiment, precursor antibodies exhibit structural integrity, correct disulfide bond formation, and correct folding. Chemical instability due to changes in the secondary or tertiary structure of an antibody can affect the antibody's activity. For example, stability, as indicated by antibody activity, may be such that, after storage for at least 12 months at 2 - 8°C, antibody activity may decrease by at most 50%, in one modality, at most 30% or even at most 10% or, in one modality, at most 5% or 1%, when compared to antibody solution prior to storage testing. Appropriate antigen binding assays can be used to determine antibody activity. 85.2. Solubility
[0202]The "solubility" of a mAb correlates with the production of correctly folded monomeric IgG. The solubility of IgG can therefore be assessed by HPLC. For example, soluble IgG (monomeric) will give rise to a single peak on the HPLC chromatograph, whereas insoluble (for example, multimeric and aggregated) will give rise to a plurality of peaks. A person skilled in the art will therefore be able to detect an increase or decrease in solubility of an IgG with routine HPLC techniques. For a more comprehensive list of analytical techniques that can be employed to analyze solubility, see Jones, A.G., Dep. Chem. Biochem. Eng., Univ. Coll. London, "Particle formation and separation in suspension crystallization processes", Chapter 4, In Process. Solid-Liquid Suspensions (P. Ayazi Shamlou, ed.) (Butterworth-Heinemann, Oxford, UK, 1993) p. 93-117; and Pearlman and Nguyen, "Analysis of protein drugs", chapter 6, In Peptide and protein drug delivery, 1st ed. [In Advances in Parenteral Sciences, vol. 4] (Lee, V.H., ed.) (Marcel Dekker, Inc., New York, 1991) p. 247-301. The solubility of a therapeutic mAb is critical to formulating the high concentration often needed for the proper dose. As described in this patent application, solubilities >100 mg/ml may be needed to accommodate the efficient dose of the antibody. For example, antibody solubility cannot be less than approximately 5 mg/mL in early research, in one modality, less than approximately 25 mg/mL in advanced process scientific stages, or in one modality, less than approximately 100 mg/mL or, in one modality, less than approximately 150 mg/mL. The intrinsic properties of a protein molecule are important to the physicochemical properties of the protein solution, eg stability, solubility, viscosity. However, one skilled in the art will recognize that there is a wide variety of excipients that can be used as additives to beneficially affect the characteristics of the final protein formulation. These excipients may include: (i) liquid solvents, co-solvents (for example alcohols such as ethanol); (ii) buffering agents (eg phosphate, acetate, citrate, amino acid buffers); (iii) sugars or sugar alcohols (for example sucrose, trehalose, fructose, raffinose, mannitol, sorbitol, dextrans); (iv) surfactants (eg polysorbate 20, 40, 60, 80, poloxamers); (v) isotonicity modifiers (eg salts such as NaCl, sugars, sugar alcohols); and (vi) others (eg, preservatives, chelating agents, antioxidants, chelating substances (eg EDTA), biodegradable polymers, carrier molecules (eg HSA, PEGs))
[0203] Viscosity is a highly important parameter with respect to antibody manufacturing and processing (eg diafiltration/ultrafiltration), final filling processes (pumping-related aspects, filtration-related aspects) and release-related aspects ( ability to be released by syringe, release by sophisticated device). Low viscosities allow the liquid antibody solution to have a higher concentration. This allows the same dose to be administered in smaller volumes. Smaller injectable volumes offer the advantage of less pain during application and the solutions do not necessarily need to be isotonic to reduce pain upon application to the patient. The viscosity of the antibody solution can be such that, at shear rates of 100 (1/sec), the viscosity of the antibody solution is below 200 mPa s, in one embodiment, below 125 mPa s, in another embodiment, below 70 mPas and in another modality below 25 mPa s or even below 10 mPa s. 85.3. production efficiency
[0204] The generation of a DVD-Ig that is efficiently expressed in mammalian cells, such as Chinese hamster ovary (CHO) cells, will require, in one embodiment, two precursor monoclonal antibodies that are themselves efficiently expressed in Chinese hamster cells. mammals. The production yield of a stable mammalian strain (ie, CHO) should be above approximately 0.5 g/L, in one modality, above approximately 1g/L, and in another modality, in the range of approximately 2 to approximately 5 g/L or more (Kipriyanov et al., Mol. Biotechnol., 12:173-201 (1999); Carroll et al., Expert Opin Biol Ther., 4:1821-1829 (2004)).
[0205]The production of antibodies and Ig fusion proteins in mammalian cells is influenced by several factors. The construction of the expression vector by incorporating strong promoters, enhancers and selection markers can maximize transcription of the gene of interest from an integrated vector copy. Identification of vector integration sites that tolerate high levels of gene transcription can increase a vector's protein expression (Wurm, F.M., Nature Biotechnol., 22(11): 1393-1398 (2004)). Furthermore, production levels are affected by the proportion of antibody heavy and light chains and by various steps in the protein assembly and secretion process (Jiang et al., Biotechnol. Prog., 22(1): 313-318 (2006)). 86. Immunogenicity
[0206]Administration of a therapeutic mAb may result in some incidence of immune response (ie, the formation of endogenous antibodies directed against the therapeutic mAb). Potential elements that could induce immunogenicity should be analyzed during the selection of precursor monoclonal antibodies, and steps to reduce this risk can be taken to optimize the precursor monoclonal antibodies prior to construction of DVD-Ig. Mouse-derived antibodies have been shown to be highly immunogenic in patients. The generation of chimeric antibodies, composed of mouse variable regions and human constants, represents a logical next step to reduce the immunogenicity of therapeutic antibodies (Morrison and Schlom, "Recombinant Chimeric Monoclonal Antibodies", chapter 1, In Important Advances in Oncology 1990 ( JB Lippincott Company, Philadelphia, 1990) pp. 3-18). Alternatively, immunogenicity can be reduced by transferring murine CDR sequences to a human antibody framework (CDR remodeling/grafting/humanization) as described for a therapeutic antibody by Riechmann et al., Nature, 332: 323-327 (1988) . Another method is called "resurfacing" or "veneering", starting with the variable domains of light and heavy chain, in which only surface-accessible scaffold amino acids are altered for humans, while the CDR and buried amino acids remain those of the precursor antibody. from rodent ( Roguska et al., Protein Eng., 9(10): 895-904 (1996)). In another type of humanization, instead of grafting the entire CDRs, one technique grafts only the "specificity determining regions" (SDRs), defined as the subset of CDR residues that are involved in antibody binding to its target (Kashmiri et al., Methods, 36(1): 25-34 (2005)). This technique requires the identification of SDRs, either by analyzing available three-dimensional structures of antibody-target complexes, or by analyzing mutations in antibody CDR residues to determine which ones interact with the target. Alternatively, fully human antibodies may show lower immunogenicity when compared to murine, chimeric or humanized antibodies.
[0207] Another approach to reducing the immunogenicity of therapeutic antibodies is the elimination of certain specific sequences that are predicted to be immunogenic. In one approach, after a first-generation biologic has been tested in humans and shown to be unacceptably immunogenic, B cell epitopes can be mapped and then altered to avoid immune detection. Another approach employs methods to predict and remove potential epitopes for T cells. Computational methods have been developed to scan and identify the peptide sequences of biological therapeutic agents with the potential to bind to MHC proteins (Desmet et al., Proteins, 58: 53 -69 (2005)). Alternatively, a human dendritic cell-based method can be used to identify epitopes for CD4+ T cells on potential protein allergens (Stickler et al., J. Immunother., 23:654-660 (2000); Morrison and Schlom, Important Adv Oncol. (1990) pp. 3-18; Riechmann et al. "Reshaping human antibodies for therapy", Nature. 332: 323-327 (1988); Roguska et al., "A comparison of two murine mAbs humanized by CDR -grafting and variable domain resurfacing", Protein Eng., 9: 895-904 (1996); Kashmiri et al., "SDR grafting--a new approach to antibody humanization", Methods, 36(1): 25-34 ( 2005); Desmet et al., "Anchor profiles of HLA-specific peptides: analysis by a novel affinity scoring method and experimental validation", Proteins, 58: 53-69 (2005); Stickler et al., "CD4+ T-cell epitope determination using unexposed human donor peripheral blood mononuclear cells", J. Immunother., 23: 654-660 (2000)). 87. Effectiveness in vivo
[0208] In order to generate a DVD-Ig molecule with the desired in vivo efficacy, it is important to generate and select mAbs with the equally desired in vivo efficacy when provided in combination. However, under some circumstances, DVD-Ig can exhibit an in vivo efficacy that cannot be achieved with the combination of two separate mAbs. For example, a DVD-Ig can bring two targets in close proximity, leading to activity that cannot be achieved by combining two separate mAbs. Additional desirable biological functions are described in this patent application in Section B3. Precursor antibodies with desirable characteristics on the DVD-Ig molecule can be selected based on factors such as pharmacokinetic half-life (t%); tissue distribution; soluble targets versus cell surface targets; and target-soluble/density-surface concentration. 88. Tissue distribution in vivo
[0209] In order to generate a DVD-Ig molecule with the desired tissue distribution in vivo, in one embodiment, the precursor mAbs with the similar desired tissue distribution profile in vivo needs to be selected. Alternatively, based on the mechanism of the dual-specific targeting strategy, it is sometimes not necessary to select precursor mAbs with the equally desired tissue distribution in vivo when provided in combination. For example, in the case of a DVD-Ig in which one binding component directs the DVD-Ig to a specific site, thereby taking the second binding component to the same targeted site. For example, one specificity of binding a DVD-Ig could target the pancreas (islet cells) and the other specificity could bring GLP1 to the pancreas to induce insulin production. 89. Isotype
[0210] In order to generate a DVD-Ig molecule with the desired properties, including, among others, isotype, effector functions, and circulating half-life, precursor mAbs that have appropriate Fc effector functions are selected, depending on the therapeutic utility and the desired therapeutic outcome. There are five major classes or heavy chain isotypes, some of which have several subtypes, and these determine the effector functions of an antibody molecule. These effector functions are located in the hinge region, in the CH2 and CH3 domains of the antibody molecule. However, residues elsewhere in an antibody molecule can also have effects on effector functions. The effector functions of the hinge Fc region include: (i) antibody dependent cellular cytotoxicity (ADCC), (ii) complement binding (C1q), activation and complement dependent cytotoxicity (CDC), (iii) phagocytosis/clearance of complexes antigen-antibody and (iv) cytokine release in some circumstances. These Fc effector functions of an antibody molecule are mediated through the interaction of the Fc region with a set of cell surface class-specific receptors. IgG1 isotype antibodies are the most active, while IgG2 and IgG4 have minimal or no effector functions. The effector functions of IgG antibodies are mediated through interactions with three structurally homologous Fc cell receptor types (and subtypes) (FcgR1, FcgRII and FcgRIII). These effector functions of an IgG1 can be eliminated by mutations at specific amino acid residues in the lower hinge region (eg, L234A, L235A) that are required for binding to FcgR and C1q. Amino acid residues in the Fc region, especially in the CH2-CH3 domains, also determine the circulating half-life of the antibody molecule. This Fc function is mediated by the binding of the Fc region to the neonatal Fc receptor (FcRn), which is responsible for recycling antibody molecules from acidic lysosomes back to the general circulation.
[0211]The presentation of active or inactive isotype by the mAb will depend on the desired therapeutic outcome for an antibody. Some examples of the use of isotypes and the desired therapeutic result are listed below: 1. If the desired outcome is the functional neutralization of a soluble cytokine, an inactive isotype can be used in this case; 2. If the desired result is clearance of a pathological protein, an active isotype can be used; 3. If the desired result is clearance of protein aggregates, an active isotype can be used; 4. If the desired result is to antagonize a surface receptor, an inactive isotype is used (Tysabri, IgG4; OKT3®, IgG1 mutant); 5. If the desired result is to eliminate target cells, an active isotype is used (Herceptin, IgG1 (and with enhanced effector functions); and 6. If the desired result is to clear circulating proteins without penetration into the central nervous system, an IgM isotype can be used (eg clearance of circulating species of Ab peptides).
[0212] The Fc effector functions of a precursor mAb can be determined by various in vitro methods well known in the art.
[0213] As discussed, the selection of isotype, and hence the effector functions, will depend on the desired therapeutic outcome. In cases where simple neutralization of a circulating target is desired, eg blocking receptor-ligand interactions, effector functions may not be necessary. In these circumstances, isotypes or mutations in the Fc region of an antibody that eliminate effector functions are desirable. In other circumstances where elimination of target cells is the therapeutic outcome, for example, elimination of tumor cells, isotypes or mutations or defucosylation in the Fc region that enhance effector functions are desirable (Presta, LG, Adv. Drug Del. Rev. , 58: 640-656 (2006); Satoh et al., Expert Opin. Biol. Ther., 6: 1161-1173 (2006). Similarly, depending on therapeutic utility, the circulating half-life of a molecule antibody can be reduced/prolonged by modulating antibody-FcRn interactions, by specific mutations introduced into the Fc region (Dall'Acqua et al., J. Biol. Chem., 281: 2351423524 (2006); Petkova et al., Int. Immunol. , 18: 1759-1769 (2006); Vaccaro et al., Proc. Natl. Acad. Sci. USA, 103: 18709-18714 (2006).
[0214] Published information on the various residues that influence the different effector functions of a normal therapeutic mAb need to be confirmed for a DVD-Ig. It may be possible that in an additional (different) format from DVD-Ig, Fc function residues, excluding those that have been identified for modulation of monoclonal antibody effector functions, may be important.
[0215] Overall, the decision as to which Fc effector functions (isotype) will be critical in the final DVD-Ig format will depend on the indication for the disease, the therapeutic target, the desired therapeutic outcome, and safety considerations. Listed below are suitable exemplary heavy and light chain constant regions including, but not limited to: IgG1 - allotype: G1mz; mutant IgG1 - A234, A235; IgG2 - allotype: G2m(n-); Kappa - Km3; and Lambda.
[0216]Receptor Fc and C1q studies: The possibility of unwanted antibody-dependent cellular cytotoxicity (ADCC) and unwanted complement-dependent cytotoxicity (CDC), by the formation of complexes by the antibody and any overexpressed target in cell membranes, can be abrogated by mutations in the hinge region (eg L234A, L235A). These substituted amino acids, present in the IgG1 hinge region of the mAb, are predicted to result in decreased mAb binding to human Fc receptors (but not FcRn), as binding to FcgR supposedly occurs within overlapping sites in the hinge region of IgG1. This characteristic of the mAb may lead to an improved safety profile over wild-type IgG-containing antibodies. The binding of the mAb to human Fc receptors can be determined by flow cytometry experiments using cell lines (eg, THP-1, K562) and a cell line constructed from CHO that expresses FcgRIIb (or other FcgRs). When compared to control IgG1 monoclonal antibodies, the mAb showed reduced binding to FcgRI and FcgRIIa, whereas binding to FcgRIIb is unaffected. The binding and activation of C1q by antigen/IgG immune complexes triggers the classic complement cascade with consequent inflammatory and/or immunoregulatory responses. The C1q binding site on IgGs was located at residues within the hinge region of IgG. Binding to C1q at increasing mAb concentrations was assessed by C1q ELISA. The results demonstrate that the mAb is not able to bind to C1q, as predicted when compared to binding a wild-type control IgG. Overall, the L234A, L235A mutation in the hinge region abolishes mAb binding to FcgRI, FcgRIIa and C1q, but does not affect the mAb's interaction with FcgRIIb. These data suggest that in vivo mAb with mutant Fc will normally interact with the inhibitory FcgRIIb, but will likely no longer interact with the activating FcgRI and FcgRIIa or with C1q.
[0217] Binding to human FcRn: The neonatal receptor (FcRn) is responsible for transporting IgG across the placenta and for controlling the catabolic half-life of IgG molecules. It might be desirable to increase the terminal half-life of an antibody to improve efficacy, to reduce the dose or frequency of administration, or to improve localization to the target. Alternatively, it could be advantageous to do the reverse, that is, to decrease the terminal half-life of an antibody to reduce whole-body exposure or to improve target-to-non-target binding ratios. Adapting the interaction between IgG and its rescue receptor, FcRn, offers a way to increase or decrease the terminal half-life of IgG. Circulating proteins, including IgG, are taken up in the liquid phase through micropinocytosis by certain cells, such as those in the vascular endothelium. IgG can bind to FcRn in endosomes under mildly acidic conditions (pH 6.0 - 6.5) and can recycle to the cell surface, where it is released under nearly neutral conditions (pH 7.0 - 7.4). Mapping of the Fc region binding site in FcRn80, 16, 17 revealed two histidine residues that are conserved among species, His310 and His435, are responsible for the pH dependence of this interaction. With phage display technology, a mutation in the Fc region of mice, which increases binding to FcRn and prolongs the half-life of mouse IgG, has been identified (see Ghetie et al., Nature Biotechnol., 15(7) : 637-640 (1997)). Mutations in the Fc region that increase the binding affinity of IgG for FcRn at pH 6.0, but not at pH 7.4, have also been identified (see, Dall'Acqua et al., J. Immunol., 169(9) : 5171-5180 (2002)). Furthermore, in one case, a similar pH-dependent increase in binding (up to 27-fold) was also observed for FcRn of rhesus monkeys, which resulted in a doubled serum half-life in rhesus monkeys when compared to that of precursor IgG (see , Hinton et al., J. Biol. Chem., 279(8): 6213-6216 (2004)). These findings indicate that it is feasible to prolong the plasma half-life of therapeutic antibodies, adapting the interaction of the Fc region with FcRn. On the other hand, mutations in the Fc region that attenuate the interaction with FcRn can reduce the half-life of the antibody. 6.10. Pharmacokinetics (PK)
[0218] In order to generate a DVD-Ig molecule with the desired pharmacokinetic profile, in one embodiment, precursor mAbs with the equally desired pharmacokinetic profile are selected. One consideration is that the immunogenic response to monoclonal antibodies (ie, "HAHA", human anti-human antibody response; "HACA", human anti-chimeric antibody response) further complicates the pharmacokinetics of these therapeutic agents. In one embodiment, monoclonal antibodies with minimal or no immunogenicity are used to construct DVD-Ig molecules such that the resulting DVD-Igs will have minimal or no immunogenicity. Some of the factors that determine the PK of a mAb include, among others, the intrinsic properties of the mAb (VH amino acid sequence); immunogenicity, binding to FcRn and Fc functions.
[0219] The PK profile of selected precursor monoclonal antibodies can be easily determined in rodents, as the Pk profile in rodents correlates well (or closely predicts) with the PK profile of monoclonal antibodies in cynomolgus monkey and man.
[0220]After precursor monoclonal antibodies with the desired PK characteristics (and other desired functional properties as discussed in this specification) are selected, DVD-Ig is constructed. Since DVD-Ig molecules contain two antigen-binding domains from two precursor monoclonal antibodies, the PK properties of DVD-Ig are also evaluated. Therefore, while determining the PK properties of DVD-Ig, PK assays that determine the PK profile based on the functionality of both antigen-binding domains derived from the two precursor monoclonal antibodies can be employed. The PK profile of a DVD-Ig can be determined. Additional factors that may affect the PK profile of DVD-Ig include antigen-binding domain (CDR) orientation, linker size, and Fc/FcRn interactions. The PK characteristics of precursor antibodies can be evaluated by looking at the following parameters: absorption, distribution, metabolism and excretion.
[0221] Absorption: For the time being, administration of therapeutic monoclonal antibodies is via parenteral routes (eg, intravenous [IV], subcutaneous [SC], or intramuscular [IM]). Absorption of a mAb into the systemic circulation after SC or IM administration from the interstitial space is primarily via the lymphatic route. Saturable, pre-systemic, proteolytic degradation can result in variable absolute bioavailability after extravascular administration. In general, increases in absolute bioavailability with increasing doses of monoclonal antibodies can be seen that are due to saturated proteolytic capacity at higher doses. The absorption process for a mAb is usually quite slow as the lymph drains slowly into the vascular system and the absorption duration can be over hours to several days. The absolute bioavailability of monoclonal antibodies after SC administration generally ranges from 50% to 100%. In the case of a blood-brain barrier (BBB) transport mediating structure that is driven by the constructed DVD-Ig, plasma circulation times can be reduced thanks to enhanced trans-cellular transport across the blood-brain barrier (BBB) to the system compartment central nervous system, where DVD-Ig is released to enable interaction via its second antigen-recognition site.
[0222]Distribution: Following IV administration, monoclonal antibodies typically follow a biphasic serum (or plasma) concentration-time profile, starting with a rapid distribution phase, followed by a slow elimination phase. In general, a biexponential pharmacokinetic model best describes this type of pharmacokinetic profile. The volume of distribution in the central compartment (Vc) of a mAb is usually equal to or slightly greater than the plasma volume (2 - 3 liters). A distinct biphasic pattern in the serum (plasma) concentration versus time profile may not be evident with other parenteral pathways, such as IM or SC, because the distribution phase of the serum (plasma)-time concentration curve is masked by the long portion of absorption. Many factors, including physicochemical properties, site-specific and target-oriented receptor-mediated uptake, tissue binding capacity, and mAb dose, can influence the biodistribution of a mAb. Some of these factors may contribute to the non-linearity in the biodistribution of a mAb.
[0223] Metabolism and Excretion: Due to molecular size, intact monoclonal antibodies are not excreted in urine via the kidney. Antibodies are primarily inactivated by metabolism (eg, catabolism). For IgG-based therapeutic monoclonal antibodies, the half-life typically ranges from hours or 1-2 days over 20 days. The clearance of a mAb can be affected by many factors, including, among others, affinity for the FcRn receptor, immunogenicity of the mAb, the degree of glycosylation of the mAb, the susceptibility of the mAb to proteolysis, and receptor-mediated clearance. 6.11. Tissue cross-reactivity pattern in human and tox species Identical staining pattern suggests that potential human toxicity can be assessed in tox species. Tox species are animals in which unrelated toxicity is studied.
[0224] Individual antibodies are selected to meet two criteria: (1) appropriate staining in tissues for known expression of the antibody target, and (2) similar staining pattern between tissues from human species and tox from the same organ.
[0225]Criterion 1: Immunizations and/or antibody selections typically employ recombinant or synthesized antigens (proteins, carbohydrates or other molecules). Binding to the natural correspondent and screening against unrelated antigens is often part of the screening funnel for therapeutic antibodies. However, screening against a multitude of antigens is often impractical. Therefore, tissue cross-reactivity studies with human tissues from all major organs serve to rule out unwanted antibody binding to any unrelated antigens.
[0226]Criterion 2: Comparative tissue cross-reactivity studies with human tissues and tox species (cynomolgus monkey, dog, possibly rodent and others, the same 36 or 37 tissues being tested as in the human study) help to validate the selection of a tox species. In typical tissue cross-reactivity studies in frozen sections of tissue, therapeutic antibodies may demonstrate predicted binding to known antigen and/or binding to a lesser degree to tissues based on low-level interactions (non-specific binding, low-level binding to similar antigens, low-level interactions based on charge, etc.). In any case, the animal species of most relevance to toxicology is the one with the highest degree of coincidence of binding to human and animal tissue.
[0227] Tissue cross-reactivity studies follow appropriate regulatory guidelines, including EC Guideline CPMP III/5271/94 "Production and quality control of mAbs" and the 1997 US FDA/CBER "Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use". Cryosections (5 μm) of human tissue, obtained at autopsy or biopsy, were fixed and dried on a glass object. Peroxidase staining of tissue sections is performed with the avidin-biotin system. FDA Guidance "Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use". Relevant references include Clarke, J. (2004), Boon, L. (2002a), Boon, L. (2002b), Ryan, A. (1999).
[0228] Tissue cross-reactivity studies are often performed in two stages, with the first including 32-tissue cryosections (typically: adrenal gland, gastrointestinal tract, prostate, bladder, heart, skeletal muscle, blood cells, kidney, skin, marrow bone, liver, spinal cord, breast, lung, spleen, cerebellum, lymph node, testes, cerebral cortex, ovary, thymus, colon, pancreas, thyroid, endothelium, parathyroid, ureter, eye, pituitary, uterus, fallopian tube and placenta) of a human donor. In the second phase, a complete cross-reactivity study is performed with up to 38 tissues (including adrenal, blood, blood vessel, bone marrow, cerebellum, brain, cervix, esophagus, eye, heart, kidney, large intestine, liver, lung, lymph node , breast, mammary gland, oviduct, oviduct, pancreas, parathyroid, peripheral nerve, pituitary, placenta, prostate, salivary gland, skin, small intestine, spinal cord, spleen, stomach, striated muscle, testis, thymus, thyroid, tonsil, ureter , urinary bladder and uterus) of three unrelated adults. Studies are typically performed at at least two dose levels.
[0229]Therapeutic antibody (ie, test item) and isotype-matched control antibody can be biotinylated for detection by the avidin-biotin complex (ABC); other methods of detection may include tertiary antibody detection for FITC-labeled test item (or otherwise) or pre-complexion with an anti-human IgG labeled for unlabeled test item.
[0230] Briefly, cryosections (around 5 μm) of human tissue, obtained in autopsy or biopsy, are fixed and dried on a glass object. Peroxidase staining of tissue sections is performed using the avidin-biotin system. Firstly (in case of a system for detecting previous complex formation), the test item is incubated with secondary biotinylated anti-human IgG and developed into an immune complex. The immune complex at the final concentrations of 2 and 10 μg/mL of test item is added to tissue sections on a glass object and then the tissue sections are reacted for 30 minutes with an avidin-biotin-peroxidase kit . Subsequently, DAB (3,3'-diaminobenzidine), a substrate for the peroxidase reaction, was applied for 4 minutes to stain the tissue. Antigen-sepharose microspheres are used as a positive control of tissue sections.
[0231] The appearance of specific staining is assessed as predicted (eg, compatible with antigen expression) or unanticipated reactivity, based on the known expression of the target antigen in question. The specific assessed staining is scored for intensity and frequency. Antigen or serum competition or blocking studies may further assist in determining whether the observed staining is specific or non-specific.
[0232]If two selected antibodies are shown to meet the selection criteria - appropriate staining in tissues, matching staining between human and animal specific tissue for toxicology - these antibodies can be selected for DVD-Ig generation.
[0233] The tissue cross-reactivity study must be repeated with the final constructed DVD-Ig, but although these studies follow the same protocol outlined in this descriptive report, their assessment is more complex, as any link can result from any of the two precursor antibodies and any unexplained binding needs to be confirmed with competition studies of the complex with the antigen.
[0234] It is quickly evident that the complex task of tissue cross-reactivity studies with a multispecific molecule such as a DVD-Ig is greatly simplified if the two precursor antibodies are selected by: (1) there are no unanticipated cross-reactivity findings in tissues and (2) appropriate similarity of tissue cross-reactivity findings between human tissues corresponding to those of animal species for toxicology. 6.12. Specificity and selectivity
[0235] In order to generate a DVD-Ig molecule with the desired specificity and selectivity, it is necessary to generate and select precursor mAbs with the equally desired specificity and selectivity profile.
[0236] Binding studies for specificity and selectivity with a DVD-Ig can be complex due to four or more binding sites, two each for each antigen. Briefly, binding studies using ELISA, BIAcore, KinExA or other interaction studies with a DVD-Ig need to monitor the binding of one, two or more antigens to the DVD-Ig molecule. While BIAcore technology is able to resolve sequenced binding, independent of multiple antigens, more traditional methods, including ELISA or more modern techniques such as KinExA, cannot. Therefore, careful characterization of each precursor antibody is critical. After each individual antibody has been characterized for specificity, the individual confirmation of retention of the specificity of the binding sites on the DVD-Ig molecule is greatly simplified.
[0237]It is quickly evident that the complex task of determining the specificity of a DVD-Ig is greatly simplified if the two precursor antibodies are selected for specificity before being combined into a DVD-Ig.
[0238] Antigen-antibody interaction studies can take many forms, including many classic studies of protein-protein interaction such as ELISA (enzyme immunosorbent assay), mass spectrometry, chemical crosslinking, SEC with light scattering, equilibrium dialysis, gel permeation, ultrafiltration, gel chromatography, large zone analytical SEC, micropreparative ultracentrifugation (sedimentation equilibrium), spectroscopic methods, titration microcalorimetry, sedimentation equilibrium (in analytical ultracentrifuge), sedimentation rate (in centrifuge) analytical), surface plasmon resonance (including BIAcore). Relevant references include Current Protocols in Protein Science, Volume 3, chapters 19 and 20, (Coligan et al., eds.) (John Wiley & Sons Inc.) and references included therein; and Current Protocols in Immunology, (Coligan et al., eds.) (John Wiley & Sons Inc.) and relevant references included therein.
[0239] Cytokine release in whole blood: The interaction of mAbs with human blood cells can be investigated by a cytokine release assay (Wing et al., Therapeutic Immunol., 2(4): 183-190 (1995); Current Protocols in Pharmacology, (Enna et al., eds.) (John Wiley & Sons Inc.); Madhusudan et al., Clin. Cancer Res., 10(19): 6528-6534 (2004); Cox et al. ., Methods, 38(4): 274-282 (2006); Choi et al., Eur. J. Immunol., 31(1): 94-106 (2001)). Briefly, various concentrations of mAb are incubated with human whole blood for 24 hours. The concentration tested should cover a wide range, including final concentrations that simulate typical blood levels in patients (including, but not limited to, 100 ng/ml - 100 μg/ml). After incubation, supernatants and cell lysates are analyzed for the presence of IL-1Rα, TNF-α, IL-1b, IL-6 and IL-8. Cytokine concentration profiles, generated for the mAb, are compared to profiles produced by a native human IgG control and LPS or PHA positive control. The cytokine profile displayed by mAb from cell supernatants and cell lysates are compared to that using control human IgG. In one embodiment, the monoclonal antibody does not interact with human blood cells to spontaneously release inflammatory cytokines.
[0240] Cytokine release studies for a DVD-Ig are complex due to four or more binding sites, two each for each antigen. Briefly, cytokine release studies as described herein measure the effect of the entire DVD-Ig molecule on whole blood or other cell systems, but fail to resolve which part of the molecule causes cytokine release. Once cytokine release has been detected, the purity of the DVD-Ig preparation needs to be ascertained, as some concomitantly purified cellular components can on their own cause cytokine release. If purity is not an issue, DVD-Ig fragmentation (including, but not limited to, removal of the Fc portion, separation of binding sites, etc.), binding site mutagenesis, or other methods may need to be employed to elucidate any observations. It is quickly evident that this complex task is greatly simplified if the two precursor antibodies are selected for not releasing cytokines before being combined into a DVD-Ig. 6.13. Cross-reactivity with other species for toxicological studies
[0241] In one embodiment, individual antibodies are selected with sufficient cross-reactivity with appropriate tox species, eg, cynomolgus monkey. Precursor antibodies must bind to the target of the orthologous species (ie, cynomolgus monkey) and elicit the appropriate response (modulation, neutralization, activation). In one modality, the cross-reactivity (affinity/potency) with the target of the orthologous species must be within 10 times that of the human target. In practice, precursor antibodies are evaluated for multiple species, including mouse, rat, dog, monkey (and other non-human primates), as well as disease model species (i.e., asthma model sheep). Acceptable cross-reactivity with tox species of precursor monoclonal antibodies enables future toxicological studies of DVD-Ig in the same species. For this reason, the two precursor monoclonal antibodies must show acceptable cross-reactivity to a common tox species, thus enabling toxicological studies of DVD-Ig in the same species.
[0242]Precursor mAbs can be selected from several mAbs capable of binding to specific targets and are well known in the art. These include, but are not limited to, IL-1β, anti-TNF antibody (U.S. Patent No. 6,258,562), anti-IL-12 and/or anti-IL-12p40 antibody (U.S. Patent No. 6,914,128); anti-IL-18 antibody (US Publication No. 2005/0147610 A1), anti-C5, anti-CBL, anti-CD147, anti-gp120, anti-VLA-4, anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L, anti CD-40 (for example, see PCT Publication No. WO 2007/124299); anti-Id, anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-beta 2, anti-HGF, anti-cMet, anti DLL-4, anti-NPR1, anti-PLGF, anti-ErbB3, anti-E-selectin, anti-Fact VII, anti-Her2/neu, anti-F gp, anti-CD11/18, anti-CD14, anti-ICAM-3, anti-RON, anti CD-19 , anti-CD80 (for example, see PCT Publication No. WO 2003/039486), anti-CD4, anti-CD3, anti-CD23, anti-beta2-integrin, anti-alpha4beta7, anti-CD52, anti-HLA DR, anti-CD22 (see, for example, US Patent No. 5,789,554), anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR alpha beta, anti-CD2, anti-Hep B, anti- CA 125, anti-EpCAM, anti-gp120, anti-CMV, anti-gpIIbIIIa, anti-IgE, anti-CD25, anti-CD33, anti-HLA, anti-IGF1,2, anti-IGFR, anti-VNRintegrin, anti -IL-1alpha, anti-IL-1beta, anti-IL-1 receptor, anti-IL-2 receptor, anti-IL-4, anti-IL-4 receptor, anti-IL5, anti-IL receptor -5, anti-IL-6, anti-IL-6R, RANKL, NGF, DKK, alphaVbeta3, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor and anti -IL-23; IL-23p19; (see, Presta, LG, "Selection, design and engineering of therapeutic antibodies", J. Allergy Clin. Immunol., 116: 731-736 (2005) and on the website http://www.path.cam.ac .uk/~mrc7/humanisation/antibodies.html).
[0243]Precursor mAbs can also be selected from various therapeutic antibodies approved for use, either in clinical studies or in development for clinical use. These therapeutic antibodies include, among others, rituximab (Rituxan®, IDEC/Genentech/Roche) (see, for example, U.S. Patent No. 5,736,137), chimeric anti-CD20 antibody approved for the treatment of Non-Hodgkin's Lymphoma; HuMax-CD20, an anti-CD20 currently under development by Genmab, anti-CD20 antibody described in US Patent No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel) and PRO70769 (PCT Publication No. WO 2004/056312 (PCT/US2003/040426), entitled "Immunoglobulin Variants and Uses Thereof"), trastuzumab (Herceptin®, Genentech) (see, for example, US Patent No. 5,677,171), humanized antibody anti-Her2/neu approved for the treatment of breast cancer; pertuzumab (rhuMab-2C4, Omnitarg®), currently being developed by Genentech; anti-Her2 antibody described in U.S. Patent No. 4,753,894; cetuximab (Erbitux®, ImClone) (U.S. Patent No. 4,943,533; PCT Publication No. WO 96/40210), chimeric anti-EGFR antibody in clinical studies for a variety of cancers; ABX-EGF (U.S. Patent No. 6,235,883), currently being developed by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S. Serial No. 10/172,317, published as US 2003/0091561, currently U.S. Patent No. 7,247,301), currently under development by Genmab; 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (U.S. Patent No. 5,558,864; Murthy et al., Arch. Biochem. Biophys., 252(2): 773-783 (1991)); ICR62 (Institute of Cancer Research), PCT Publication No. WO 95/20045; 549-560 (1987); Rodeck et al., J. Cell Biochem., 35(4):315-320 (1987); Kettleborough et al., Protein Eng., 4(7): Modjtahedi et al., J. Cell Biophys., 22(1-3):129-146 (1993); Modjtahedi et al., Br. J. Cancer, 67(2):247-253 (1993); Modjtahedi et al., Br. J. Cancer, 73(2):228-2335 (1996); Modjtahedi et al., Int. J. Cancer, 105(2):273-280 (2003)); TheraCIM hR3 (YM Biosciences, Canada and Center for Molecular Immunology, Cuba (US Patent No. 5,891,996; US Patent No. 6,506,883; Mateo et al., Immunotechnology, 3(1):71-81(1997)); mAb -806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al., Proc. Natl. Acad. Sci. USA., 100(2): 639-644 (2003)); KSB-102 (KS Biomedix ); MR1-1 (IVAX, National Cancer Institute) (PCT Publication No. WO 01/62931); and SC100 (Scancell) (PCT Publication No. WO 01/88138); alentuzumab (Campath®, Millennium), humanized mAb currently approved for the treatment of B-cell chronic lymphocytic leukemia; muromonab-CD3 (Orthoclone OKT3®), anti-CD3 antibody developed by Ortho Biotech/Johnson & Johnson, ibritumomab tiuxetano (Zevalin®), anti-CD20 antibody developed by IDEC/Schering AG, gemtuzumab ozogamycin (Mylotarg®), anti-CD33 antibody (p67 protein) developed by Celltech/Wyeth, alefacept (Amevive®), anti-LFA-3 Fc, fusion developed by Biogen, abciximab (Reo Pro®), developed by Centocor/Lilly, basiliximab (Simulect®), developed by Novartis, palivizumab (Synagis®), developed by Medimmune, infliximab (Remicade®), anti-TNFalpha antibody developed by Centocor, adalimumab (Humira®), anti-TNFalpha antibody developed by Abbott Laboratories, Humicade®, anti-TNFalpha antibody developed by Celltech, golimumab (CNTO-148), fully human antibody against TNF developed by Centocor, etanercept (Enbrel®), p75 TNF-receptor Fc fusion developed by Immunex/Amgen, lenercept, p55TNF-Fc receptor fusion previously developed by Roche, ABX-CBL, anti-CD147 antibody being developed by Abgenix, ABX-IL8, anti-IL8 antibody under development by Abgenix, ABX-MA1, anti antibody -MUC18 being developed by Abgenix, Pemtumomab (R1549, 90Y-muHMFG1), anti-MUC1 antibody under development by Antisoma, Therex (R1550), anti-MUC1 antibody being developed by Antisoma, AngioMab (AS1405), being developed developed by Antisoma, HuBC-1, being developed by Antisoma, Thioplatin (AS1407) being developed by Antisoma, Antegren® (natalizumab), anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta- 7 being developed by Biogen, mAb against VLA-1, anti-VLA-1 integrin antibody being developed by Biogen, mAb against LTBR, anti-beta lymphotoxin receptor (LTBR) antibody being developed by Biogen, CAT-152, anti- TGF-β2 being developed by Cambridge Antibody Technology, ABT 874 (J695), anti-IL-12 p40 antibody being developed by Abbott Laboratories, CAT-192, anti-TGFβ1 antibody being developed by Cambridge Antibody Technology and Genzyme, CAT-213, anti-Eotaxin1 antibody being developed by Cambridge Antibody Technology, LymphoStat-B®, anti-Blys antibody being developed by Cambridge Antibody Technology and Human Genome Sciences Inc., TRAIL-R1mAb, anti-TRAIL-R1 antibody being developed by Cambridge Antibody Technology and Human Genome Science ces, Inc., Avastin® bevacizumab, rhuMAb-VEGF), anti-VEGF antibody being developed by Genentech, anti-HER family receptor antibody being developed by Genentech, Anti-Tissue Factor (ATF), anti-tissue factor antibody being developed by Genentech, Xolair® (Omalizumab), anti-IgE antibody being developed by Genentech, Raptiva® (Efalizumab), anti-CD11a antibody being developed by Genentech and Xoma, Antibody MLN-02 (formerly LDP-02), being developed by Genentech and Millennium Pharmaceuticals, HuMax CD4, anti-CD4 antibody being developed by Genmab, HuMax-IL15, anti-IL15 antibody being developed by Genmab and Amgen, HuMax-Inflam, being developed by Genmab and Medarex, HuMax-Cancer, anti-heparanase antibody I being developed by Genmab, Medarex and Oxford GcoSciences, HuMax-Lymphoma being developed by Genmab and Amgen, HuMax-TAC being developed by Genmab, IDEC-131 and anti-CD40L antibody being developed by IDEC Pharma ceuticals, IDEC-151 (Clenoliximab), anti-CD4 antibody being developed by IDEC Pharmaceuticals, IDEC-114, anti-CD80 antibody being developed by IDEC Pharmaceuticals, IDEC-152, anti-CD23 being developed by IDEC Pharmaceuticals, anti-factor antibodies macrophage migration (MIF) being developed by IDEC Pharmaceuticals, BEC2, anti-idiotypic antibody antibody being developed by ImClone, IMC-1C11, anti-KDR antibody being developed by ImClone, DC101, anti-flk-1 antibody being developed by ImClone , anti-VE cadherin antibodies being developed by ImClone, CEA-Cide® (labetuzumab), anti-carcinoembryonic antigen (CEA) antibody being developed by Immunomedics, LymphoCide® (Epratuzumab), anti-CD22 antibody being developed by Immunomedics, AFP-Cide , being developed by Immunomedics, MyelomaCide, being developed by Immunomedics, LkoCide, being developed by Immunomedics, ProstaCide, being developed by Immunomedics, MD X-010, anti-CTLA4 antibody being developed by Medarex, MDX-060, anti-CD30 antibody being developed by Medarex, MDX-070 being developed by Medarex, MDX-018 being developed by Medarex, Osidem® (IDM-1) and anti-Her2 antibody being developed by Medarex and Immuno-Designed Molecules, HuMax®-CD4, anti-CD4 antibody being developed by Medarex and Genmab, HuMax-IL15, anti-IL15 antibody being developed by Medarex and Genmab, CNTO 148, anti antibody -TNFα being developed by Medarex and Centocor/Johnson & Johnson, CNTO 1275, anti-cytokine antibody being developed by Centocor/Johnson & Johnson, MOR101 and MOR102, anti-intercellular adhesion molecule-1 antibodies (ICAM-1) (CD54) being developed by MorphoSys, MOR201, anti-fibroblast growth factor receptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion® (visilizumab), anti-CD3 antibody being developed by Protein Design Labs, HuZAF®, anti antibody - interferon gamma being developed by Protein Design Labs, Anti-α 5β1 Integrin, being developed by Protein Design Labs, anti-IL-12, being developed by Protein Design Labs, ING-1, anti-Ep-CAM antibody being developed by Xoma, Xolair® ( Omalizumab), humanized anti-IgE antibody developed by Genentech and Novartis and MLN01, anti-Beta2 integrin antibody being developed by Xoma. In another embodiment, therapeutic agents include KRN330 (Kirin); antibody to huA33 (A33, Ludwig Institute for Cancer Research); CNTO 95 (alpha V integrins, Centocor); MEDI-522 (alpha Vβ3 integrin, Medimmune); volociximab (alpha Vβ1 integrin, Biogen/PDL); human mAb 216 (B-cell glycosylated epitope, NCI); BiTE MT103 (CD19 x CD3 bispecific, Medimmune); 4G7xH22 (BcellxFcgammaR1 bispecific, Medarex/Merck KGa); rM28 (CD28 x MAPG bispecific, European Patent No. EP 1 444 268); MDX447 (EMD 82633) (CD64 x EGFR bispecific, Medarex); Catumaxomab (removab) (EpCAM x bispecific anti-CD3, Trion/Fres); Ertumaxomab (HER2/CD3 bispecific, Fresenius Biotech); oregovomab (OvaRex) (CA-125, ViRexx); Rencarex® (WX G250) (carbonic anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507) (CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab (Rituxan) (CD20, Genentech); veltuzumab (hA20) (CD20, Immunomedics); Epratuzumab (CD22, Amgen); lumiliximab (IDEC 152) (CD23, Biogen); Muromonab-CD3 (CD3 ortho); HuM291 (CD3 fc receptor, PDL Biopharma); HeFi-1, CD30, NCI); MDX-060 (CD30, Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, Seattle Genentics); SGN-33 (Lintuzumab) (CD33, Seattle Genentics); Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis); SGN-40 (CD40, Seattle Genentics); Campath1h (Alemtuzumab) (CD52, Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38, Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293 (TRC093/D93) (cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma); ipilimumab (MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab (Ticilimumab, CP-675.2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab) (DR4 agonist TRAIL-R1, Human Genome Science / Glaxo Smith Kline); AMG-655 (DR5, Amgen); Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-ETR2 (lexatumumab) (DR5 agonist TRAIL-R2, HGS); Cetuximab (Erbitux) (EGFR, ImClone); IMC-11F8, (EGFR, ImClone); Nimotuzumab (EGFR, YM Bio); Panitumumab (Vectabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANT Immunotherapeutics); adecatumumab (MT201) (Epcam, Merck); edrecolomab (Panorex, 17-1A) (Epcam, Glaxo/Centocor); MORAb-003 (folate receptor α, Morphotech); KW-2871 (GD3 ganglioside, Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX1307) (hCGb, Celldex); Trastuzumab (Herceptin) (HER2, Celldex); Pertuzumab (rhuMAb 2C4) (HER2 (DI), Genentech); apolizumab (HLA-DR beta chain, PDL Pharma); AMG-479 (IGF-1R, Amgen); anti-IGF-1R R1507 (IGF1-R, Roche); CP 751871 (IGF1-R, Pfizer); IMC-A12 (IGF1-R, ImClone); BIIB022 (IGF-1R, Biogen); Mik-beta-1 (IL-2Rb (CD122), Hoffman LaRoche); CNTO 328 (IL6, Centocor); Anti-KIR (1-7F9) (Killer Cell Ig-Like Receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute of Cancer Research); hCBE-11 (LTβR, Biogen); HuHMFGI (MUC1, Antisome/NCl); RAV12 (N-linked carbohydrate epitope, Raven); CAL (parathyroid hormone-related protein (PTH-rP), University of California); CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1, Medarex/Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa, ImClone); bavituximab (phosphatidylserine, Peregrine); huJ591 (PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research Foundation); GC1008 ((pan) TGFb (IgG4) inhibitor, Genzyme); Infliximab (Remicade) (TNFa, Centocor); A27.15 (Transferrin receptor, Salk Institute, INSERM, PCT Publication No. WO 2005/111082); E2.3 (transferrin receptor, Salk Institute); Bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab, PCT Publication No. WO 2000/034337, University of Texas); IMC-18F1 (VEGFR1, ImClone); IMC-1121 (VEGFR2, ImClone). C. Construction of DVD-Ig™ binding proteins
[0244] A multispecific multispecific immunoglobulin-like double variable domain binding protein (DVD-Ig™) is designed in such a way that two different light chain variable domains (VL) from two different precursor monoclonal antibodies are linked in tandem directly or by intermediated by a short linker by recombinant DNA techniques, followed by the light chain constant domain. Likewise, the heavy chain comprises two different heavy chain (VH) variable domains linked in tandem, followed by the CH1 constant domain and the Fc region.
[0245]The variable domains can be obtained by recombinant DNA techniques from a precursor antibody generated by any of the methods described herein. In one embodiment, the variable domain is a murine heavy or light chain variable domain. In another embodiment, the variable domain is a heavy or light chain grafted or humanized CDR variable domain. In one embodiment, the variable domain is a human heavy or light chain variable domain.
[0246] In one embodiment, the first and second variable domains are directly linked to each other using recombinant DNA techniques. In another embodiment the variable domains are linked via a linker sequence. In one modality, two variable domains are linked. Three or more variable domains can also be linked directly or via a linker sequence. Variable domains can bind to the same antigen or can bind to different antigens. DVD-Ig molecules of the invention may include an immunoglobulin variable domain and a variable domain that does not belong to immunoglobulins, such as ligand binding domain of a receptor, active domain of an enzyme. DVD-Ig molecules can also comprise two more non-Ig domains.
[0247] The linker sequence can be a single amino acid or a linker polypeptide comprising two or more amino acid residues joined by peptide bonds. In one embodiment, a linker sequence selected from the group consisting of GGGGSG (SEQ ID NO:26), GGSGG (SEQ ID NO:27), GGGGSGGGGS (SEQ ID NO:28), GGSGGGGSG (SEQ ID NO:223), GGSGGGGSGS (SEQ ID NO:29), GGSGGGGSGGGGS (SEQ ID NO:30), GGGGSGGGGSGGGG (SEQ ID NO:31), GGGGSGGGGSGGGGS (SEQ ID NO:32), ASTKGP (SEQ ID NO:33), ASTKGPSVFPLAP (SEQ ID NO :34), TVAAP (SEQ ID NO:35), RTVAAP (SEQ ID NO:224), TVAAPSVFIFPP (SEQ ID NO:36), RTVAAPSVFIFPP (SEQ ID NO:225), AKTTPKLEEGEFSEAR (SEQ ID NO:37), AKTTPKLEEGEFSEARV (SEQ ID NO:38), AKTTPKLGG (SEQ ID NO:39), SAKTTPKLGG (SEQ ID NO:40), SAKTTP (SEQ ID NO:41), RADAAP (SEQ ID NO:42), RADAAPTVS (SEQ ID NO: 43), RADAAAAGGPGS (SEQ ID NO:44), RADAAAAGGGGSGGGGSGGGGSGGGS (SEQ ID NO:45), SAKTTPKLEEGEFSEARV (SEQ ID NO:46), ADAAP (SEQ ID NO:47), ADAAPTVSIFPP (SEQ ID NO:48), QPKAAP ( SEQ ID NO:49), QPKAAPSVTLFPP (SEQ ID NO:50), AKTTPP (SEQ ID NO:51), AKTTPPSVTPLAP (SEQ ID NO:52), AKTTAP (SEQ ID NO:53), AKTTAPSVYPLAP (SEQ ID NO:54 ), GENKVEYAPALMALS (SEQ ID NO:55), GPAKELTPLKEAKVS (SEQ ID NO:56) and GHEAAAVMQVQYPAS (SEQ ID NO:57). The choice of linker sequences is based on the analysis of the crystal structure of various Fab molecules. There is a natural flexible linkage between the variable domain and the CH1/CL constant region in the Fab or antibody molecular structure. This natural linkage comprises approximately 10-12 amino acid residues, contributed by 4-6 residues from the C-terminus of the V domain and 4-6 residues from the N-terminus of the CL/CH1 domain. The DVD-Igs described herein can be generated using 5-6 amino acid residues from the N-terminus or 11-12 amino acid residues from CL or CH1 as a linker in the light chain or heavy chain of DVD-Ig, respectively. The N-terminal residues of the CL or CH1 domains, especially the first 5-6 amino acid residues, adopt a loop conformation without strong secondary structures and can therefore act as flexible linkers between the two variable domains. The N-terminal residues of the CL or CH1 domains are a natural extension of the variable domains as they are part of Ig sequences and therefore greatly minimize any immunogenicity potentially arising from linkers and junctions.
[0248]Other linker sequences can include any sequence of any length of the CL/CH1 domain, but not all residues of the CL/CH1 domain; for example, the first 5-12 amino acid residues of the CL/CH1 domains; light chain linkers can be from CK or CX; and heavy chain linkers can be derived from the CH1 of any isotypes, including Cy1, Cy2, Cy3, Cy4, Cα1, Cα2, Cδ, Cε and Cμ. Linker sequences can also be derived from other proteins such as Ig-like proteins, (e.g. TCR, FcR, KIR); G/S-based sequences; sequences derived from the hinge region; and other natural sequences from other proteins.
[0249] In one embodiment, a constant domain is linked to the two linked variable domains using recombinant DNA techniques. In one embodiment, a sequence comprising heavy chain in tandem linked domains is linked to a heavy chain constant domain and a sequence comprising light chain in tandem linked variable domains is linked to a light chain constant domain. In one embodiment, the constant domains are human heavy chain constant domains and human light chain constant domains, respectively. In one embodiment, the DVD heavy chain is further linked to an Fc region. The Fc region can be a native sequence Fc region or a variant Fc region. In another embodiment, the Fc region is a human Fc region. In another embodiment, the Fc region includes Fc region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.
[0250]In a more preferred embodiment, two heavy chain DVD polypeptides and two light chain DVD polypeptides are combined to form a DVD-Ig molecule. Detailed description of specific DVD-Ig molecules capable of binding to specific target antigens, such as IL-1β, and methods for producing them are provided in the Examples section below. D. Production of DVD-Ig binding proteins
[0251] DVD-Ig binding proteins of the present invention can be produced by any of a number of techniques known in the art, including, for example, expression from host cells, in which expression vector(s) that encode (m) DVD-Ig heavy chains and DVD-Ig light chains is (are) transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly employed for introducing exogenous DNA into a prokaryotic or eukaryotic host cell, eg, electroporation, calcium phosphate-mediated precipitation, DEAE-transfection. dextran and the like. While it is possible to express the DVD-Ig proteins of the invention in prokaryotic or eukaryotic host cells, DVD-Ig proteins are expressed in eukaryotic cells, eg mammalian host cells, because it is more likely than these eukaryotic cells (and especially mammalian cells) ), than prokaryotic cells, arrange themselves and secrete a properly folded and immunologically active DVD-Ig protein.
Exemplary mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77, 77 :4216-4220 (1980), used with a selectable marker DHFR, for example, as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982)), NS0 myeloma cells, cells, COS and SP2 and PER.C6 cells. When recombinant expression vectors encoding DVD-Ig proteins are introduced into mammalian host cells, DVD-Ig proteins are produced by culturing the host cells for a sufficient period of time to allow expression of the DVD-Ig proteins in the host cells or by secretion of DVD proteins into the culture medium in which the host cells are grown. DVD-Ig proteins can be recovered from the culture medium using standard protein purification methods.
[0253] In an exemplary system for recombinant expression of DVD-Ig proteins of the invention, a recombinant expression vector, encoding both DVD-Ig heavy chain and DVD-Ig light chain, is introduced into dhfr- cells. CHO by calcium phosphate-mediated transfection. Within the recombinant expression vector, the DVD-Ig heavy and light chain genes are each operably linked to regulatory elements, CMV enhancer/AdMLP promoter, to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which enables selection of CHO cells that have been transfected with the vector by selection with methotrexate/amplification. Selected transforming host cells are cultured to allow expression of DVD-Ig heavy and light chains and intact DVD-Ig protein is recovered from the culture medium. Standard molecular biology techniques are employed to prepare the recombinant expression vector, transfect host cells, select transformants, cultivate host cells, and recover DVD-Ig protein from the culture medium. Still further, the invention provides a method for synthesizing a DVD-Ig protein of the invention, comprising culturing a host cell of the invention in a suitable culture medium until a DVD-Ig protein of the invention is synthesized. The method may further include isolating the DVD-Ig protein from the culture medium.
[0254] An important feature of DVD-Ig is that it can be produced and purified in a manner similar to a conventional antibody. DVD-Ig production results in a single homogeneous main product with the desired dual-specific activity, without any modification to the constant region sequence or chemical modifications of any kind. Other previously described methods for generating "bispecific", "multispecific" and "multivalent multispecific" complete binding proteins do not lead to a single primary product, but rather to the intracellular or secreted production of a mixture of inactive, monospecific, assembled complete binding proteins, multispecific, multivalent and multivalent complete binding proteins with combination of different binding sites. By way of example, based on the design described by Miller and Presta (PCT Publication No. WO 2001/077342, there are 16 possible heavy and light chain combinations. Consequently, it is likely that only 6.25% of the protein is in the desired active form and not a single major product or single primary product when compared to the other 15 possible combinations. Separating the desired fully active forms of the protein from the inactive and partially active forms of the protein with standard chromatography techniques typically employed in bulk manufacturing scale, has yet to be demonstrated.
[0255] Surprisingly, the design of the "double-specific multivalent complete binding proteins" of the present invention leads to a variable double domain light chain and a variable double domain heavy chain that assemble primarily in the form of the "binding proteins double-specific multivalent completes” desired.
[0256]At least 50%, at least 75%, and at least 90% of assembled and expressed DVD-Ig molecules are of the desired dual-specific tetravalent protein. This aspect of the invention especially enhances the commercial utility of the invention. Therefore, the present invention includes a method for expressing a variable double domain light chain and a variable double domain heavy chain in a single cell, leading to a single primary product of a "double-specific tetravalent complete binding protein".
[0257] The present invention provides methods for expressing a variable double domain light chain and a variable double domain heavy chain in a single cell, leading to a "primary product" of a "double-specific tetravalent complete binding protein", wherein the "primary product" represents greater than 50% of all assembled protein, comprising a variable double domain light chain and a variable double domain heavy chain.
[0258] The present invention provides methods for expressing a variable double domain light chain and a variable double domain heavy chain in a single cell, leading to a "primary product" of a "double-specific tetravalent complete binding protein", wherein the "primary product" represents more than 75% of all assembled protein, comprising a variable double domain light chain and a variable double domain heavy chain.
[0259] The present invention provides methods for expressing a variable double domain light chain and a variable double domain heavy chain in a single cell, leading to a "primary product" of a "double-specific tetravalent complete binding protein", wherein the "primary product" represents more than 90% of all assembled protein, comprising a double variable domain light chain and a double variable domain heavy chain. 6. Production of IL-1β binding proteins and binding protein-producing cell lines
[0260] Preferably, the IL-1β binding proteins, including anti-IL-1β antibodies, of the present invention, exhibit high capacity to reduce or to neutralize the activity of IL-1β, for example, as assessed by anyone of various in vitro and in vivo assays known in the art. Preferably, the IL-1/3 binding proteins of the present invention also exhibit high capacity to reduce or neutralize IL-1/3 activity.
[0261] In preferred embodiments, a binding protein or its antigen-binding portion binds to human IL-1β, wherein the binding protein or its antigen-binding portion dissociates from human IL-1β with a constant to koff dissociation rate of approximately 0.1s-1 or less, as determined by surface plasmon resonance, or inhibit human IL-1β activity with IC50 of approximately 1 x 10-6M or less. Alternatively, the binding protein or its antigen-binding portion can dissociate from human IL-1β with a constant for the koff dissociation rate of approximately 1 x 10-2s-1 or less, as determined by surface plasmon resonance, or can inhibit human IL-1β activity with IC50 of approximately 1 x 10 -7M or less. Alternatively, the binding protein or its antigen-binding portion can dissociate from human IL-1β with a constant for the koff dissociation rate of approximately 1 x 10-3s-1 or less, as determined by surface plasmon resonance, or can inhibit human IL-1β activity with IC50 of approximately 1 x 10-8M or less. Alternatively, the binding protein or its antigen-binding portion can dissociate from human IL-1β with a dissociation rate constant of approximately 1 x 10-4s-1 or less, as determined by surface plasmon resonance, or can inhibit human IL-1β activity with IC50 of approximately 1 x 10-9M or less. Alternatively, the binding protein or its antigen-binding portion can dissociate from human IL-1β with a dissociation rate constant of approximately 1 x 10-5s-1 or less, as determined by surface plasmon resonance, or can inhibit human IL-1β activity with IC50 of approximately 1 x 10-10M or less. Alternatively, the binding protein or its antigen-binding portion can dissociate from human IL-1β with a dissociation rate constant of approximately 1 x 10-5s-1 or less, as determined by surface plasmon resonance, or can inhibit human IL-1β activity with IC50 of approximately 1 x 10-11M or less.
[0262] In certain embodiments, the binding protein comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. Preferably, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In addition, the antibody may comprise a light chain constant region, either kappa light chain constant region or lambda light chain constant region. Preferably, the antibody comprises a kappa light chain constant region. Alternatively, the antibody portion can be, for example, a single-chain Fab fragment or Fv fragment.
[0263] Substitutions of amino acid residues in the Fc portion to alter the effector function of antibodies are known in the prior art (Winter et al., U.S. Patent Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions, for example, cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases, these effector functions are desirable for therapeutic antibodies, but in others, they might be unnecessary or even harmful, depending on the therapeutic goals. Certain human IgG isotypes, especially IgG1 and IgG3, mediate ADCC and CDC by binding to FCYRS and complement C1q, respectively. Neonatal Fc receptors (FcRn) are the critical components that determine the circulating half-life of antibodies. In yet another embodiment, at least one amino acid residue is substituted in the antibody constant region, e.g., the antibody Fc region, such that the antibody effector functions are altered.
[0264] An embodiment provides a labeled binding protein, wherein an antibody or antibody portion of the invention is derived or linked to another functional molecule (eg, another peptide or protein). For example, a tagged binding protein of the invention can be derived by functionally linking an antibody or antibody part of the invention (by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other entities, such as another antibody (eg, bispecific antibody or diabody), a detectable agent, cytotoxic agent, pharmaceutical agent, and/or a protein and/or peptide that can mediate the association of the antibody or part of the antibody with another molecule (such as the core region of streptavidin or a polyhistidine tag).
[0265] Useful detectable agents from which a binding protein, such as an antibody or antibody part of the invention, can be derived include fluorescent compounds. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. An antibody can also be derived with detectable enzymes such as alaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When derived from a detectable enzyme, the antibody is detected by adding other reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product which is detectable. An antibody can also be derived from biotin and detected by indirectly measuring binding to avidin or streptavidin.
[0266] Another embodiment of the invention provides a crystallized binding protein. Preferably, the invention relates to whole anti-IL-1β antibody crystals and fragments thereof, as described herein, and to formulations and compositions containing these crystals. In one embodiment, the crystallized binding protein has a longer half-life in vivo than the soluble version of the binding protein. In another embodiment, the binding protein retains biological activity after crystallization.
[0267] The crystallized binding protein of the invention can be produced according to methods known in the art and as disclosed in PCT Publication No. WO 02/072636, incorporated herein by reference in this patent application.
[0268] Another embodiment of the invention provides a glycosylated binding protein, wherein the antibody or its antigen-binding portion comprises one or more carbohydrate residues. Nascent protein production in vivo can undergo further processing, known as post-translational modification. Specifically, sugar residues (glycosyl) can be added enzymatically, a process known as glycosylation. The resulting proteins, carrying covalently linked oligosaccharide side chains, are known as glycosylated proteins or glycoproteins.
[0269] Natural antibodies are glycoproteins with one or more carbohydrate residues in the Fc domain as well as in the variable domain. Carbohydrate residues in the Fc domain have an important effect on the effector function of the Fc domain, with minimal effect on antigen binding or antibody half-life (Jefferis, R., Biotechnol. Prog., 21: 11-16 (2005)). On the other hand, variable domain glycosylation may have an effect on the antigen-binding activity of the antibody. Variable domain glycosylation can have a negative effect on antibody binding affinity, probably due to steric hindrance (Co et al., Mol. Immunol., 30: 1361-1367 (1993)), or result in higher affinity by the antigen (Wallick et al., J. Exp. Med., 168:1099-1109 (1988); Wright et al., EMBO J., 10:2717-2723 (1991)).
[0270] Another aspect of the present invention is directed to the generation of mutants in glycosylation sites, in which the O- or N-linked glycosylation site of the binding protein has undergone a mutation. The person skilled in the art is able to generate such mutants using well-known standard technologies. Mutants in the glycosylation site that retain biological activity, but show increased or decreased binding activity, represent another object of the present invention.
[0271] In yet another embodiment, the glycosylation of the antibody or antigen-binding portion of the invention is modified. For example, an aglycosylated antibody can be produced (i.e., the antibody lacking glycosylation). Glycosylation can be altered to, for example, increase antibody affinity for antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites inserted into the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more glycosylation sites in the variable region, thereby eliminating glycosylation at that site. Such aglycosylation can increase antibody affinity for antigen. This approach is described in detail in PCT Publication No. WO 2003/016466 and in U.S. Patent Nos. 5,714,350 and 6,350,861.
[0272] Additionally or alternatively, a modified binding protein of the invention can be produced that has an altered type of glycosylation, such as a hypofucosylated antibody with reduced amounts of fucosyl residues (see, Kanda et al., J. Biotechnol., 130( 3): 300-310 (2007)), or an antibody with more structures bifurcated with GlcNAc. These altered glycosylation patterns have been shown to increase the ADCC capacity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation mechanism. Cells with altered glycosylation mechanism have been described in the state of the art and can be used as host cells to express the recombinant antibodies of the invention and thus produce an antibody with altered glycosylation. See, for example, Shields et al., J. Biol. Chem., 277: 26733-26740 (2002); Umana et al., "Engineered glycoforms of an IgG1 antineuroblastoma with optimized antibody-dependent cellular cytotoxic activity", Nat. Biotechnol., 17: 176-180 (1999), as well as European Publication No. EP 1 176 195; PCT Publication Nos. WO 03/035835 and WO 99/54342.
[0273] Protein glycosylation depends on the amino acid sequence of the protein of interest as well as the host cell in which the protein is expressed. Different organisms can produce different glycosylation enzymes (eg, glycosyltransferases and glycosidases) and have different substrates (nucleotide sugars) available. In view of such factors, the glycosylation pattern of proteins and the composition of glycosyl residues may differ depending on the host system in which the particular protein is expressed. Glycosyl residues useful in the invention can include, among others, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. Preferably, the glycosylated binding protein comprises glycosyl residues such that the glycosylation pattern is human.
[0274] It is known to those skilled in the art that diverse protein glycosylation can result in different characteristics in the protein. For example, the effectiveness of a therapeutic protein produced in a host microorganism, such as yeast, and glycosylated using the endogenous yeast pathway may be less than that of the same protein expressed in a mammalian cell, such as the CHO cell line. Such glycoproteins can also be immunogenic in humans and have shown reduced half-life in vivo after administration. Specific receptors in humans and other animals can recognize specific glycosyl residues and promote rapid clearance of the protein from the bloodstream. Other adverse effects may include alterations in protein folding, solubility, susceptibility to proteases, transit, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity or allergenicity. Thus, a practicing practitioner may prefer a therapeutic protein with a specific composition and pattern of glycosylation, for example, composition and pattern of glycosylation identical or at least similar to those produced in human cells or species-specific cells of the intended animal. .
[0275]Expressing glycosylated proteins different from that of a host cell can be achieved by genetically modifying the host cell to express heterologous glycosylation enzymes. With techniques known in the art, a practitioner in the art will generate antibodies or antigen-binding portions thereof exhibiting human protein glycosylation. For example, yeast strains have been genetically engineered to express unnatural glycosylation enzymes such that glycosylated proteins (glycoproteins) produced in these yeast strains exhibit protein glycosylation identical to that of animal cells, especially human cells (US Publication Nos. 2004/0018590 and 2002/0137134).
[0276] In addition to binding proteins, the present invention is also directed to anti-idiotypic (anti-Id) antibodies specific for such binding proteins of the invention. An anti-Id antibody is an antibody that recognizes unique determinants generally associated with the antigen-binding region of another antibody. Anti-Id can be prepared by immunizing an animal with the binding protein or a CDR containing a region thereof. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an anti-Id antibody. It is quickly evident that it may be easier to generate anti-idiotypic antibodies against the two or more precursor antibodies incorporated into a DVD-Ig molecule; and confirm binding with studies by methods well recognized in the art (eg BIAcore, ELISA) to verify that anti-idiotypic antibodies to the idiotype of each precursor antibody also recognize the idiotype (eg the binding site to antigen) in the context of DVD-Ig. Anti-idiotypic antibodies specific for each of the two or more antigen-binding sites of a DVD-Ig serve as ideal reagents for measuring concentrations of DVD-Ig from a human DVD-Ig in patient serum. For example, DVD-Ig concentration assays can be established using a "sandwich format for the ELISA assay" with an antibody against a first antigen-binding region, coated onto the solid phase (eg, BIAcore chip, plate of ELISA, etc.), rinse with wash buffer, incubation with a serum sample, another rinse step, and finally incubation with another anti-idiotypic antibody against the other antigen-binding site, the one labeled with an enzyme for quantifying the binding reaction. In one embodiment, for a DVD-Ig with more than two different binding sites, anti-idiotypic antibodies against the two outermost binding sites (the most distal and proximal of the constant region) will not only help to determine the concentration of DVD-Ig in human serum, but will also document the integrity of the molecule in vivo. Each anti-Id antibody can also be used as an "immunogen" to induce an immune response in yet another animal. using a so-called anti-anti-Id antibody.
[0277] Additionally, it will be recognized by the person skilled in the art that a protein of interest can be expressed using a library of host cells genetically constructed to express various glycosylation enzymes, such that host cells that are part of the library produce the protein of interest with patterns glycosylation variants. A practitioner can then select and isolate the protein of interest with specific new glycosylation patterns. Preferably, the protein with a specially selected new glycosylation pattern exhibits improved or altered biological properties. 7. Uses of IL-1β binding proteins
[0278] Considering their ability to bind to human IL-1β, the IL-1β binding proteins, or antigen-binding portions thereof, of the invention can be used to detect IL-1β (eg, in biological sample, such as serum or plasma, employing a conventional immunoassay, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry. The invention provides a method for detecting IL-1β in a biological sample, comprising bringing a biological sample into contact with a binding protein, or antigen-binding portion, of the invention and detecting the binding protein (or antigen-binding portion) bound to IL-1β or the unbound binding protein (or binding portion), thereby detecting IL-1β in biological sample. The binding protein is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups s, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescein dichlorotriazinylamine, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho or 153Sm.
[0279] Alternatively to binding protein labeling, human IL-1β can be analyzed in biological fluids by a competition immunoassay using rh IL-1β patterns labeled with a detectable substance and an unlabelled IL-1β binding protein . In this assay, the biological sample, labeled rh IL-1β standards, and human IL-1β binding protein are combined, and the amount of labeled rh IL-1β standard bound to the unlabeled antibody is determined. The amount of human IL-1β in the biological sample is inversely proportional to the amount of labeled rh IL-1β standard bound to the IL-1β binding protein. Likewise, human IL-1β can also be analyzed in biological fluids by a competition immunoassay, using rh IL-1β standards labeled with a detectable substance and an unlabeled human IL-1β binding protein.
[0280] The binding proteins and binding portions to IL-1β of the invention are preferably capable of neutralizing human IL-1β activity in vitro and in vivo. Thus, such binding proteins and their IL-1β binding portions of the invention can be used to inhibit the activity of human IL-1β, for example, in a cell culture containing human IL-1β, in humans and others. mammals having IL-1β with which an antibody of the invention cross-reacts. In one embodiment, the invention provides a method for inhibiting the activity of human IL-1/3, comprising contacting human IL-1β with an IL-1β binding protein, or binding portion thereof, of the invention in such a way that the activity of human IL-1β is inhibited. For example, in a cell culture containing or suspected of containing human IL-1β, an IL-1β binding protein, or binding portion thereof, of the invention can be added to the culture medium to inhibit IL-1β activity. human in culture.
[0281] In another embodiment, the invention provides a method for reducing human IL-1β activity in a subject, advantageously in a subject suffering from a disease or disorder in which IL-1β activity is detrimental. The invention provides methods for reducing IL-1/3 activity in a subject suffering from such a disease or disorder, which method comprises administering to the subject an antibody or antibody portion of the invention such that the IL-1/3 activity in the subject is reduced . Preferably, the IL-1/3 is human IL-1/3 and the subject is human. Alternatively, the subject may be a mammal expressing an IL-1β to which an antibody of the invention is capable of binding. Still further, the subject may be a mammal into which IL-1/3 has been introduced (for example, by administration of IL-1/3 by an expression of IL-1/3 transgenes). An IL-1β binding protein of the invention can be administered to a human subject for therapeutic purposes. Furthermore, a binding protein of the invention can be administered to a non-human mammal that expresses an IL-1β to which the antibody is capable of binding for veterinary purposes or as an animal model of human disease. With respect to the latter, such animal models may be useful to assess the therapeutic efficacy of antibodies of the invention (e.g., dose tests and administration time cycles).
[0282] In this descriptive report, the term "disorder in which IL-1β activity is harmful" is intended to include diseases and other disorders in which the presence of IL-1β in an individual suffering from the disorder has been demonstrated or suspected to be responsible for the pathophysiology of the disorder or a factor that contributes to the worsening of the disorder. Thus, a disorder in which IL-1β activity is detrimental is a disorder in which reduced IL-1β activity is predicted to alleviate the symptoms and/or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of IL-1β in a biological fluid of an individual suffering from the disorder (for example, an increase in the concentration of IL-1β in the individual's serum, plasma, synovial fluid, etc. ), which can be detected, for example, using an anti-IL-1/3 antibody as described above. Non-limiting examples of disorders that can be treated with the antibodies of the invention include those discussed in the section below pertaining to pharmaceutical compositions of the antibodies of the invention.
[0283] The DVD-Igs of the invention can bind to IL-1β only or to multiple antigens (eg human IL-1β and another antigen other than IL-1β). Therefore, a DVD-Ig can block or reduce the activity of hu IL-1β and the activity of another target antigen. Such other target antigens may include soluble targets (eg IL-1α) and cell surface receptor targets (eg VEGFR, EGFR).
[0284] Such other antigens include, but are not limited to, targets listed in publicly available databases, which include those made available on the Internet web and which are incorporated herein by reference in this patent application. These target databases include:
[0285] Therapeutic targets (http://xin.cz3.nus.edu.sg/group/cjttd/ttd.asp); Cytokines and cytokine receptors (http://www.cytokinewebfacts.com/, http://www.copewithcytokines.de/cope.cgi and http://cmbi.bjmu.edu.cn/cmbidata/cgf/CGF_Database/cytokine .medic.kumam oto-u.ac.jp/CFC/indexR.html); Chemokines (http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html); Chemokine Receptors and GPCRs (http://csp.medic.kumamoto-u.ac.jp/CSP/Receptor.html, http://www.gpcr.org/7tm/); Olfactory receptors (http://senselab.med.yale.edu/senselab/ORDB/default.asp); Receivers (http://www.iuphar-db.org/iuphar-rd/list/index.htm); Cancer targets (http://cged.hgc.jp/cgi-bin/input.cgi); Secreted proteins as potential targets for antibodies (http://spd.cbi.pku.edu.cn/); Protein kinases (http://spd.cbi.pku.edu.cn/) and Human CD Markers (http://content.labvelocity.com/tools/6/1226/CD_table_final_locked.pdf) and (Zola et al. , "CD molecules 2005: human cell molecules differentiation", Blood, 106: 3123-3126 (2005)).
[0286]DVD-Igs useful as therapeutic agents to simultaneously block two or more different targets, i.e. human IL-1β and one or more different IL-1β target antigens, and enhance efficacy/safety and/or increase coverage to the patient. Such targets can include soluble targets (TNF) and cell surface receptor targets (VEGFR and EGFR).
[0287] Additionally, DVD-Igs of the invention can be employed for tissue-specific release (targeted to a tissue marker and a disease mediator to improve local PK and thus increase efficacy and/or decrease toxicity), including release intracellular (targeted to an internalizing receptor and an intracellular molecule), release into the brain (targeted to the transferrin receptor and a mediator of disease in the central nervous system for passage across the blood-brain barrier). DVD-Ig can also serve as a transporter protein to carry an antigen to a specific location, through binding to a non-neutralizing epitope of that antigen, and also to increase the half-life of the antigen. In addition, DVD-Ig can be designed to physically attach to medical devices implanted in patients or to target these medical devices (see Burke et al., "Zotarolimus eluting stents", Adv. Drug Deliv. Rev., 58 (3): 437-446 (2006); Hildebrand et al., "Surface coatings for biological activation and functionalization of medical devices", Surface and Coatings Technology, 200(22-23): 6318-6324 (2006); Wu et al., "Drug/device combinations for local drug therapies and infection prophylaxis", Biomaterials, 27: 2450-2467 (2006); Marques et al., "Mediation of the Cytokine Network in the Implantation of Orthopedic Devices", chapter 21, In Biodegradable Systems in Tissue Engineering and Regenerative Medicine, (Reis et al., eds.) (CRC Press LLC, Boca Raton, 2005) pp. 377-397). Briefly, proper targeting of cell types to the medical implant site can promote healing and restore normal tissue function. Alternatively, inhibition of mediators (including, but not limited to, cytokines), released upon device implantation, by a DVD-coupled or directed to a device is also provided. For example, stents have been used for years in interventional cardiology to release blocked arteries and to improve blood flow to the heart muscle. However, traditional bare metal stents are known to cause restenosis (re-narrowing of the artery in a treated area) in some patients and can lead to blood clots. Recently, an anti-CD34 antibody-coated stent has been described that reduces restenosis and prevents the occurrence of blood clots by capturing progenitor endothelial cells (EPC) that circulate throughout the blood. Endothelial cells are cells that line blood vessels, allowing blood to flow smoothly. The EPCs adhere to the rigid surface of the stent, forming a smooth layer that not only promotes healing but also prevents restenosis and clot formation, complications previously associated with the use of stents (Aoki et al., J. Am. Coll. Cardiol., 45(10): 1574-1579 (2005)). In addition to improving outcomes for patients who need stents, there are also implications for patients who require cardiovascular bypass surgery. For example, a prosthetic vascular tube (artificial artery) coated with anti-EPC antibodies would eliminate the need to use arteries in patients' legs or arms for grafts in bypass surgery. This will reduce surgery and anesthesia time, which in turn will reduce the number of deaths from coronary artery surgery. DVD-Ig are designed in such a way that they bind to a cell surface marker (such as CD34) as well as a protein (or an epitope of any type, including but not limited to proteins, lipids and polysaccharides) that has been coated over the implanted device to facilitate cell recruitment. Such approaches can also be applied to other medical implants in general. Alternatively, DVD-Igs can be coated onto medical devices and upon implantation and release all device DVDs (or any other need that may require additional fresh DVD-Igs, including aging and denaturation of already loaded DVD-Ig) , the device could be recharged by systemic administration of fresh DVD-Ig to the patient, where DVD-Ig is designed to bind to a target of interest (a cytokine, a cell surface marker (such as CD34) etc.) with one set of target binding sites coated on the device (including a protein, an epitope of any type such as lipids, polysaccharides and polymers) with the other. This technology offers the advantage of extending the usefulness of coated implants. A. Use of DVD-Igs in various diseases
[0288] The DVD-Ig molecules of the invention are also useful as therapeutic molecules to treat various diseases. These DVD molecules can bind to one or more targets involved in a specific disease. Examples of these targets in various diseases are described below. Human autoimmune and inflammatory response
[0289] In one aspect, a DVD-Ig binding protein of the invention is capable of binding to human IL-1β and to one or more antigens that have been implicated in autoimmune and inflammatory responses in general, including C5, CCL1 (I -309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2 / eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b ), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11 (I-TAC / IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78 / LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 , CX3CR1, IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCY1 ( endothelial monocyte activating cytokine), SPP1, TNF, TNFSF5, IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9 R, ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAK1, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF4, TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z, CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, CXCL12, CXCR4 SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2, BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A , IL1R1, IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL11RA , IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA, LTB, LTB4R, LTB4R2, LTPB, MIF, NP, PDGFB, TBX21, TDGF1, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11 TNF, TNFSF4 VEGF, ZFPM2 and RNF110 (ZNF144). Asthma
[0290]Allergic asthma is characterized by the presence of eosinophilia, goblet cell metaplasia, changes in epithelial cells, airway hyperreactivity (AHR) and expression of Th2 and Th1 cytokines, as well as by elevated serum levels of IgE. It is now widely accepted that airway inflammation is the fundamental factor underlying the pathogenesis of asthma, involving a complex participation of inflammatory cells such as T cells, B cells, eosinophils, mast cells and macrophages and their secreted mediators, including cytokines and chemokines. Corticosteroids are currently the most important anti-inflammatory treatment for asthma, however, their mechanism of action is non-specific, and there is concern about safety, especially in the population of young patients. The development of more specific and more targeted therapies is therefore warranted.
[0291] Animal models, such as the mouse OVA-induced asthma model, in which inflammation and AHR can be assessed, are known in the art and can be used to determine the capacity of various DVD-Ig molecules to treat asthma. Animal models that study asthma are described in Coffman et al., J. Exp. Med., 201(12): 1875-1879 (2005); Lloyd et al., Adv. Immunol., 77: 263-295 (2001); Boyce et al., J. Exp. Med., 201(12): 1869-1873 (2005); and Snibson et al., Clin. Exp. Allergy, 35(2): 146-152 (2005). In addition to routine safety assessments of these target pairs, specific tests for the degree of immunosuppression may be warranted and useful in selecting the best target pairs (see Luster et al., Toxicology, 92(1-3): 229-243 (1994); Descotes, J., Develop. Biol. Standard., 77: 99-102 (1992); Hart et al., J. Allergy Clin. Immunol., 108(2): 250257 (2001)).
[0292] One aspect of the invention pertains to DVD-Ig molecules capable of binding to IL-1β and to one or more, for example, two targets selected from the group consisting of IL-4, IL-5, IL- 8, IL-9, IL-13, IL-18, IL-5R(α), TNFSF4, IL-4R(a), interferon α, eotaxin, TSLP, PAR-2, PGD2 and IgE. One modality includes a dual-specific anti-IL-1β/IL-1α DVD-Ig as a beneficial therapeutic agent for the treatment of asthma. Rheumatoid arthritis (RA)
[0293] Rheumatoid arthritis (RA), a systemic disease, is characterized by a chronic inflammatory reaction in the joint synovium and is associated with cartilage degeneration and juxta-articular bone erosion. Many pro-inflammatory cytokines, including TNF, chemokines and growth factors are expressed in diseased joints. Systemic administration of anti-TNF antibody or sTNFR fusion protein to mouse models of AR has been shown to be anti-inflammatory and protective of joints. Several cytokines, including IL-1β, have been implicated in RA. Clinical investigations in which TNF activity in RA patients was blocked by intravenously administered infliximab (Harriman et al., "Summary of clinical trials in rheumatoid arthritis using infliximab, an anti-TNFalpha treatment", Ann. Rheum. Dis. , 58 (Suppl 1): I61-I64 (1999)), a chimeric anti-TNF mAb, provided evidence that TNF regulates IL-6, IL-8, MCP-1 and VEGF production, cell recruitment inflammatory and immune effects to joints, angiogenesis and reduced blood levels of matrix metalloproteinases 1 and 3. A better understanding of the inflammatory pathway in rheumatoid arthritis has led to the identification of other therapeutic targets involved in rheumatoid arthritis. Promising treatments such as interleukin-6 antagonists (MRA antibody against IL-6 receptor, developed by Chugai, Roche (see Nishimoto et al., Arthritis Rheum., 50(6): 1761-1769 (2004)), CTLA4Ig (abatacept, Genovese et al., "Abatacept for rheumatoid arthritis refractory to tumor necrosis factor alpha inhibition" N. Engl. J. Med., 353: 1114-1123 (2005)) and anti-B cell therapy (rituximab, Okamoto et al., "Rituximab for rheumatoid arthritis," N. Engl. J. Med., 351: 1909 (2004)), have already been tested in randomized controlled trials over the past year. , as IL-15 and IL-18, has been identified using animal models of RA (therapeutic antibody, HuMax-IL_15, AMG 714, see Baslund et al., Arthritis Rheum., 52(9): 2686-2692 (2005)) Dual-specific antibody therapy, combining anti-TNF and another mediator such as IL-1β, has great potential to increase clinical efficacy and/or patient coverage. ear TNF and VEGF can potentially eradicate inflammation and angiogenesis, both of which are involved in the pathophysiology of AR. A DVD-Ig binding protein capable of blocking IL-1α and IL-1β is considered. In addition to routine safety assessments of these target pairs, specific tests for the degree of immunosuppression may be warranted and useful to select the best target pairs (see Luster et al., Toxicology, 92(1-3): 229-243 (1994); Descotes et al., Develop. Biol. Standard., 77: 99-102 (1992); Hart et al., J. Allergy Clin. Immunol., 108(2): 250-257 (2001)) . Whether a DVD-Ig molecule will be useful for the treatment of rheumatoid arthritis can be assessed using preclinical animal models of AR, such as the mouse collagen-induced arthritis model. Other useful models are also well known in the art (see Brand, D.D., Comp. Med., 55:114-122 (2005)). Based on the cross-reactivity of precursor antibodies to human and mouse orthologs (eg, reactivity to human and mouse TNF, human and mouse IL-15, etc.), validation studies in the mouse CIA model can be conducted with DVD-Ig molecules derived from “matching surrogate antibody”. Briefly, a DVD-Ig based on two (or more) mouse target-specific antibodies can be matched as far as possible to the characteristics of human precursor or humanized antibodies used for the construction of human DVD-Ig (similar affinity, similar potency of neutralization, similar half-life, etc.).
[0294] In one embodiment, a DVD-Ig of the invention that binds to human IL-1β and to a target other than IL-1β can also be used to treat other diseases in which IL-1β participates. Such diseases include, but are not limited to, SLE, multiple sclerosis (MS), sepsis, various neurological diseases and cancers (including cervical, breast, gastric). A more extensive list of diseases and disorders in which IL-1β participates is also provided below.
[0295] One embodiment of the invention pertains to DVD-Ig molecules capable of binding to human IL-1β and to one or more targets selected from the group consisting of IL-1α, TNFa, IL-12, TWEAK, IL- 23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNFβ, CD45RB, CD200, IFN-y, GM-CSF, FGF, C5, CD52, sclerostin and CCR2. Systemic lupus erythematosus (SLE)
[0296] The immunopathogenic hallmark of SLE is the activation of polyclonal B cells, which leads to hyperglobulinemia, the production of autoantibodies and the formation of immune complexes. The fundamental abnormality appears to be the fact that T cells do not suppress the banned B cell clones, caused by generalized dysregulation of T cells. Additionally, the interaction of B and T cells is facilitated by several cytokines, such as IL-10, as well as by costimulatory molecules such as CD40 and CD40L, B7 and CD28 and CTLA-4, which initiate the second signal. These interactions, associated with compromised clearance of immune complexes and apoptotic material by phagocytic cells, perpetuate the immune response with resulting tissue damage.
[0297] In one aspect, a DVD-Ig binding protein of the invention is capable of binding to human IL-1β and to one or more of the following antigens that have been implicated in SLE: B cell-targeted therapies: CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGS1, SLA2, CD81, IFNB1, IL10, TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA and NT5E.; costimulatory signals: CTLA4 or B7.1/B7.2; inhibition of B cell survival: BlyS, BAFF; Complement inactivation: C5; Cytokine modulation: The overriding principle is that the ultimate biological response in any tissue results from a balance between local levels of pro-inflammatory or anti-inflammatory cytokines (see Sfikakis et al., Curr. Opin. Rheumatol., 17:550- 557 (2005)). SLE is considered a Th-2 driven disease with documented serum elevations of IL-4, IL-6, IL-10. DVD-Igs capable of binding to one or more targets selected from the group consisting of IL-4, IL-6, IL-10, IFN-α and TNF-α are also being considered. The combination of targets discussed here will increase therapeutic efficacy for SLE, which can be tested in some preclinical models of lupus (see, Peng S.L., Methods Mol. Med., 102: 227-272 (2004)). Based on the cross-reactivity of precursor antibodies to human and mouse orthologs (eg, reactivity to human and mouse CD20, human and mouse white interferon, etc.), validation studies in the mouse lupus model can be conducted with DVD-Ig molecules derived from "matching surrogate antibody". Briefly, a DVD-Ig based on two (or more) mouse target-specific antibodies can be matched as far as possible to the characteristics of human precursor or humanized antibodies used for the construction of human DVD-Ig (similar affinity, similar potency of neutralization, similar half-life, etc.). Multiple Sclerosis (MS)
[0298] Multiple sclerosis (MS) is a complex human autoimmune disease with predominantly unknown etiology. Immunological destruction of myelin basic protein (MBP) throughout the nervous system is the main pathology of multiple sclerosis. MS is a disease of complex pathologies, involving infiltration by CD4+ T cells and CD8+ T cells and response within the central nervous system. The expression in the central nervous system of cytokines, reactive nitrogen species and costimulatory molecules have all been described in MS. Of important consideration are the immunological mechanisms that contribute to the development of autoimmunity. Specifically, the expression of antigens, cytokine and leukocyte interactions and regulatory T cells, which help to balance/modulate other T cells, such as Th1 and Th2 cells, are important areas for the identification of therapeutic targets.
[0299]IL-12 is a pro-inflammatory cytokine produced by APC that promotes the differentiation of effector Th1 cells. IL-12 is produced in the developing lesions of patients with MS as well as in animals affected by EAE. It has been previously shown that interfering with IL-12 pathways effectively prevents EAE in rodents, and that in vivo neutralization of IL-12p40 with an anti-IL-12 mAb has beneficial effects in the myelin-induced EAE model in common tamarins. TWEAK is a member of the TNF family, constitutively expressed in the central nervous system (CNS), with pro-inflammatory, proliferative or apoptotic effects, depending on cell types. Its receptor, Fn14, is expressed in the CNS by endothelial cells, reactive astrocytes and neurons. TWEAK and Fn14 mRNA expression increased in the spinal cord during experimental autoimmune encephalitis (EAE). Anti-TWEAK antibody treatment of oligodendrocyte myelin glycoprotein (MOG)-induced EAE in C57BL/6 mice resulted in reduced disease severity and leukocyte infiltration when the mice were treated after the presentation phase.
[0300] One aspect of the invention pertains to molecules of DVD-Ig molecules capable of binding to IL-1β and to one or more, for example, two targets selected from the group consisting of IL-12, TWEAK, IL-23 , CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200, IFNgamma, GM-CSF, FGF, C5, CD52, osteopontin and CCR2. One modality includes an anti-IL-1β /TWEAK dual-specific DVD-Ig as a beneficial therapeutic agent for the treatment of MS.
[0301] Several animal models to assess the usefulness of DVD-Ig molecules in the treatment of MS are known in the prior art (see Steinman et al., Trends Immunol., 26(11): 565-571 (2005); Lublin et al., Springer Semin Immunopathol., 8(3): 197-208 (1985); Genain et al., J. Mol. Med., 75(3): 187-197 (1997); J. Exp. Med., 189(7): 1033-1042 (1999); Owens et al., Neurol. Clin., 13(1): 51-73 (1995); and 't Hart et al., J Immunol., 175(7): 4761-4768 (2005)). Based on the cross-reactivity of precursor antibodies to human and mouse orthologs (eg reactivity to human and mouse IL-1β, human and mouse TWEAK, etc.), validation studies in the mouse lupus model can be conducted with DVD-Ig molecules derived from “matching surrogate antibody”. Briefly, a DVD-Ig based on two (or more) mouse target-specific antibodies can be matched as far as possible to the characteristics of human precursor or humanized antibodies used for the construction of human DVD-Ig (similar affinity, similar potency of neutralization, similar half-life, etc.). The same concept applies to animal models in other rodent species, where a DVD-Ig derived from “matched surrogate antibody” would be selected for the planned pharmacology and possibly safety studies. In addition to routine safety assessments of these specific pairs of targets, tests specific to the degree of immunosuppression may be justified and useful to select the best pairs of targets (see Luster et al., Toxicology, 92(1-3): 229- 243 (1994); Descotes et al., Develop. Biol. Standard., 77: 99-102 (1992); Jones, R., "Rovelizumab -ICOS Corp", IDrugs, 3(4):442-446 (2000) )). sepsis
[0302]The pathophysiology of sepsis is initiated by external membrane components of gram negative organisms (lipopolysaccharide [LPS], lipid A, endotoxin) and gram positive organisms (lipoteic acid, peptidoglycan). These outer membrane components are capable of binding to the CD14 receptor on the surface of monocytes. Due to the recently described toll-like receptors, a signal is then transmitted to the cell, leading to the final production of the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1). Overwhelming inflammatory and immune responses are essential features of septic shock, in addition to playing a central role in the pathogenesis of tissue damage, multiple organ failure, and sepsis-induced death. Cytokines, especially tumor necrosis factor (TNF) and interleukin (IL-1), have been shown to be critical mediators of septic shock. These cytokines exert a direct toxic effect on tissues; additionally, they activate phospholipase A2. These and other effects increase the concentration of platelet activating factor, promote nitric oxide synthase activity, promote tissue infiltration by neutrophils, and promote neutrophil activity.
[0303] The treatment of sepsis and septic shock remains a clinical conundrum, and recent prospective studies with biological response modifiers (ie, anti-TNF, anti-MIF) targeting the inflammatory response have shown only modest clinical benefit. Recently, interest has shifted to therapies aimed at reversing the associated periods of immune suppression. Studies in experimental animals and critically ill patients have shown that increased apoptosis of lymphoid organs and some parenchymal tissues contribute to this immune suppression, anergy and organ systems dysfunction. During septicemic syndromes, lymphocyte apoptosis can be triggered by the absence of IL-2 or by the release of glucocorticoids, granzymes, or the so-called “death” cytokines: tumor necrosis factor alpha or Fas ligand. Apoptosis proceeds via self-activation of cytosolic and/or mitochondrial caspases, which may be influenced by pro- and anti-apoptotic members of the Bcl-2 family. In experimental animals, not only treatment with apoptosis inhibitors can prevent apoptosis lymphoid cells, but also improve the outcome. Although clinical studies with anti-apoptotic agents are still a long way off in view of the technical difficulties largely associated with their administration and targeting to tissues, inhibition of lymphocyte apoptosis represents an attractive therapeutic target for the septic patient. Likewise, a dual-specific agent targeting an inflammatory mediator and an apoptotic agent may increase the benefit achieved. One aspect of the invention pertains to DVD-Igs capable of binding to IL-1β and to one or more targets involved in sepsis, selected from the group consisting of TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4, IL-10, IL-1B, NFKB1, PROC, TNFRSF1A, CSF3, CCR3, IL1RN, MIF, NFKB1, PTAFR, TLR2, TLR4, GPR44, HMOX1, HMG-B1, midkine IRAK1, NFKB2, SERPINA1, SERPINA1 and TREM1. The efficacy of such DVD-Igs for sepsis can be evaluated in preclinical animal models known in the prior art (see, Buras et al., Nat. Rev. Drug Discov., 4(10): 854-865 (2005) ); and Calandra et al., Nature Med., 6(2):164-170 (2000)). Neurological disorders and neurodegenerative diseases
[0304] Neurodegenerative diseases are chronic, in which case they are usually age-dependent, or acute (eg, stroke, traumatic brain injury, spinal cord injury, etc.). These diseases are characterized by the progressive loss of neuronal functions (neuronal cell death, demyelination), loss of mobility and memory loss. The emerging knowledge about the mechanisms underlying chronic neurodegenerative diseases (eg Alzheimer's disease, AD) shows a complex etiology, and the contribution of a variety of factors has been recognized to their development and progression, eg age, glycemic status , production and multimerization of amyloids, accumulation of advanced glycation end products (AGEs) that bind to their RAGE receptor (AGE receptor), increased oxidative stress in the brain, decreased cerebral blood flow, neuroinflammation, including cytokine release and inflammatory chemokines, neuronal dysfunction and microglial activation. Therefore, these chronic neurodegenerative diseases represent a complex interaction between multiple cell types and mediators. Therapeutic strategies for these diseases are limited and mainly consist of blocking inflammatory processes with non-specific anti-inflammatory agents (eg, corticosteroids, COX inhibitors) or agents to prevent the loss of neurons and/or synaptic functions. These treatments cannot stop the progression of the disease. Recent studies suggest that more targeted therapies, such as with antibodies against soluble Aβ peptide (including the oligomeric Aβ forms), may not only help stop disease progression, but also help maintain memory. These preliminary observations suggest that specific therapies targeting more than one disease mediator (eg, Aβ and a pro-inflammatory cytokine such as TNF) may provide even better therapeutic efficacy for chronic neurodegenerative diseases than observed that target a single mechanism of disease (eg, soluble Aβ only) (see Shepherd et al., Neuropathol. Appl. Neurobiol., 31: 503511(2005); Nelson, RB, Curr. Pharm. Des., 11: 3335-3352 (2005) ); Klein, WL, Neurochem. Int., 41: 345-352 (2002); Janelsins et al., "Early correlation of microglial activation with enhanced tumor necrosis factor-alpha and monocyte chemoattractant protein-I expression specifically within the entorhinal cortex of triple transgenic Alzheimer's disease mice", J. Neuroinflammation, 2(23): 1-12 (2005); Soloman, B., Curr. Alzheimer. Res., 1:149-163 (2004); Klyubin et al., Nature Med., 11:556-561 (2005); Arancio et al., EMBO J., 23: 4096-4105 (2004); Bornemann et al., Am. J. Pathol., 158: 63-73 (2001); Deane et al., Nature Med., 9:907-913 (2003); and Masliah et al., Neuron, 46: 857-868 (2005)).
[0305] The DVD-Ig molecules of the invention can bind to IL-1β and to one or more targets involved in chronic neurodegenerative diseases such as Alzheimer's. These targets include, among others, any mediator, soluble or on the cell surface, implicated in the pathogenesis of AD, eg AGE (S100 A, amphotericin), pro-inflammatory cytokines (eg IL-1), chemokines (eg , MCP 1), molecules that inhibit nerve regeneration (eg, Nogo, RGM A), molecules that enhance neurite outgrowth (neurotrophins) and molecules capable of mediating transport across the blood-brain barrier (eg, transferrin receptor, insulin receptor or RAGE). The effectiveness of DVD-Ig molecules can be validated in preclinical animal models, such as transgenic mice that overexpress the amyloid precursor protein or RAGE and that develop symptoms similar to those of Alzheimer's disease. Additionally, DVD-Ig molecules can be constructed and tested for efficacy in animal models, and the best therapeutic DVD-Ig can be selected for testing in human patients. DVD-Ig molecules can also be used to treat other neurodegenerative diseases such as Parkinson's disease. Alpha-synuclein is involved in the pathology of Parkinson's disease. A DVD-Ig capable of targeting IL-1β and LINGO-1, alpha-synuclein and/or inflammatory mediators such as TNF, IL-17, MCP-1 may improve effective therapy for Parkinson's disease and are contemplated in the invention . Neuronal regeneration and spinal cord injury
[0306] Despite increased knowledge about pathological mechanisms, spinal cord injury (SCI) is still a devastating condition and represents a medical indication characterized by high medical need. Most spinal cord injuries are contusion or compression injuries, and the primary injury is usually followed by secondary injury mechanisms (inflammatory mediators, eg, cytokines and chemokines) that worsen the initial injury and result in significant enlargement of the lesion. injured area, sometimes more than 10 times. These primary and secondary mechanisms in SCI are very similar to those that occur in brain injury by other means, for example, stroke. There is no satisfactory treatment, and high-dose methylprednisolone (MP) bolus injection is the only therapy used within a narrow time window of 8 hours after injury. This treatment, however, is only intended to prevent secondary injury, without causing any significant functional recovery. It is highly criticized for its lack of unequivocal efficacy and serious adverse effects, such as immunosuppression with subsequent infections and severe histopathological muscle alterations. There are no other approved drugs, biological agents or small molecules that stimulate the potential for endogenous regeneration, but therapeutic principles and promising drug candidates have shown efficacy in animal models of IBS in recent years. To a large extent, the lack of functional recovery in human SCI is caused by factors that inhibit neurite outgrowth, at sites of injury, in scar tissue, in myelin-associated cells, as well as in injury-associated cells. These factors are myelin-associated proteins, NogoA, OMgp and MAG, RGM A, scar-associated CSPG (chondroitin sulfate proteoglycans) and inhibitory factors in reactive astrocytes (some semaphorins and ephrins). However, at the injury site, not only growth inhibitory molecules are found, but also neurite growth-stimulating factors such as neurotrophins, laminin, L1 and others. This bringing together of neurite growth inhibitory and growth-promoting molecules may explain why blocking isolated factors such as NogoA or RGM A resulted in significant functional recovery in rodent models of SCI, as a reduction in inhibitory influences could shift the growth inhibition balance for growth promotion. However, the recoveries observed with blocking a single neurite outgrowth inhibitor molecule were not complete. In order to achieve faster and sharper recoveries, block two neurite growth inhibitory molecules, for example, Nogo and RGM A, or block a neurite growth inhibitory molecule and enhance the functions of a neurite outgrowth promoting molecule, for example, Nogo and neurotrophins, or blocking a neurite outgrowth inhibitory molecule, for example, Nogo and a pro-inflammatory molecule, for example, TNF, may be desirable (see McGee et al., Trends Neurosci., 26: 193 -198 (2003); Domeniconi et al., J. Neurol. Sci., 233: 43-47 (2005); Makwana et al., FEBS J., 272: 2628-2638 (2005); Dickson, BJ, Science , 298: 1959-1964 (2002); Teng et al., J. Neurosci. Res., 79: 273-278 (2005); Karnezis et al., Nature Neurosci., 7: 736 (2004); Xu et al. ., J. Neurochem., 91: 1018-1023 (2004)).
[0307] In one aspect, a DVD-Ig that binds to human IL-1β may also bind to one or two of the target pairs, such as NgR and RGM A; NogoA and RGM A; MAG and RGM A; OMgp and RGM A; RGM A and RGM B; CSPGs and RGM A; aggrecan, midkine, neurocan, versican, phosphacan, Te38 and TNF-α; antibodies specific to Aβ globulomer, combined with antibodies that promote the budding of dendrites and axons are provided. Dendritic pathology is a very early sign of AD, and NOGO A is known to restrict dendrite growth. It is possible to combine such ab type with any of the candidate Abs for SCI (myelin proteins). Other targets for DVD-Ig can include any combination of NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG or OMgp. Additionally targets can include any mediator, soluble in cell surface, implicated in neurite inhibition, e.g., Nogo, OMgp, MAG, RGM A, semaphorins, ephrins, soluble Aβ, pro-inflammatory cytokines (e.g., IL-1 ), chemokines (eg MIP 1a), molecules that inhibit nerve regeneration. The efficacy of anti-nogo / anti-RGM A DVD-Ig molecules or the like may be validated in preclinical animal models of spinal cord injury. Furthermore, these DVD-Ig molecules can be constructed and tested for efficacy in animal models, and the best therapeutic DVD-Ig can be selected for testing in human patients. Furthermore, DVD-Ig molecules can be constructed that target two distinct ligand-binding sites on a single receptor, for example, Nogo receptor, which binds to three ligands, Nogo, OMgp and MAG and RAGE, the which binds to Aβ and S100A. Furthermore, neurite outgrowth inhibitors, eg, nogo and nogo receptor, also participate in preventing regeneration in immunological diseases such as multiple sclerosis. Inhibition of the nogo-nogo receptor interaction has been shown to improve recovery in animal models of multiple sclerosis. Therefore, DVD-Ig molecules that can block the function of an immune mediator, for example, an IL-12-like cytokine and a neurite outgrowth inhibitor molecule, for example, Nogo or RGM, may offer faster and greater efficacy than blocking an immune molecule or a neurite outgrowth inhibitor molecule alone.
[0308] In general, antibodies do not cross the blood-brain barrier (BBB) in an efficient and relevant manner. However, in certain neurological diseases, eg stroke, traumatic brain injury, multiple sclerosis, etc., the BBB may be compromised and allow greater penetration of DVD-Igs and antibodies into the brain. In other neurological conditions, where BBB extravasation is not occurring, it is possible to employ targeting to endogenous transport systems, including carrier-mediated transporters, such as glucose and amino acid carriers, and to cellular structures/receptors that mediate Receptor-mediated transcytosis in the vascular endothelium of the BBB, thus enabling the transport of DVD-Ig across the BBB. Structures in the BBB that enable such transport include, among others, the insulin receptor, the insulin receptor, LRP and RAGE. Additionally, strategies make possible the use of DVD-Igs also as vehicles to transport potential drugs to the CNS, including low molecular weight drugs, nanoparticles and nucleic acids ( Coloma et al., Pharm. Res., 17(3):266 -274 (2000); Boado et al., Bioconjug. Chem., 18(2):447-455 (2007)). cancer disorders
[0309] Monoclonal antibody therapy has emerged as an important therapeutic modality for cancer (von Mehren et al., Ann. Rev. Med., 54: 343369 (2003)). Antibodies can exert antitumor effects by inducing apoptosis, redirecting cytotoxicity, and interfering with ligand-receptor interactions, or preventing the expression of proteins that are critical to the neoplastic phenotype. Additionally, antibodies can target components of the tumor microenvironment, disrupting vital structures such as the formation of tumor-associated vasculature. Antibodies can also target receptors whose ligands are growth factors, such as epidermal growth factor. The antibody thus inhibits natural ligands that stimulate cell growth, preventing them from binding to targeted tumor cells. Alternatively, the antibodies can induce an anti-idiotypic network, complement-mediated cytotoxicity or antibody-dependent cellular cytotoxicity (ADCC). The use of dual-specific antibody, targeting two distinct tumor mediators, will likely provide additional benefit when compared to monospecific therapy.
[0310] In another embodiment, a DVD-Ig that binds to human IL-1β of the invention may also be able to bind to another target involved in cancer diseases, including, among others: IGFR, IGF, VGFR1, PDGFRb, PDGFRa , IGF1,2, ERB3, CDCP, 1BSG2, ErbB3, CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10 , FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2, IL12A, IL1 , IL1B, IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1R, IL2, BCL2, CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2B , GNRH1, IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2 , INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL, TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1, IGF1, IGF2, INHA, INSL3 , INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3, NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2, NR1D2, NR1H2, NR1H4, NR1I2, NR2C1, NR2C2, NRE2 NR2 NRE , NR4A1, NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1, BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10, FGF11 , FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2, IGFBP3, IGFBP6, IL12A, IL1A , IL2, IL24, INHA, INSL3, INSL4, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3 , SHBG, TGFA, TIMP3, CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164 , COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3, CYB5, CYC1, DAB2IP, DES, DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584, FLJ25530, GAGEB1 , GAGEC1, GGT1 , GSTP1, HIP1, HUMCYT2A, IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC1 ANGPT , ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR, LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB1, VEGF, VEGFC, ANGPTL3, BAI1, COL4A3, IL8, LAMA5, NRP1, NRP2 , STAB1, ANGPTL4, PECAM1, PF4, PROK2, SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA , EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1 , CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B (p27Kip1), CDKN2A (p16INK4a), COL6A1, CTNNB1 (b-catenin), CTSB (cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3, GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130), ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c -Jun), MKI67 (Ki-6 7), NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin), SERPINE1 (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1 ), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53, AZGP1 (zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1), CLDN7 (claudin-7), CLU ( clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin), KLF5 (GC Box BP), KRT19 (keratin 19 ), KRTHB6 (hair-specific keratin type II), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophyllin B), SCGB2A1 (mammaglobin 2 ), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2, THBS4 and TNFAIP2 (B94), RON, c-Met, CD64, DLL4, PLGF, CTLA4, phosphatidylserine, ROBO4, CD80, CD22, CD40, CD23, CD28, CD55, CD38, CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137, DR4, DR5, RANKL, VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1, EPHA2, EpCAM, PGE2, NKG2D, LPA, SIP, APRIL , BCMA, MAPG, FLT3, PDGFR alpha, PDGFR beta, ROR1, PSMA, PSCA, SCD1 and CD59. D. Pharmaceutical Compositions
[0311] The invention also provides pharmaceutical compositions comprising an antibody (including a DVD-Ig described herein), or antigen-binding portion thereof, of the invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising antibodies of the invention are intended for use in, among others, diagnosing, detecting or monitoring a disorder, in preventing, treating, controlling or ameliorating a disorder or one or more of its symptoms and/or in research. In a specific embodiment, a composition comprises one or more antibodies of the invention. In another embodiment, the pharmaceutical composition comprises one or more antibodies of the invention and one or more prophylactic or therapeutic agents other than antibodies of the invention for treating a disorder in which IL-1/3 activity is detrimental. In one embodiment, prophylactic or therapeutic agents are known to be useful to or have been or are currently being used in preventing, treating, controlling or ameliorating a disorder or one or more of its symptoms. According to these embodiments, the composition may further comprise a carrier, diluent or excipient.
[0312] Antibodies and antibody portions of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody or antibody portion of the invention and a pharmaceutically acceptable carrier. In this specification, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or effectiveness of the antibody or antibody portion.
[0313] Various delivery systems are known and can be used to administer one or more antibodies of the invention or a combination of one or more antibodies of the invention and a prophylactic agent or therapeutic agent useful to prevent, control, treat or ameliorate a disorder or one or more of its symptoms, for example, encapsulation in liposomes, microparticles, recombinant cells capable of expressing the antibody or fragment fragment, receptor-mediated endocytosis (see, for example, Wu and Wu, J. Biol. Chem., 262 :4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector. Methods for administering a prophylactic or therapeutic agent of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral ). Furthermore, pulmonary administration can be employed, for example, by use of an inhaler or nebulizer and formulation with an aerosolizing agent. See, for example, U.S. Patent Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5 855 913 and 5 290 540; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346 and WO 99/66903, the contents of which are incorporated herein in their entirety by reference in this application. In one embodiment, an antibody or antibody portion of the invention, combination therapy or a composition of the invention is administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Massachusetts). In a specific embodiment, prophylactic or therapeutic agents of the invention are administered intramuscularly, intravenously, intratumorally, orally, intranasally, pulmonary or subcutaneously. Prophylactic or therapeutic agents can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through the epithelial or mucocutaneous lining (e.g., oral mucosa, rectal mucosa and intestinal mucosa, etc.) and can be administered together with other biologically active agents. Administration can be systemic or local.
[0314] In one embodiment, the specific binding of antibody-coupled carbon nanotubes (CNTs) to tumor cells in vitro, followed by their highly specific ablation with near infrared light (NIR), can be used to target tumor cells. For example, biotinylated polar lipids can be used to prepare stable, biocompatible, non-cytotoxic dispersions of CNT which are then fixed to one or two different avidin Neutralite-derived DVD-Igs, targeted against one or more tumor antigens (eg, CD22) (Chakravarty et al., Proc. Natl. Acad. Sci. USA, 105: 8697-8702 (2008)).
[0315] In a specific embodiment, it may be desirable to administer the prophylactic or therapeutic agents of the invention locally to the area in need of treatment; this can be achieved, for example and without any limitation, by local infusion, by injection or by means of an implant, said implant being of porous or non-porous material, including membranes and matrices such as Sialastic membranes, polymers, fibrous matrices (eg Tissuel®) or collagen matrices. In one embodiment, an effective amount of one or more antagonist antibodies of the invention is administered locally to the affected area of an individual to prevent, treat, control and/or ameliorate a disorder or symptom thereof. In another embodiment, an effective amount of one or more antibodies of the invention is administered locally to the affected area in combination with an effective amount of one or more therapies (e.g., one or more prophylactic or therapeutic agents) other than an antibody of the invention , of an individual to prevent, treat, control and/or ameliorate a disorder or one or more of its symptoms.
[0316] In another modality, the prophylactic or therapeutic agent may be released into a controlled release or sustained release system. In one embodiment, a pump can be used to achieve controlled or sustained release (see Langer, supra; Sefton, MV, CRC Crit. Rev. Biomed. Eng., 14: 201240 (1987); Buchwald et al., Surgery, 88: 507-516 (1980); Saudek et al., N. Engl. J. Med., 321: 574-579 (1989)). In another embodiment, polymeric materials can be used to effect controlled or sustained release of the therapies of the invention (see, for example, Goodson, JM, Chapter 6, In Medical Applications of Controlled Release, Vol. II, Applications and Evaluation, (Langer and Wise, eds.) (CRC Press, Inc., Boca Raton, 1984) pp. 115-138; Langer and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. Phys., C23(1): 61- 126 (1983); see also Levy et al., Science, 228:190-192 (1985); During et al., Ann. Neurol., 25:351-356 (1989); Howard et al., J. Neurosurg ., 71:105-112 (1989)); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid) , polyglycolides (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA) and polyorthoesters. In an exemplary embodiment, the polymer used in a sustained release formulation is inert, free of leaching impurities, storage stable, sterile and biodegradable. In yet another embodiment, a controlled or sustained release system can be placed in proximity to the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
[0317] Controlled release systems are discussed in the review by Langer (Science, 249:1527-1533 (1990)). Any technique known to the person skilled in the art can be used to produce sustained release formulations comprising one or more therapeutic agents of the invention. See, for example, U.S. Patent No. 4,526,938, PCT Publication No. WO 91/05548, PCT Publication No. WO 96/20698; Ning et al., "Intratumoral radioimmunotherapy of a human colon cancer xenograft using a sustained-release gel", Radiotherapy Oncol., 39: 179-189 (1996); Song et al., "Antibody Mediated Lung Targeting of Long-Circulating Emulsions", PDA J. Pharm. Sci.Technol., 50:372-377 (1996); Cleek et al., "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application", Proceed. Int'l. Symp. Control. Rel. Bioact. Mater., 24: 853-854 (1997); and Lam et al., "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery", Proceed. Int'l. Symp. Control Rel. Bioact. Mater., 24: 759-760 (1997), the contents of which are incorporated herein in their entirety by reference in this patent application.
[0318] In a specific embodiment, where the composition of the invention is a nucleic acid encoding a prophylactic or therapeutic agent, the nucleic acid can be administered in vivo to promote the expression of its encoded prophylactic or therapeutic agent, by its construction as part of an appropriate nucleic acid expression vector, and its administration in such a way that it becomes intracellular, for example, by use of a retroviral vector (see US Patent No. 4,980,286), or by direct injection or by use of microparticle bombardment (eg, genetic weapon; Biolistic®, DuPont) or coating with lipids or cell surface receptors or by transfection agents, or by administering them in connection with a homeobox-type peptide, which is known to penetrate the nucleus (see, for example, Joliot et al., Proc. Natl. Acad. Sci. USA, 88: 1864-1868 (1991)). Alternatively, a nucleic acid can be introduced intracellularly and incorporated into host cell DNA for expression by homologous recombination.
[0319] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., by inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. When necessary, the composition can also include a solubilizing agent and a local anesthetic, such as lidocaine, to lessen pain at the injection site.
[0320] If the compositions of the invention are intended for topical administration, the compositions may be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion or other form well known to the skilled person. Subject. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pennsylvania (1995). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms, comprising a carrier or one or more excipients compatible with topical application and having dynamic viscosity preferably greater than water, are typically employed. Suitable formulations include, among others, solutions, suspensions, emulsions, creams, ointments, powders, ointments, dressings and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents , buffers or salts) to influence various properties, such as, for example, the osmotic pressure. Other suitable topical dosage forms include aerosol preparations, in which the active ingredient, preferably combined with an inert solid or liquid carrier, is packaged in a mixture with pressurized volatile (eg gaseous propellant such as FREON®) or in a vial flexible. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms, if desired. Examples of such additional agents are well known in the art.
[0321] If the method of the invention comprises intranasal administration of a composition, the composition can be formulated as an aerosol, spray, mist or in the form of drops. Specifically, prophylactic or therapeutic agents for use in accordance with the present invention can be conveniently delivered in aerosol spray presentation form from pressurized packs or a nebulizer with the use of a suitable propellant (eg, dichlorodifluoromethane, trichlorofluoromethane , dichlorotetrafluorethane, carbon dioxide or other suitable gas). In the case of pressurized aerosol, the unit dose can be determined by a valve provided to deliver a metered amount. Capsules and cartridges (compounded, for example, of gelatin), for use in an inhaler or insufflator, may be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch.
[0322] If the method of the invention comprises oral administration, the compositions can be formulated orally in the form of tablets, capsules, pills, gel capsules, solutions, suspensions and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (for example pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (for example lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (for example potato starch or sodium starch glycolate); or wetting agents (eg, sodium lauryl sulfate). Tablets can be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, among others, solutions, syrups or suspensions, or they may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations can be obtained by conventional means with pharmaceutically acceptable additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (eg lecithin or acacia); non-aqueous vehicles (eg almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (for example, methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents, as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release or sustained release of one or more prophylactic or therapeutic agents.
[0323] The method of the invention may comprise pulmonary administration, for example, by the use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent. See, for example, U.S. Patent Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5 855 913; and 5,290,540; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346 and WO 99/66903, the contents of which are incorporated herein in their entirety by reference in this application. In a specific embodiment, an antibody of the invention, combination therapy and/or composition of the invention is administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Massachusetts).
[0324] The method of the invention may comprise administering a composition formulated for parenteral administration by injection (eg, by bolus injection or continuous infusion). Injectable formulations may be presented in unit dose form (eg, in ampoules or multi-dose containers) with an added preservative. The compositions may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulation aids such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle (eg, sterile pyrogen-free water) before use.
[0325] The methods of the invention may further comprise administering compositions formulated as depot preparations. Such long-acting formulations can be administered by implant (eg, implanted subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, compositions may be formulated with suitable polymeric or hydrophobic materials (eg, in emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (eg, in sparingly soluble salt form).
[0326] The methods of the invention encompass the administration of compositions formulated in neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, acetic, oxalic, tartaric, etc., acid. and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. hydroxides.
[0327] In general, the ingredients of the compositions are provided separately or mixed in unit dose form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent . When the mode of administration is by infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the method of administration is by injection, an ampoule of sterile water for injections or saline solution may be provided so that the ingredients can be mixed prior to administration.
[0328] Specifically, the invention also provides that one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the invention are packaged in a hermetically sealed container, such as an ampoule or sachet indicating the amount of the agent. In one embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the invention are provided as a sterile lyophilized dry powder or water-free concentrate in a hermetically sealed container that can be reconstituted (eg, with water or saline solution). ) to the appropriate concentration for administration to an individual. Preferably, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the invention are provided as a sterile dry powder lyophilized in a hermetically sealed container in a unit dose of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25mg, at least 35mg, at least 45mg, at least 50mg, at least 75mg or at least 100mg. Prophylactic agents or lyophilized therapeutic agents or pharmaceutical compositions of the invention must be stored at a temperature between 2 °C and 8 °C in their original container, and prophylactic or therapeutic agents or pharmaceutical compositions of the invention must be administered within 1 week, preferably within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours or within 1 hour of being reconstituted. In an alternative embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the invention are provided in liquid form in a hermetically sealed container, indicating the amount and concentration of the agent. Preferably, the liquid form of the administered composition is provided in a hermetically sealed container, containing at least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg /ml or at least 100 mg/ml. The liquid form must be stored at a temperature between 2 °C and 8 °C in its original container.
[0329] Antibodies and antibody parts of the invention can be incorporated into a pharmaceutical composition suitable for parenteral administration. Preferably, the antibody or antibody parts will be prepared in an injectable solution containing 0.1 - 250 mg/ml of antibody. The solution for injection can be made up in a flint or amber vial, ampoule or pre-filled syringe. The buffer can be L-histidine (1 - 50 mM), ideally 5 - 10mM, at pH 5.0 to 7.0 (ideally pH 6.0). Other suitable buffers include, among others, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0 - 300 mM (ideally 150 mM for liquid dosage form). Cryoprotectants can be included for the lyophilized dosage form, mainly sucrose 0 - 10% (ideally 0.5 - 1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents can be included for lyophilized dosage form, mainly mannitol 1 - 10% (ideally 2 - 4%). Stabilizers can be used in liquid and lyophilized dosage forms, mainly L-methinone 1 - 50 mM (ideally 5 - 10 mM). Other suitable bulking agents include glycine, arginine, polysorbate-80 may be included 0 - 0.05% (ideally 0.005 - 0.01%). Suitable surfactants include, among others, polysorbate 20 and BRIJ surfactants. The pharmaceutical composition comprising an antibody or antibody portion of the invention, prepared in an injectable solution for parenteral administration, may further comprise an agent useful as an adjuvant, such as those used to enhance the absorption or dispersion of a therapeutic protein (e.g., antibody ). An especially useful adjuvant is hyaluronidase (such as Hylenex® recombinant human hyaluronidase). The addition of hyaluronidase to the injectable solution improves human bioavailability after parenteral administration, especially subcutaneous. In addition, it allows for larger volumes at the injection site (ie, greater than 1 ml) with less pain and discomfort and minimal incidence of injection site reactions (see, PCT Publication No. WO 2004/078140 and US Publication No. 2006/104968 ).
[0330] The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid pharmaceutical forms, such as liquid solutions (for example, injectable and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and the therapeutic application. Typical preferred compositions are those in the form of injectable solutions or for infusion, such as compositions similar to those used for passive immunization of humans with other antibodies. The preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In an exemplary embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.
[0331]Therapeutic compositions should typically be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome or other organized structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (ie, antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as needed, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile lyophilized powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and spray drying, which result in powdered active ingredient plus any additional desired ingredient from a sterile solution previously filtered from it. Proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, or by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
The binding proteins of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous injection, intravenous injection or infusion. As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired results. In certain embodiments, the active compound can be prepared with a vehicle that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and poly(lactic acid). Many methods for preparing such formulations are proprietary or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, (J.R. Robinson, ed.) (Marcel Dekker, Inc., New York, 1978).
[0333] In certain embodiments, an antibody or antibody portion of the invention may be administered orally, for example, with an inert diluent or assimilable edible carrier. The compound (and other ingredients, if desired) can also be enclosed in a hard or soft gelatin-encased capsule, compressed into tablets, or incorporated directly into the individual's diet. For oral therapeutic administration, the compounds can be incorporated with excipients and used in the form of tablets for ingestion, buccal tablets, dragees, capsules, elixirs, suspensions, syrups, wafers and the like. To administer a compound of the invention by administration other than parenteral administration, it may be necessary to coat the compound or administer it concurrently with a material to prevent its inactivation.
[0334]Supplementary active compounds may also be incorporated into the compositions. In certain embodiments, an antibody or antibody portion of the invention is co-formulated and/or co-administered with one or more additional therapeutic agents that are useful to treat disorders in which IL-1/3 activity is detrimental. For example, an anti-human IL-1/3 antibody or antibody portion of the invention can be co-formulated and/or co-administered with one or more additional antibodies that bind to other targets (e.g., antibodies that bind to other cytokines or that bind to bind to cell surface molecules). Furthermore, one or more antibodies of the invention can be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies can advantageously employ lower doses of the therapeutic agents administered, thus avoiding possible toxicities or complications associated with the various monotherapies.
[0335] In certain embodiments, an antibody against IL-1β, or fragment thereof, is linked to a vehicle known in the art that extends its half-life. These vehicles include, among others, the Fc domain, polyethylene glycol and dextran. Such vehicles are described, for example, in U.S. Patent Application Serial No. 09/428,082 (now U.S. Patent No. 6,660,843), the contents of which are incorporated herein by reference in this application for all purposes.
[0336] In a specific embodiment, nucleic acid sequences, comprising nucleotide sequences encoding an antibody of the invention or other prophylactic or therapeutic agent of the invention, are administered to treat, prevent, control or ameliorate a disorder or one or more of its symptoms through gene therapy. Gene therapy refers to therapy performed by administering to an individual an expressed or to be expressed nucleic acid. In this embodiment of the invention, the nucleic acids produce their antibody or encoded prophylactic or therapeutic agent of the invention that mediates a prophylactic or therapeutic effect.
[0337] Any of the methods for gene therapy available in the prior art can be used in accordance with the present invention. For general reviews of gene therapy methods, see Goldspiel et al., Clinical Pharm., 12: 488-505 (1993); Wu et al., "Delivery systems for gene therapy", Biotherapy, 3:87-95 (1991); Tolstoshev,P., Ann. Rev. Pharmacol. Toxicol., 32:573-596 (1993); Mulligan, R.C., Science, 260: 926-932 (1993); and Morgan and Anderson, "Human Gene Therapy", Ann. Rev. Biochem., 62:191-217 (1993); Robinson, C., Trends Biotechnol., 11:155 (1993). Methods commonly known in the prior art of recombinant DNA technology, which can be used, are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, New York (1990). Detailed description of various gene therapy methods is disclosed in US Publication No. 2005/0042664 A1, incorporated herein by reference in this patent application.
[0338] Members of the IL-1 family (IL-1β and IL-1a) play a key role in the pathology associated with a variety of disorders involving immune and inflammatory elements. An IL-1 binding protein described herein can be administered to an individual to treat such disorders. In one embodiment, a disorder that can be treated by a method of the invention, comprising administering to an individual an IL-1 binding protein described herein, includes, but is not limited to, diabetes; uveitis; neuropathic pain; osteoarthritic pain; inflammatory pain; rheumatoid arthritis; osteoarthritis; juvenile chronic arthritis; septic arthritis; Lyme arthritis; psoriatic arthritis; reactive arthritis; spondyloarthropathy; systemic lupus erythematosus (SLE); Crohn's disease; ulcerative colitis; inflammatory bowel disease; autoimmune diabetes; insulin dependent diabetes mellitus; thyroiditis; asthma; allergic diseases; psoriasis; dermatitis; scleroderma; graft versus host disease; transplant organ rejection; acute immune disease associated with organ transplantation; chronic immune disease associated with organ transplantation; sarcoidosis; atherosclerosis; disseminated intravascular coagulation (DIC); Kawasaki disease; Grave's disease; nephrotic syndrome; chronic fatigue syndrome; Wegener's granulomatosis; Henoch-Schoenlein purple; microscopic renal vasculitis; active chronic hepatitis; autoimmune uveitis; septic shock; toxic shock syndrome; septicemic syndrome; cachexia; infectious diseases; parasitic diseases; acute transverse myelitis; Huntington's chorea; Parkinson's disease; Alzheimer's disease; stroke; primary biliary cirrhosis; hemolytic anemia; malignancies; cardiac insufficiency; myocardial infarction; Addison's disease; type I sporadic polyglandular deficiency; type II polyglandular deficiency (Schmidt's syndrome); acute respiratory distress syndrome (ARDS); alopecia; alopecia areata; seronegative arthropathy; arthropathy; Reiter's disease; psoriatic arthropathy; arthropathy in ulcerative colitis; enteropathic synovitis; arthropathy associated with chlamydia infection; Yersinia and Salmonella; spondyloarthropathy; atheromatous disease/arteriosclerosis; atopic allergy; autoimmune bullous disease; pemphigus vulgaris; pemphigus foliaceus; pemphigoid; linear IgA disease; autoimmune hemolytic anemia; Coombs positive hemolytic anemia; acquired pernicious anemia; juvenile pernicious anemia; myalgic encephalitis/Royal Free disease; chronic mucocutaneous candidiasis; giant cell arteritis (GCA); primary sclerosing hepatitis; autoimmune cryptogenic hepatitis; acquired immunodeficiency syndrome (AIDS); diseases related to acquired immunodeficiency; Hepatitis B; hepatitis C; common variable immunodeficiency (common variable hypogammaglobulinemia); dilated cardiomyopathy; female infertility; ovarian failure; premature ovarian failure; fibrotic lung disease; cryptogenic fibrosing alveolitis; post-inflammatory interstitial lung disease; interstitial pneumonitis; interstitial lung disease associated with connective tissue disease; pulmonary disease associated with mixed connective tissue disease; interstitial lung disease associated with systemic sclerosis; interstitial lung disease associated with rheumatoid arthritis; pulmonary disease associated with systemic lupus erythematosus; lung disease associated with dermatomyositis/polymyositis; lung disease associated with Sjogren's syndrome; lung disease associated with ankylosing spondylitis; diffuse pulmonary vasculitis; pulmonary disease associated with hemosiderosis; drug-induced interstitial lung disease; fibrosis; radiation fibrosis; bronchiolitis obliterans; chronic eosinophilic pneumonia; lymphocytic infiltrative lung disease; post-infectious interstitial lung disease; gouty arthritis; autoimmune hepatitis; type 1 autoimmune hepatitis (classic or lupoid autoimmune hepatitis); type 2 autoimmune hepatitis (anti-LKM antibody hepatitis); autoimmunity-mediated hypoglycemia; type B insulin resistance associated with acanthosis nigricans; hypoparathyroidism; osteoarthritis; primary sclerosing cholangitis; type 1 psoriasis; type 2 psoriasis; idiopathic leukopenia; autoimmune neutropenia; kidney disease NOS; glomerulonephritis; microscopic renal vasculitis; Lyme disease; discoid lupus erythematosus; idiopathic male infertility; male infertility associated with nitric oxide; autoimmunity against sperm; multiple sclerosis (all subtypes, including primary progressive, secondary progressive, relapsing-remitting); sympathetic ophthalmia; pulmonary hypertension secondary to connective tissue disease; Goodpasture's syndrome; pulmonary manifestation of polyarteritis nodosa; acute rheumatic fever; rheumatoid spondylitis; Still's disease; systemic sclerosis; Sjorgren's syndrome; Takayasu's disease/arteritis; autoimmune thrombocytopenia (AITP); idiopathic thrombocytopenia; autoimmune thyroid disease; hyperthyroidism; autoimmune hypothyroidism with goiter (Hashimoto's disease); autoimmune atrophic hypothyroidism; primary myxedema; phacogenic uveitis; primary vasculitis; vitiligo; acute liver disease; chronic liver disease; alcoholic cirrhosis; alcohol-induced liver damage; cholestasis; idiosyncratic liver disease; drug-induced hepatitis; non-alcoholic steatohepatitis; allergy; group B Streptococci (GBS) infection; mental disorders (eg depression and schizophrenia); diseases mediated by Type Th2 lymphocytes and Type Th1 cells; acute and chronic pain (different forms of pain); cancer (such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate, and rectal cancer); hematopoietic malignancies; leukemia; lymphoma; abetalipoproteinemia; acrocyanosis; acute and chronic parasitic or infectious processes; acute leukemia; acute lymphoblastic leukemia (ALL); ALL of T cells; ALL from FAB; acute myeloid leukemia (AML); acute or chronic bacterial infection; acute pancreatitis; acute renal failure; adenocarcinomas; atrial ectopic beats; AIDS dementia complex; alcohol-induced hepatitis; allergic conjunctivitis; allergic contact dermatitis; allergic rhinitis; allograft rejection; alpha-1 antitrypsin deficiency; amyotrophic lateral sclerosis; anemia; angina pectoris; anterior horn cell degeneration; anti-CD3 therapy; antiphospholipid syndrome; antireceptor hypersensitivity reactions; aortic and peripheral aneurysms; aortic dissection; arterial hypertension; arteriosclerosis; arteriovenous fistula; ataxia; atrial fibrillation (sustained or paroxysmal); atrial flutter; atrioventricular block; B-cell lymphoma; bone graft rejection; bone marrow transplant rejection (BMT); bundle branch blockage; Burkitt's lymphoma; burns; cardiac arrhythmias; cardiac stun syndrome; cardiac tumors; cardiomyopathy; inflammatory response in cardiopulmonary bypass surgery; cartilage transplant rejection; cortical cerebellar degenerations; cerebellar disorders; chaotic or multifocal atrial tachycardia; disorders associated with chemotherapy; chronic myelocytic leukemia (CML); chronic alcoholism; chronic inflammatory pathologies; chronic lymphocytic leukemia (CLL); chronic obstructive pulmonary disease (COPD); chronic salicylate poisoning; colorectal carcinoma; Congestive heart failure; conjunctivitis; contact dermatitis; pulmonale color; coronary artery disease; Creutzfeldt-Jakob disease; sepsis with negative culture; cystic fibrosis; disorders associated with cytokine therapy; boxer's dementia; demyelinating diseases; dengue hemorrhagic fever; dermatitis; dermatological conditions; diabetes mellitus; diabetic atherosclerotic disease; diffuse Lewy body disease; dilated congestive cardiomyopathy; basal ganglia disorders; Down syndrome in old age; movement disorders induced by drugs that block dopamine receptors in the central nervous system; drug sensitivity; eczema; encephalomyelitis; endocarditis; endocrinopathy; epiglottitis; Epstein-Barr virus infection; erythromelalgia; extrapyramidal and cerebellar disorders; familial hemophagocytic lymphohistiocytosis; fetal thymus implant rejection; Friedreich's ataxia; functional disorders of the peripheral arteries; fungal sepsis; gas gangrene; gastric ulcer; glomerular nephritis; rejection of grafts from any organ or tissue; gram negative sepsis; gram positive sepsis; granulomas due to intracellular organisms; hairy cell leukemia; Hallervorden-Spatz disease; Hashimoto's thyroiditis; hay fever; heart transplant rejection; hemochromatosis; hemodialysis; hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura; bleeding; hepatitis A; His bundle arrhythmias; HIV infection/HIV neuropathy; Hodgkin's disease; hyperkinetic movement disorders; hypersensitivity reactions; hypersensitivity pneumonitis; hypertension; hypokinetic movement disorders; assessment of the hypothalamic-pituitary-adrenal axis; idiopathic Addison's disease; idiopathic pulmonary fibrosis (IPF); antibody-mediated cytotoxicity; asthenia; infantile spinal muscular atrophy; inflammation of the aorta; influenza A; exposure to ionizing radiation; iridocyclitis/uveitis/optic neuritis; ischemia-reperfusion injury; ischemic stroke; juvenile rheumatoid arthritis; juvenile spinal muscular atrophy; Kaposi's sarcoma; kidney transplant rejection; legionellosis; leishmaniasis; leprosy; lesions of the corticospinal system; lipedema; liver transplant rejection; lymphedema; malaria; malignant lymphoma; malignant histiocytosis; malignant melanoma; meningitis; meningococcemia; migraine-like headache associated with metabolic syndrome; idiopathic migraine-type headache; mitochondrial multisystem disease; mixed connective tissue disease; monoclonal gammopathy; multiple myeloma; degenerations of multiple systems (Menzel; Dejerine-Thomas; Shy-Drager; and Machado-Joseph); myasthenia gravis; mycobacterium avium intracellulare; mycobacterium tuberculosis; myelodysplastic syndrome; myocardial infarction; myocardial ischemic disorders; nasopharyngeal carcinoma; neonatal chronic lung disease; nephritis; nephrosis; neurodegenerative diseases; neurogenic muscle atrophies I; neutropenic fever; non-Hodgkin's lymphoma; occlusion of the abdominal aorta and its branches; occlusive arterial disorders; OKT3® therapy; orchitis/epididymitis; orchitis/vasectomy reversal procedures; organomegaly; osteoporosis; rejection of pancreas transplantation; pancreatic carcinoma; paraneoplastic syndrome/hypercalcemia of malignancy; rejection of parathyroid transplantation; pelvic inflammatory disease; perennial rhinitis; pericardial disease; peripheral atherosclerotic disease; peripheral vascular disorders; peritonitis; pernicious anemia; Pneumocystis carinii pneumonia; pneumonia; POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes); post-perfusion syndrome; post-pump syndrome; post-MI cardiotomy syndrome; pre eclampsia; progressive supranuclear palsy; primary pulmonary hypertension; radiotherapy; Raynaud's phenomenon; Raynaud's disease; Refsum's disease; regular tachycardia with narrow QRS; renovascular hypertension; reperfusion injury; restrictive cardiomyopathy; sarcomas; senile chorea; senile dementia of the Lewy bodies type; seronegative arthropathies; shock; sickle cell anemia; skin allograft rejection; skin disorder syndrome; small bowel transplant rejection; solid tumors; specific arrhythmias; spinal ataxia; spinocerebellar degenerations; streptococcal myositis; structural lesions of the cerebellum; subacute sclerosing panencephalitis; syncope; syphilis of the cardiovascular system; systemic anaphylaxis; systemic inflammatory response syndrome; systemic onset juvenile rheumatoid arthritis; telangiectasia; thromboangiitis obliterans; thrombocytopenia; toxicity; transplants; trauma/hemorrhage; type III hypersensitivity reactions; type IV hypersensitivity; unstable angina; uremia; urosepsis; heart valve diseases; varicose veins; venous diseases; venous thrombosis; ventricular fibrillation; virus and fungal infections; viral encephalitis/aseptic meningitis; virus-associated hemophagocytic syndrome; Wernicke-Korsakoff syndrome; Wilson's disease; rejection of xenografts from any organ or tissue; acute coronary syndromes; idiopathic acute polyneuritis; acute inflammatory demyelinating polyradiculoneuropathy; acute ischemia; Adult Still's disease; alopecia areata; anaphylaxis; anti-phospholipid antibody syndrome; aplastic anemia; arteriosclerosis; atopic eczema; atopic dermatitis; autoimmune dermatitis; autoimmune disorder associated with Streptococcus infection; autoimmune enteropathy; autoimmune hearing loss; autoimmune lymphoproliferative syndrome (ALPS); autoimmune myocarditis; autoimmune premature ovarian failure; blepharitis; bronchiectasis; bullous pemphigoid; cardiovascular disease; catastrophic antiphospholipid syndrome; celiac disease; cicatricial pemphigoid; clinically isolated syndrome (CIS) with risk for multiple sclerosis; conjunctivitis; childhood-onset psychiatric disorder; dacryocystitis; dermatomyositis; diabetic retinopathy; disc herniation; disc prolapse; drug-induced immune hemolytic anemia; endocarditis; endometriosis; endophthalmitis; episcleritis; erythema multiforme; erythema multiforme major; gestational pemphigoid; Guillan-Barré syndrome (GBS); hay fever; Hughes syndrome; idiopathic Parkinson's disease; idiopathic interstitial pneumonia; IgE-mediated allergy; immune hemolytic anemia; inclusion body myositis; infectious eye inflammatory disease; inflammatory demyelinating disease; inflammatory heart disease; inflammatory kidney disease; iritis; keratitis; keratoconjunctivitis sicca; Kussmaul's disease or Kussmaul-Meier's disease; Landry's palsy; Langerhans cell histiocytosis; reticular livedo; macular degeneration; microscopic polyangiitis; Morbus Bechterev; motor neuron diseases; mucous membrane pemphigoid; multiple organ failure; myasthenia gravis; myelodysplastic syndrome; myocarditis; nerve root diseases; non-A non-B hepatitis; optic neuritis; osteolysis; pauciarticular JRA; peripheral arterial occlusive disease (PAOD); peripheral vascular disease (PVD); peripheral arterial disease (PAD); phlebitis; polyarteritis nodosa (or perarteritis nodosa); polychondritis; polymyalgia rheumatica; poliosis; polyarticular JRA; polyendocrine deficiency syndrome; polymyositis; polymyalgia rheumatica (PMR); post-pump syndrome; Primary Parkinsonism; Secondary Parkinsonism; prostatitis; pure red series aplasia; primary adrenal insufficiency; recurrent neuromyelitis optica; restenosis; rheumatic heart disease; SAPHO (synovitis, acne, pustulosis, hyperostosis and osteitis); secondary amyloidosis; shock lung; scleritis; sciatica pain; secondary adrenal insufficiency; connective tissue disease associated with silicone; Sneddon-Wilkinson dermatosis; ankylosing spondylitis; Stevens-Johnson syndrome (SJS); systemic inflammatory response syndrome; temporal arteritis; toxoplasmic retinitis; toxic epidermal necrolysis; transverse myelitis; TRAPS (Tumor Necrosis Factor Receptor Type 1 Associated Periodic Syndrome (TNFR)); type B insulin resistance associated with acanthosis nigricans; type 1 allergic reaction; type II diabetes; urticaria; usual interstitial pneumonia (UIP); vernal conjunctivitis; viral retinitis; Vogt-Koyanagi-Harada syndrome (VKH syndrome); wet macular degeneration; wound healing; arthropathy associated with infection by Yersinia and Salmonella.
The binding proteins of the invention can be used to treat humans suffering from autoimmune diseases, especially those associated with inflammation, rheumatoid arthritis (RA), osteoarthritis, psoriasis, multiple sclerosis (MS) and other autoimmune diseases.
[0340] An antibody or antibody portion of the invention may also be administered with one or more additional therapeutic agents useful in the treatment of autoimmune and inflammatory diseases.
[0341] In one embodiment, diseases that can be treated or diagnosed with the compositions and methods of the invention include, among others, primary and metastatic cancers, including carcinomas of the breast, colon, rectum, oropharynx, rhinopharynx, esophagus, stomach, pancreas , liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urothelium), female genital tract (including cervix, uterus, ovaries, as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal gland and pituitary) and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissue, as well as Kaposi's sarcoma), brain, nerve, eye and meningeal tumors (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, schwannomas and meningiomas), solid tumors from hematopoietic malignancies such as leukemias and lymphomas (Hodgkin's and non-Hodgkin's lymphomas).
[0342] In another embodiment, an antibody of the invention or its antigen-binding portion is used to treat cancer or in the prevention of metastasis of a tumor. This treatment may involve administering the antibody or its antigen-binding portion alone or in combination with another therapeutic agent or treatment, such as radiotherapy and/or a chemotherapeutic agent.
[0343] Antibodies of the invention, or antigen-binding portions thereof, can be combined with agents that include, but are not limited to, antineoplastic agents, radiotherapy, chemotherapy such as DNA alkylating agents, cisplatin, carboplatin, anti-tubulin agents, paclitaxel , docetaxel, taxol, doxorubicin, gemcitabine, gemzar, anthracyclines, adriamycin, topoisomerase I inhibitors, topoisomerase II inhibitors, 5-fluorouracil (5-FU), leucovorin, irinotecan, receptor tyrosine kinase inhibitors (eg erlotinib, gefitinib), COX-2 inhibitors (eg celecoxib), kinase inhibitors and siRNAs.
[0344] A binding protein of the invention can also be administered with one or more additional therapeutic agents useful in the treatment of various diseases.
[0345] Antibodies of the invention, or antigen-binding portions thereof, can be used alone or in combination to treat such diseases. It is to be understood that the antibodies of the invention, or antigen-binding portions thereof, can be used alone or in combination with an additional agent, for example a therapeutic agent, said additional agent being selected by the skilled person for its intended purpose. For example, the additional agent can be a therapeutic agent recognized in the art as useful to treat the disease or condition being treated by the antibody of the present invention. The additional agent can also be an agent that imparts a beneficial attribute to the therapeutic composition, for example, an agent that affects the viscosity of the composition.
[0346] It should further be understood that the combinations to be included in this invention are those combinations useful for its intended purpose. The agents presented below are for illustrative purposes and are not intended to be limited. The combinations, which are part of this invention, can be the antibodies of the present invention and at least one additional agent selected from the lists below. The combination may also include more than one additional agent, for example two or three additional agents if the combination is such that the composition formed can perform its intended function.
[0347] Preferred combinations are with non-steroidal anti-inflammatory drug(s), also called NSAIDS, which include drugs such as ibuprofen. Other preferred combinations are with corticosteroids, including prednisolone; the well-known side effects of steroid use can be reduced or even eliminated by adjusting the steroid dose needed when patients are treated in combination with the anti-IL-1β antibodies of this invention. Non-limiting examples of therapeutic agents for rheumatoid arthritis, with which an antibody or antibody portion of the invention can be combined, include, but are not limited to, the following: cytokine suppressing anti-inflammatory drug(s) ( CSAIDs); antibodies against or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 8, IL-15, IL-16, IL-18, IL-21, interferons, EMAP-II, GM-CSF, FGF and PDGF. Antibodies of the invention, or antigen-binding portions thereof, can be combined with antibodies against cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1) , CD86 (B7.2), CD90, CTLA or their linkers, including CD154 (gp39 or CD40L).
[0348] Preferred combinations of therapeutic agents may interfere at different points in the subsequent autoimmune and inflammatory cascade; preferred examples include TNF antagonists such as chimeric, humanized or human antibodies against TNF, D2E7, (PCT Publication No. WO 97/29131), CA2 (RemicadeTM), CDP 571 and soluble p55 or p75 TNF receptors, derivatives thereof, ( p75TNFR1gG (EnbrelTM) or p55TNFR1gG (Lenercept) and also TNFα converting enzyme inhibitors (TACE); likewise, IL-1 inhibitors (interleukin-1 converting enzyme inhibitors, IL-1RA etc.) may be effective for the same reason. Other preferred combinations include Interleukin 11. Still other preferred combinations are important participants in the immune response, which may act in parallel, in dependence on or in tune with the function of h IL-1β. CD4 Not Depleting Yet other preferred combinations include antagonists of the costimulatory pathway of CD80 (B7.1) or CD86 (B7.2), including antibodies, soluble receptors or antagonist ligands.
[0349] Antibodies of the invention, or their antigen-binding portions, can also be combined with agents such as methotrexate, 6-MP, azathioprine, sulfasalazine, mesalazine, olsalazine, chloroquinine/hydroxychloroquine, pencilamine, aurothiomalate (intramuscular and oral), azathioprine, colchicine, corticosteroids (oral, inhalable and local injection), beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines (theophylline, aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporine, FK , mycophenolate mofetil, leflunomide, NSAIDs, eg ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents that interfere with signaling by proinflammatory cytokines such as TNF -a or IL-1 (for example, IRAK, NIK, IKK, p38 or MAP kinases inhibitors), IL-1β converting enzyme inhibitors, TNFα-converting enzyme (TACE) inhibitors, T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin-converting enzyme inhibitors, soluble cytokine receptors and their derivatives (by example, p55 or p75 soluble TNF receptors and p75TNFRIgG derivatives (EnbrelTM and p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, sIL-6R), anti-inflammatory cytokines (e.g. IL-4, IL-10 , IL-11, IL-13 and TGFβ), celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen, valdecoxib, sulfasalazine, methylprednisolone, meloxicam, methylprednisolone acetate, gold sodium thiomalate, aspirin, trianolone , propoxyphene/apap napsylate, folate, nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium, oxaprozine, oxycodone hcl, hydrocodone/apap bitartrate, diclofenac sodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol hcl, salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium, prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin, glucosamine sulf/chondroin, amitriptyline hcl, sulfadiazine, hcl/oxycododine misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, anti-IL15, BIRB-796, SCIO-469, VX-702, AMG -548, VX-740, Roflumilast, IC-485, CDC-801 and Mesopram. Preferred combinations include methotrexate or leflunomide and, in moderate or severe cases of rheumatoid arthritis, cyclosporine.
[0350] Additional non-limiting agents that can also be used in combination with a binding protein to treat rheumatoid arthritis (RA) include, but are not limited to, the following: non-steroidal anti-inflammatory drug(s) ( NSAIDs); cytokine suppressive anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNFα antibody; Celltech/Bayer); cA2/infliximab (anti-TNFα chimeric antibody; Centocor); 75 kdTNFR-IgG/etanercept (75 kD TNF-IgG receptor fusion protein; Immunex; see, for example, Moreland et al. (Abstract No. 813), Arthritis Rheum., 37: S295 (1994); Baumgartner et al. ., J. Invest. Med., 44(3): 235A (March, 1996); 55 kdTNF-IgG (55 kD TNF-IgG receptor fusion protein; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (primatized anti-CD4 antibody that does not cause depletion; IDEC/SmithKline; see, for example, Kaine et al. (Abstract No. 195), Arthritis Rheum., 38: S185 (1995)); DAB 486-IL-2 and /or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; see, for example, Sewell et al., Arthritis Rheum., 36(9): 1223-1233 (September, 1993)); Tac (humanized anti-IL-2Rα; Protein Design Labs/Roche); IL-4 (anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering ); IL-4 agonists; IL-10 and/or IL-4 (for example, agonist antibodies); IL-1RA (IL-1 receptor antagonist; Synergen/Amgen); anakinra (Kineret®/Amgen); TNF-bp/s-TNF (soluble TNF binding protein; see, for example, Evans et al. (Abstract No. 1540), Arthritis Rheum., 39(9) (supplement): S284 (1996)); Kapadia et al., Amer. J. Physiol. - Heart and Circulatory Physiology, 268: H517-H525 (1995)); RP73401 (Type IV phosphodiesterase inhibitor; see, for example, Chikanza et al. (Abstract No. 1527), Arthritis Rheum., 39(9) (supplement): S282 (1996)); MK-966 (COX-2 inhibitor; see, for example, Erich et al. (Abstract Nos. 328 and 329), Arthritis Rheum., 39(9) (supplement): S81 (1996)); Iloprost (see, for example, Scholz, P. (Abstract No. 336), Arthritis Rheum., 39(9) (supplement): S82 (1996)); methotrexate; thalidomide (see, for example, Lee et al. (Abstract No. 1524), Arthritis Rheum., 39(9) (supplement): S282 (1996)) and thalidomide-related drugs (for example, Celgen); leflunomide (anti-inflammatory and cytokine inhibitor; see, for example, Finnegan et al. (Abstract No. 627), Arthritis Rheum., 39(9) (supplement): S131 (1996)); Thoss et al., Inflamm. Res., 45: 103-107 (1996)); tranexamic acid (inhibitor of plasminogen activation; see, for example, Ronday et al. (Abstract No. 1541), Arthritis Rheum., 39(9) (supplement): S284 (1996)); T-614 (cytokine inhibitor; see, for example, Hara et al. (Abstract No. 1526), Arthritis Rheum., 39(9) (supplement): S282 (1996)); prostaglandin E1 (see, for example, Moriuchi et al. (Abstract No. 1528), Arthritis Rheum., 39(9) (supplement): S282 (1996)); Tenidap (non-steroidal anti-inflammatory drug; see, for example, Guttadauria, M. (Abstract No. 1516), Arthritis Rheum., 39(9) (supplement): S280 (1996)); Naproxen (non-steroidal anti-inflammatory drug; see, for example, Fiebich et al., Neuro Report, 7: 1209-1213 (1996)); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidal anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine (see, for example, Farr et al. (Abstract No. 1519), Arthritis Rheum., 39(9) (supplement): S281 (1996)); Azathioprine (see, for example, Hickey et al. (Abstract No. 1521), Arthritis Rheum., 39(9) (supplement): S281 (1996)); ICE inhibitor (interleukin-1β converting enzyme inhibitor); zap-70 and/or lck inhibitor (tyrosine kinase inhibitor zap-70 or lck); VEGF inhibitor and/or VEGF-R inhibitor (vascular endothelial cell growth factor or vascular endothelial cell growth factor receptor inhibitors; angiogenesis inhibitors); corticosteroid anti-inflammatory drugs (eg, SB203580); TNF convertase inhibitors; anti-IL-12 antibodies; anti-IL-18 antibodies; interleukin-11 (see, for example, Keith Jr. et al. (Abstract No. 1613), Arthritis Rheum., 39(9) (supplement): S296 (1996)); interleukin-13 (see, for example, Bessis et al. (Abstract No 1681), Arthritis Rheum., 39(9) (supplement): S308 (1996)); interleukin-17 inhibitors (see, for example, Lotz et al. (Abstract No. 559), Arthritis Rheum., 39(9) (supplement): S120 (1996)); gold; penicillamine; chloroquine; chlorambucil; hydroxychloroquine; cyclosporine; cyclophosphamide; total lymphoid irradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins; peptides and collagens administered orally; disodium lobenzarit; Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligo-deoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulfate; minocycline; anti-IL2R antibodies; marine and botanical lipids (fatty acids from fish and plant seeds; see, for example, DeLuca et al., Rheum. Dis. Clin. North Am., 21: 759-777 (1995)); auranophin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immunoglobulin; zileuton; azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose (therafectin); cladribine (2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see, Bruncko et al., J. Med. Chem., 50(4), 641-662 (2007)); antiviral agents and immunomodulators.
[0351] In one embodiment, the binding protein, or antigen-binding portion thereof, is administered in combination with one or more of the following agents for the treatment of rheumatoid arthritis (RA): small molecule KDR inhibitor, small molecule KDR inhibitor Tie-2; methotrexate; prednisone; celecoxib; folic acid; hydroxychloroquine sulfate; rofecoxib; etanercept; infliximab; leflunomide; naproxen; valdecoxib; sulfasalazine; methylprednisolone; ibuprofen; meloxicam; methylprednisolone acetate; gold sodium thiomalate; aspirin; azathioprine; triamcinolone acetonide; propoxyphene napsylate/apap; folate; nabumetone; diclofenac; piroxicam; etodolac; sodium diclofenac; oxaprozine; oxycodone hcl; hydrocodone/aapp bitartrate; diclofenac sodium/misoprostol; fentanyl; anakinra, recombinant human; tramadol hcl; salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen; alendronate sodium; prednisolone; morphine sulfate; lidocaine hydrochloride; indomethacin; glucosamine/chondroitin sulfate; cyclosporine; amitriptyline hcl; sulfadiazine; oxycodone hcl/acetaminophen; olopatadine hcl; misoprostol; naproxen sodium; omeprazole; mycophenolate mofetil; cyclophosphamide; rituximab; IL-1 TRAP; MRA; CTLA4-IG; IL-18BP; IL-12/23; anti-IL 18; anti-IL 15; BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485; CDC-801; and mesopram.
[0352] Non-limiting examples of therapeutic agents for inflammatory bowel disease with which a binding protein of the invention can be combined include the following: budeside; epidermal growth factor; corticosteroids; cyclosporine, sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1β mAbs; anti-IL-6 mAbs; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; antibodies against or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL- 17, IL-18, EMAP-II, GM-CSF, FGF and PDGF. Antibodies of the invention, or antigen-binding portions thereof, can be combined with antibodies against cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands. Antibodies of the invention, or antigen-binding portions thereof, may also be combined with agents such as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, e.g. ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists; antithrombotic agents, complement inhibitors, adrenergic agents, agents that interfere with signaling by pro-inflammatory cytokines such as TNFα or IL-1 (eg IRAK, NIK, IKK, p38 or MAP kinases inhibitors), enzyme inhibitors IL-1β converting enzyme inhibitors, TNFα converting enzyme inhibitors, T cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and their derivatives (for example, soluble TNF receptors p55 or p75, sIL-1RI, sIL-1RII, sIL-6R) and anti-inflammatory cytokines (for example, IL-4, IL-10, IL-11, IL-13 and TGFβ ) and bcl-2 inhibitors.
[0353] Examples of therapeutic agents for Crohn's disease, in which a binding protein can be combined, include the following: TNF antagonists, e.g. anti-TNF antibodies, Adalimumab (PCT Publication No. WO 97/29131; HUMIRA® ), CA2 (REMICADE), CDP 571, TNFR-Ig constructs, (p75TNFRIgG (ENBREL®) and p55TNFRIgG (Lenercept™)) PDE4 inhibitors and inhibitors. Antibodies of the invention, or antigen-binding portions thereof, can be combined with corticosteroids, for example, budesonide and dexamethasone. The binding proteins of the invention, or antigen-binding portions thereof, can also be combined with agents such as sulfasalazine, 5-aminosalicylic acid and olsalazine and agents that interfere with the synthesis and action of proinflammatory cytokines such as IL -1, for example, IL-1 and IL-1ra converting enzyme inhibitors. Antibodies of the invention, or their antigen-binding portion, can also be used with T cell signaling inhibitors, for example, tyrosine kinase inhibitor 6-mercaptopurines. Binding proteins of the invention, or antigen-binding portions thereof, can be combined with IL-11. Binding proteins of the invention, or antigen-binding portions thereof, can be combined with mesalamine, prednisone, azathioprine, mercaptopurine, infliximab, methylprednisolone sodium succinate, atrop diphenoxylate/sulfate, loperamide hydrochloride, methotrexate, omecin, ciprozole dextrose-water, hydrocodone/apap bitartrate, tetracycline hydrochloride, fluocinonide, metronidazole, thimerosal/boric acid, cholestyramine/sucrose, ciprofloxacin hydrochloride, hyoscyamine sulfate, meperidine hydrochloride, midhydrofenachloride/acetazolam hydrochloride, halidenehydrochloride/acetazolam promethazine, sodium phosphate, trimethoprim/sulfamethoxazole, celecoxib, polycarbophil, propoxyphene napsylate, hydrocortisone, multivitamins, disodium balsalazide, codeine/apap phosphate, colesevelam hcl, cyanocobalamin, folic acid, levofloxacin, methylpredrumacin.
[0354] Non-limiting examples of therapeutic agents for multiple sclerosis (MS) with which binding proteins of the invention can be combined include the following: corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-β1a (AVONEX; Biogen); interferon-β1b (BETASERON; Chiron/Berlex); interferon α-n3) (Interferon Sciences/Fujimoto), interferon-α (Alpha Wassermann/J&J), interferon β1A-IF (Serono/Inhale Therapeutics), Peginterferon β 2b (Enzon/Schering-Plough), Copolymer 1 (Cop-1 ; COPAXONE; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; clabribine; antibodies against or antagonists of other human cytokines or growth factors and their receptors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15 , IL-16, IL-18, EMAP-II, GM-CSF, FGF and PDGF. Binding proteins of the invention can be combined with antibodies against cell surface molecules such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. Binding proteins of the invention can also be combined with agents such as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, e.g. ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents complement inhibitors, adrenergic agents, agents that interfere with signaling by proinflammatory cytokines such as TNFα or IL-1 (eg IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-converting enzyme inhibitors 1β, TACE inhibitors, T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin-converting enzyme inhibitors, soluble cytokine receptors and their derivatives (eg, soluble receptors of p55 or p75 TNF, sIL-1RI, sIL-1RII, sIL-6R), anti-inflammatory cytokines (e.g. IL-4, IL-10, IL-13 and TGFβ) and inhibited res of bcl-2.
[0355]Examples of therapeutic agents for multiple sclerosis with which binding proteins of the invention can be combined include interferon-β, for example, IFNβ1a and IFNβ1b; copaxone; corticosteroids; caspase inhibitors, for example caspase-1 inhibitors; IL-1 inhibitors; TNF inhibitors; and antibodies against CD40 and CD80 ligand.
[0356] The binding proteins of the invention can also be combined with agents such as alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone, xaliproden hydrochloride, fampridine, glatiramer acetate, natalizumab, sinabidol, a-immunokine NNSO3, ABR-215062 , AnergiX.MS, chemokine receptor antagonists, BBR-2778, calagualin, CPI-1189, LEM (liposome-encapsulated mitoxantrone), THC.CBD (cannabinoid agonist) MBP-8298, mesopram (PDE4 inhibitor), MNA-715 , anti-IL-6 receptor antibody, neurovax, pirfenidone allotrap 1258 (RDP-1258), sTNF-R1, talampanel, teriflunomide, TGF-beta2, tiplimotide, VLA-4 antagonists (e.g. TR-14035, VLA4 Ultrahaler , Antegran-ELAN/Biogen), interferon gamma antagonists, IL-4 agonists.
[0357] Non-limiting examples of therapeutic agents for angina with which binding proteins of the invention can be combined include the following: aspirin, nitroglycerin, isosorbide mononitrate, metoprolol succinate, atenolol, metoprolol tartrate, anlodipine besylate, hydrochloride diltiazem, isosorbide dinitrate, clopidogrel bisulfate, nifedipine, atorvastatin calcium, potassium chloride, furosemide, simvastatin, verapamil hcl, digoxin, propranolol hydrochloride, carvedilol, lisinopril, spironolactone, hydrochlorothiazide, ramoxapril, and sodium , heparin sodium, valsartan, sotalol hydrochloride, fenofibrate, ezetimibe, bumetanide, losartan potassium, lisinopril/hydrochlorothiazide, felodipine, captopril, bisoprolol fumarate.
[0358] Non-limiting examples of therapeutic agents for ankylosing spondylitis with which binding proteins of the invention can be combined include the following: ibuprofen, diclofenac and misoprostol, naproxen, meloxicam, indomethacin, diclofenac, celecoxib, rofecoxib, sulfasalazine, methotrexate , minocycline, prednisone, etanercept, infliximab.
[0359] Non-limiting examples of therapeutic agents for asthma with which binding proteins of the invention can be combined include the following: albuterol, salmeterol/fluticasone, montelukast sodium, fluticasone propionate, budesonide, prednisone, salmeterol xinafoate, levalbuterol hcl, albuterol/ipratropium sulphate, prednisolone sodium phosphate, triamcinolone acetonide, beclomethasone dipropionate, ipratropium bromide, azithromycin, pirbuterol acetate, prednisolone, theophylline anhydrous, methylprednisolone sodium succinate, clarfithromycin, clarfithromycin, clarithromycin, sodium succinate, clarfithromycin, vaccine Influenza, methylprednisolone, amoxicillin trihydrate, flunisolide, allergy injection, cromolyn sodium, fexofenadine hydrochloride, flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, adjunctive inhaler device, guaifenesin, dexamethasine sodium phosphate, hyclone/hoxycycline, moxif d-methorphan, p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine hydrochloride, mometasone furoate, salmeterol xinafoate, benzonatate, cephalexin, pe/hydrocodone/chlorfenir, cetirizine hcl/pseudoephed, phenylephrine/cod/promethazine, codeine/promethazine/clorfendone, cefprozil, cefprozil , nedocromil sodium, terbutaline sulfate, epinephrine, methylprednisolone, metaproterenol sulfate.
[0360] Non-limiting examples of therapeutic agents for COPD with which binding proteins of the invention can be combined include the following: albuterol/ipratropium sulfate, ipratropium bromide, salmeterol/fluticasone, albuterol, salmeterol xinafoate, fluticasone propionate, prednisone, theophylline anhydrous, methylprednisolone sodium succinate, montelukast sodium, budesonide, formoterol fumarate, triamcinolone acetonide, levofloxacin, guaifenesin, azithromycin, beclomethasone dipropionate, levalbuterol sodium triamcinolone, amxacillin, fluxonicin , flunisolide/menthol, chlorpheniramine/hydrocodone, metaproterenol sulfate, methylprednisolone, mometasone furoate, p-ephedrine/cod/chlorphenir, pirbuterol acetate, p-ephedrine/loratadine, terbutaline sulfate, tiotropium bromide, (R,R) - formoterol, TgAAT, Cilomilast, Roflumilast.
[0361] Non-limiting examples of therapeutic agents for HCV with which binding proteins of the invention can be combined include the following: Interferon-alpha-2a, Interferon-alpha-2b, Interferon-alpha con1, Interferon-alpha-n1, interferon pegylated alpha-2a, pegylated interferon alpha-2b, ribavirin, peginterferon alpha-2b + ribavirin, ursodeoxycholic acid, glycyrrhizic acid, thymalfasin, Maxamine, VX-497 and any compounds that are used to treat HCV through intervention with the following targets: HCV polymerase, HCV protease, HCV helicase, HCV IRES (internal ribosome entry site).
[0362] Non-limiting examples of therapeutic agents for idiopathic pulmonary fibrosis with which binding proteins of the invention can be combined include the following: prednisone, azathioprine, albuterol, colchicine, albuterol sulfate, digoxin, interferon gamma, methylprednisolone sod succ, lorazepam , furosemide, lisinopril, nitroglycerin, spironolactone, cyclophosphamide, ipratropium bromide, actinomycin d, alteplase, fluticasone propionate, levofloxacin, metaproterenol sulfate, morphine sulfate, oxycodone hcl, potassium chloride, interferon, triamcinolone, aceton -alpha, methotrexate, mycophenolate mofetil, Interferon-gamma-1β.
[0363] Non-limiting examples of therapeutic agents for myocardial infarction with which binding proteins of the invention can be combined include the following: aspirin, nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin sodium, clopidogrel bisulfate, carvedilol, atenolol, morphine sulphate, metoprolol succinate, sodium warfarin, lisinopril, isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril, tenecteplase, enalapril maleate, torsemide, retavase, losartan potassium, quinapril buplatme, albinidase, carb, alprite amiodarone hydrochloride, tirofiban hcl m-hydrate, diltiazem hydrochloride, captopril, irbesartan, valsartan, propranolol hydrochloride, fosinopril sodium, lidocaine hydrochloride, eptifibatide, cefazoline sodium, interferon-lactin sulfate, atropine acid, aminocathalone and sodium sulfate , potassium chloride, docusate sodium, dobutamine hcl, alprazolam, pravastatin sodium, atorvasta calcium tin, midazolam hydrochloride, meperidine hydrochloride, isosorbide dinitrate, epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin, ezetimibe/simvastatin, avasimibe, cariporida.
[0364] Non-limiting examples of therapeutic agents for psoriasis with which binding proteins of the invention can be combined include the following: KDR inhibitor small molecule, Tie-2 inhibitor small molecule, calcipotriene, clobetasol propionate, triamcinolone acetonide, propionate of halobetasol, tazarotene, methotrexate, fluocinonide, betamethasone diprop augmented, fluocinolone acetonide, acitretin, tar shampoo, betamethasone valerate, mometasone furoate, ketoconazole, pramoxine/fluocinolone, hydrocortisone betamethalide, hydrocortisone valerate, flucortisone emoll, fluticasone propionate, azithromycin, hydrocortisone, moisturizing formula, folic acid, desonide, pimecrolimus, coal tar, diflorasone diacetate, etanercept folate, lactic acid, methoxsalen, hc/bismuth subgal/znox/resor, methylprednisolone prednisone, sunscreen, halcinonide, salicylic acid, anthralin, clocortolone pivalate, extra coal tar, coal tar/salicylic acid, coal tar/salicylic acid/sulfur, deoxymethasone, diazepam, emollient, fluocinonide/emollient, mineral oil/ricinol oil/na lact, mineral oil/peanut oil, petroleum/myristate of isopropyl, psoralen, salicylic acid, soap/tribromsalan, thimerosal/boric acid, celecoxib, infliximab, cyclosporine, alefacept, efalizumab, tacrolimus, pimecrolimus, PUVA, UVB, sulfasalazine.
[0365] Non-limiting examples of therapeutic agents for psoriatic arthritis with which binding proteins of the invention can be combined include the following: methotrexate, etanercept, rofecoxib, celecoxib, folic acid, sulfasalazine, naproxen, leflunomide, methylprednisolone acetate, indomethacin, hydroxychloroquine sulfate, prednisone, sulindac, betamethasone diprop augmented, infliximab, methotrexate, folate, triamcinolone acetonide, diclofenac, dimethylsulfoxide, piroxicam, diclofenac sodium, ketoprofen, meloxicam, methylprednisolone, diclofenolone, diclofenac, cyclostolime fluocinonide, glucosamine sulfate, gold sodium thiomalate, hydrocodone/apap bitartrate, ibuprofen, risedronate sodium, sulfadiazine, thioguanine, valdecoxib, alefacept, efalizumab and bcl-2 inhibitors.
[0366] Non-limiting examples of therapeutic agents for psoriatic restenosis with which binding proteins of the invention can be combined include the following: sirolimus, paclitaxel, everolimus, tacrolimus, Zotarolimus, acetaminophen.
[0367] Non-limiting examples of therapeutic agents for psoriatic sciatica with which binding proteins of the invention can be combined include the following: hydrocodone/apap bitartrate, rofecoxib, cyclobenzaprine hcl, methylprednisolone, naproxen, ibuprofen, oxycodone hcl/acetoxybene, c. , valdecoxib, methylprednisolone acetate, prednisone, codeine/apap phosphate, tramadol hcl/acetaminophen, metaxalone, meloxicam, methocarbamol, lidocaine hydrochloride, diclofenac sodium, gabapenthine, dexamethasone, carisopromethamine, diapamethocin, ketorolac oxycodone hcl, tizanidine hcl, diclofenac sodium/misoprostol, propoxyphene napsylate/apap, asa/oxycod/oxycodone ester, ibuprofen/hydrocodone bit, tramadol hcl, etodolac, propoxyphene hcl, amitriptyline hcl, carisoprodone/sulfasine , multivitamins, naproxen sodium, orphenadrine citrate, temazepam.
[0368] Examples of therapeutic agents for psoriatic SLE (lupus) with which binding proteins of the invention can be combined include the following: NSAIDS, for example, diclofenac, naproxen, ibuprofen, piroxicam, indomethacin; COX2 inhibitors, for example Celecoxib, rofecoxib, valdecoxib; antimalarials, for example, hydroxychloroquine; steroids, for example prednisone, prednisolone, budesonide, dexamethasone; cytotoxics, for example azathioprine, cyclophosphamide, mycophenolate mofetil, methotrexate; PDE4 inhibitors or purine synthesis inhibitor, for example Cellcept. Binding proteins of the invention can also be combined with agents such as sulfasalazine, 5-aminosalicylic acid, olsalazine, Imuran and agents that interfere with the synthesis, production or action of pro-inflammatory cytokines such as IL-1, e.g. caspases as inhibitors of the converting enzyme of IL-1β and IL-1ra. Binding proteins of the invention can also be used with T cell signaling inhibitors, for example tyrosine kinase inhibitors; or molecules directed against T cell activation, e.g. CTLA-4-IgG or anti-B7 family antibodies, anti-PD-1 family antibodies. Binding proteins of the invention can be combined with IL-11 or anti antibodies -cytokine, e.g. fotolizumab (anti-IFNg antibody) or anti-receptor antibodies, e.g. anti-IL-6 receptor antibody and antibodies against B cell surface molecules. Antibodies of the invention or its antigen-binding portion may also be used with LJP 394 (abethymus), agents that deplete or inactivate B cells, eg Rituximab (anti-CD20 antibody), limfostat-B (anti-BliS-antibody), TNF antagonists, eg anti-antibodies - TNF, Adalimumab (PCT Publication No. WO 97/29131; HUMIRA®), CA2 (REMICADE®), CDP 571, TNFR-Ig constructs, (p75TNFRIgG (ENBREL®) and p55TNFRIgG (LENERCEPT®)) and bcl-inhibitors 2, because overexpression of bcl-2 in transgenic mice has been shown to cause a similar phenotype that of lupus (see Marquina et al., J. Immunol., 172(11): 7177-7185 (2004)), therefore its inhibition is expected to have therapeutic effects.
Pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody portion of the invention. "Therapeutically effective amount" refers to an effective amount, in doses and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antibody portion can be determined by one of ordinary skill in the art and may vary according to factors such as the disease state, age, sex and weight of the individual and the capacity of the antibody or portion antibody elicits a desired response in the individual. Therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. "Prophylactically effective amount" refers to an amount effective, in doses and for periods of time necessary, to achieve the desired prophylactic effect. Typically, since a prophylactic dose is used in individuals before or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
[0370]Dose regimens can be adjusted to provide the desired optimal response (eg, therapeutic or prophylactic response). For example, a single bolus can be given, multiple divided doses can be given over time, or the dose can be proportionately reduced or increased as indicated by the requirements of the therapeutic situation. It is especially advantageous to formulate parental compositions in unit dose form to facilitate administration and uniformity of dose. Unit dose form, in this specification, refers to physically distinct units suitable as unit doses for the mammals to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the pharmaceutical vehicle required. The specification for the unit dose forms of the invention is dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved and (b) the limitations inherent in the state of the art in pharmaceutical manipulation of such active ingredient for the treatment of sensitivity in individuals.
[0371]It should be noted that dose values may vary with the type and severity of the condition to be relieved. It should also be understood that for any given individual, the specific dosage regimen should be adjusted over time according to individual need and the professional judgment of the person responsible for administering or supervising the administration of the compositions, and that the dose ranges presented in this descriptive report are examples only and are not intended to limit the scope or practice of the claimed composition. Diagnosis
[0372] The present invention also provides diagnostic applications. These will be elucidated in more detail below. IL-1β-binding antibodies of the invention can be employed in any of a variety of formats to detect IL-1β in vivo, in vitro or ex vivo (ie, in cells or tissues that have been obtained from a living individual. , subjected to a procedure and then returned to the individual). DVD-Igs of the invention offer the additional advantage of being able to bind an IL-1β epitope in addition to other antigens or epitopes in various diagnostic and detection assay formats. I. Test method
[0373] The present invention also provides a method to determine the presence, amount or concentration of IL-1β or its fragment ("analyte") in a test sample, using at least one anti-IL-binding protein. 1β or its antigen-binding portion, including a DVD-Ig, as described herein. Any suitable assay as known in the prior art can be used in the method. Examples include, but are not limited to, immunoassay, such as a sandwich immunoassay (e.g., monoclonal, polyclonal and/or DVD-Ig antibody sandwich immunoassay, or any variation thereof (e.g. monoclonal/DVD-Ig, DVD-Ig/polyclonal , etc.), including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (eg, Quantikine ELISA assays, R&D Systems, Minneapolis, Minnesota)), immunoassay by competitive inhibition (eg, forward and reverse), fluorescent polarization immunoassay (FPIA), multiplied enzyme immunoassay technique (EMIT), bioluminescence resonance energy transfer (BRET) and homogeneous chemiluminescent assay, etc. In an immunoassay, in an immunoassay SELDI-based, a capture reagent that specifically binds to an analyte (or fragment thereof) of interest is attached to the surface of a mass spectrometry probe, such as a p-chip array. preactivated protein. The analyte is then specifically captured on the biochip, and the captured analyte is detected by mass spectrometry. Alternatively, the analyte (or its fragment) can be eluted from the capture reagent and detected by traditional MALDI (matrix-assisted laser desorption/ionization) or by SELDI. A chemiluminescent microparticle immunoassay, especially one employing the ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park, Illinois), is an example of an exemplary immunoassay.
[0374] Methods well known in the art for collecting, handling and processing urine, blood, serum and plasma and other body fluids are used in the practice of the present invention, for example, when an anti-IL-1β binding protein, as described herein, is employed as an immunodiagnostic reagent and/or in an analyte immunoassay kit. The test sample may comprise groups other than the analyte of interest, such as antibodies, antigens, haptens, hormones, drugs, enzymes, receptors, proteins, peptides, polypeptides, oligonucleotides and/or polynucleotides. For example, the sample can be a whole blood sample obtained from an individual. It may be necessary or desired that a test sample, especially whole blood, be treated prior to the immunoassay as described herein, for example, with a pretreatment reagent. Even in cases where pre-treatment is not necessary (eg in most urine samples), Pre-treatment can optionally be carried out (eg as part of a scheme on a commercial platform).
[0375] The pretreatment reagent may be any suitable for use with the immunoassay and kits of the invention. The pretreatment optionally comprises: (a) one or more solvents (for example methanol and ethylene glycol) and optionally salt, (b) one or more solvents and salt and optionally detergent, (c) detergent or (d) detergent and salt. Pretreatment reagents are known in the art and such pretreatment can be employed, for example, as used for testing on Abbott TDx, AxSYM® and ARCHITECT® analyzers (Abbott Laboratories, Abbott Park, Illinois), as per described in the literature (see, for example, Yatscoff et al., "Abbott TDx Monoclonal Antibody Assay Evaluated for Measuring Cyclosporine in Whole Blood", Clin. Chem., 36: 1969-1973 (1990); and Wallemacq et al., " Evaluation of the New AxSYM Cyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and EMIT Cyclosporine Assays", Clin. Chem., 45: 432-435 (1999)), and/or as commercially available. Additionally, pretreatment can be performed as described in U.S. Patent No. 5,135,875; European Publication No. EP 0 471 293; PCT Publication No. WO 2008/082984; and Abbott US Publication No. 2008/0020401 (incorporated in its entirety for its teaching regarding pretreatment by reference in this application). The pretreatment reagent can be a heterogeneous or homogeneous agent.
[0376]With the use of a heterogeneous pretreatment reagent, the pretreatment reagent precipitates the analyte-binding protein (eg, the protein that can bind an analyte or its fragment) present in the sample. Such a pretreatment step comprises removing any analyte binding protein by separating the precipitated analyte binding protein from the supernatant of the mixture formed by adding the pretreatment agent to the sample. In such an assay, the supernatant from the mixture without any binding protein is used in the assay, proceeding directly to the capture antibody step.
[0377]With the use of a homogeneous pretreatment reagent, there is no such separation step. The entire mixture of test sample and pretreatment reagent is contacted with a specific binding partner labeled for the analyte (or its fragment), such as a labeled anti-analyte antibody (or its antigenically reactive fragment). The pretreatment reagent employed for such an assay is typically diluted into the pretreated test sample mixture, either prior to or during capture by the first specific binding partner. Despite this dilution, some amount of the pretreatment reagent is still present (or remains) in the test sample mixture during capture. According to the invention, an exemplary labeled specific binding partner may be a DVD-Ig (or fragment, variant or variant fragment thereof).
[0378]In a heterologous format, after the test sample is obtained from an individual, a first mixture is prepared. The mixture contains the test sample being evaluated for the analyte (or fragment thereof) and a first specific binding partner, wherein the first specific binding partner and any analyte contained in the test sample form a first specific binding partner complex- analyte. Preferably, the first specific binding partner is an anti-analyte antibody or fragment thereof. The first specific binding partner can be a DVD-Ig (or fragment, variant or variant fragment thereof) as described herein. The order in which the test sample and the first specific binding partner are added to form the mixture is not critical. Preferably, the first specific binding partner is immobilized on a solid phase. The solid phase used in the immunoassay (for the first specific binding partner and optionally the second specific binding partner) can be any solid phase known in the prior art, such as, among others, magnetic particle, microsphere, test tube , microtiter plate, cuvette, membrane, scaffold molecule, film, filter paper, disk and chip.
[0379] After the mixture containing the first specific binding partner-analyte complex is formed, any unbound analyte is removed from the complex using any technique known in the prior art. For example, unbound analyte can be washed away. Desirably, however, the first specific binding partner is present in excess of any analyte present in the test sample, such that all analyte present in the test sample has been bound by the first specific binding partner.
[0380]After any unbound analyte has been removed, a second specific binding partner is added to the mixture to form a first specific binding partner-analyte-second specific binding partner complex. The second specific binding partner is preferably an anti-analyte an antibody that binds to an epitope different from that on the analyte that is bound by the first specific binding partner. Furthermore, also preferably, the second specific binding partner is labeled or contains a detectable label as described above. The second specific binding partner can be a DVD-Ig (or fragment, variant or variant fragment thereof) as described herein.
[0381] Any suitable detectable marker as known in the prior art can be used. For example, the detectable label can be radioactive label (such as 3H, 125I, 35S, 14C, 32P and 33P), enzyme label (such as horseradish peroxidase, alkaline phosphatase, glucose 6-phosphate dehydrogenase and the like), chemiluminescent label (such as esters, thio esters or sulfonamides of acridinium, luminol, isoluminol, phenanthridinium esters and the like), fluorescent label (such as fluorescein (eg, 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein , 6-hexachloro-fluorescein; 6-tetrachlorofluorescein, fluorescein isothiocyanate and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (eg, cadmium selenide coated with zinc sulfide), thermometric marker or reaction marker immuno with polymerase. An introduction to markers, marking procedures and marker detection is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, NY (1997) and in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), which is a combined manual and catalog published by Molecular Probes, Inc., Eugene, Oregon. A fluorescent label can be used in FPIA (see, for example, U.S. Patent Nos. 5,593,896; 5,573,904; 5,496,925; 5,359,093 and 5,352,803 ). An acridinium compound can be used as a detectable marker in a homogeneous or heterogeneous chemiluminescent assay (see, for example, Adamczyk et al., Bioorg. Med. Chem. Lett., 16: 1324-1328 (2006); Adamczyk et al., Bioorg.Med.Chem.Lett., 14:2313-2317 (2004); Adamczyk et al., Biorg.Med.Chem.Lett., 14:3917-3921 (2004); and Adamczyk et al.Org.Lett. , 5:37793782 (2003)).
[0382] An exemplary acridinium compound is acridinium-9-carboxamide. Methods for preparing acridinium 9-carboxamides are described in Mattingly, J. Biolumin. Chemilumin., 6: 107-114 (1991); Adamczyk et al., J.Org.Chem., 63:56365639 (1998); Adamczyk et al., Tetrahedron, 55: 10899-10914 (1999); Adamczyk et al. Org. Lett., 1:779-781 (1999); Adamczyk et al., Bioconjugate Chem., 11: 714-724 (2000); Adamczyk and Mattingly, In Luminescence Biotechnology: Instruments and Applications; (Dyke, K.V., ed.) (CRC Press: Boca Raton, 2002) p. 77-105; Adamczyk et al. Org. Lett., 5:3779-3782 (2003); and U.S. Patent Nos. 5,468,646, 5,543,524 and 5,783,699. Another preferred acridinium compound is aryl ester of acridinium-9-carboxylate. An example of an acridinium-9-carboxylate aryl ester is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available from Cayman Chemical, Ann Arbor, Michigan). Methods for preparing aryl acridinium-9-carboxylate ester are described in McCapra et al., Photochem. Photobiol., 4:1111-21 (1965); Razavi et al., Luminescence, 15: 245-249 (2000); Razavi et al., Luminescence, 15: 239-244 (2000); and U.S. Patent No. 5,241,070. Additional details regarding the aryl ester of acridinium-9-carboxylate and its use are set forth in US Publication No. 2008/0248493.
[0383]Chemiluminescent assays (for example, using acridinium as described above or other chemiluminescent agents) can be performed according to the methods described in Adamczyk et al., Anal. Chim. Acta, 579(1): 61-67 (2006). Although any suitable assay format can be used, a microplate chemiluminometer (Mithras LB-940, Berthold Technologies USA., LLC, Oak Ridge, Tennessee) allows multiple small volume samples to be quickly analyzed.
[0384] The order in which the test sample and specific binding partner(s) are added to form the chemiluminescence assay mixture is not critical. If the first specific binding partner is detectably labeled with a chemiluminescent agent, such as an acridinium compound, detectably labeled first specific binding partner-analyte complexes form. Alternatively, if a second specific binding partner is used and the second specific binding partner is detectably labeled with a chemiluminescent agent, such as an acridinium compound, detectably labeled first specific binding partner-analyte-second specific binding partner complexes if form. Any unbound specific binding partner, whether labeled or not, can be removed from the mixture using any techniques known in the art, such as washing.
[0385] Hydrogen peroxide can be generated in situ in the mixture or provided or supplied to the mixture (for example, the source of the hydrogen peroxide being one or more buffers or other solutions known to contain hydrogen peroxide) before, simultaneously or after the addition of an acridinium compound described above. Hydrogen peroxide can be generated in situ in a number of ways, as would be evident to one skilled in the art.
[0386]When the simultaneous or subsequent addition of at least one basic solution to the sample, a detectable signal, namely, chemiluminescent signal, indicative of the presence of the analyte, is generated. The basic solution contains at least one base and a pH greater than or equal to 10, preferably greater than or equal to 12. Examples of basic solutions include, among others, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium carbonate, calcium hydroxide, calcium carbonate and calcium bicarbonate. The amount of base solution added to the sample depends on the concentration of the base solution. Based on the concentration of base solution used, the skilled person can quickly determine the amount of base solution to be added to the sample.
[0387]The chemiluminescent signal generated can be detected with routine techniques known to those versed in the subject. Based on the intensity of the generated signal, the amount of analyte in the sample can be quantified. Specifically, the amount of analyte in the sample is proportional to the intensity of the generated signal. The amount of analyte present can be quantified by comparing the amount of light generated to a standard curve for the analyte, or by comparing it to a reference standard. The standard curve can be generated with serial dilutions or solutions of known concentrations of analyte by mass spectroscopy, gravimetric methods and other techniques known in the art. Although the foregoing is described with an emphasis on the use of an acridinium compound, one skilled in the art can readily adapt this description to the use of other chemiluminescent agents.
[0388]Analyte immunoassays can generally be conducted using any format known in the art, such as, among others, sandwich format. Specifically, in an immunoassay format, at least two antibodies are employed to separate and quantify the analyte, such as human analyte or its fragment in a sample. More specifically, the at least two antibodies bind to different epitopes on an analyte (or its fragment), forming an immunocomplex, which is called a “sandwich.” In general, in immunoassays, one or more antibodies can be used to capture the analyte. (or its fragment) in the test sample (these antibodies are often called "capture" antibody or "capture" antibodies), and one or more antibodies can be used to bind a detectable (namely, quantifiable) marker to the sandwich ( these antibodies are often referred to as the "detection antibody", the "detection antibodies", the "conjugate" or the "conjugates".) Thus, in the context of a sandwich-type immunoassay format, a DVD-Ig (or fragment thereof) , variant or variant fragment), as described herein, can be used as a capture antibody, detection antibody, or both. For example, a DVD-Ig containing a domain capable of binding a first epitope on an analyte (or s). or fragment) can be used as a capture antibody and/or another DVD-Ig containing a domain capable of binding a second epitope on an analyte (or its fragment) can be used as a detection antibody. In this regard, a DVD-Ig containing a first domain capable of binding a first epitope in an analyte (or its fragment) and a second domain capable of binding a second epitope (or its fragment) can be used as capture antibody and/or detection antibody. Alternatively, a DVD-Ig ha containing a first domain capable of binding an epitope in a first analyte (or its fragment) and a second domain capable of binding an epitope in a second analyte (or its fragment) can be used as capture antibody and/or detection antibody to detect and optionally quantify two or more analytes. In case an analyte is present in a sample in more than one form, such a monomeric form and a dimeric/multimeric form, which can be homomeric or heteromeric, a DVD-Ig, containing a domain capable of binding to an epitope that is exposed only in monomeric form, and other DVD-Ig containing a domain capable of binding to an epitope in a different part of a dimeric/multimeric form can be used as capture antibodies and/or detection antibodies, thus enabling detection and optional quantification of different forms of a given analyte. Furthermore, employing DVD-Igs with different affinities on a single DVD-Ig and/or between DVD-Igs can serve an advantage in terms of avidity. In the context of immunoassays as described herein, it may generally be useful or desired to incorporate one or more linkers into the structure of a DVD-Ig. When present, the linker ideally should be of sufficient length and structural flexibility to allow binding to one epitope via the internal domains as well as binding to another epitope via the external domains. In this sense, if a DVD-Ig can bind to two different analytes and one analyte is larger than the other, desirably the larger analyte is bound by the external domains.
[0389] In general terms, a sample, being tested for (for example, suspected of containing) an IL-1β protein (or its fragment), may be contacted with at least one antibody (or antibodies) of capture and at least one detection antibody (which can be a second detection antibody or a third detection antibody or even a successively numbered antibody, e.g. as when the capture and/or detection antibody comprises multiple antibodies), simultaneously or in sequence and in any order. For example, the test sample may first be contacted with at least one capture antibody and then (in sequence) with at least one detection antibody. Alternatively, the test sample may first be contacted with at least one detection antibody and then (in sequence) with at least one capture antibody. Alternatively, the sample can be contacted simultaneously with a capture antibody and a detection antibody.
[0390] In the sandwich-type assay format, a sample suspected of containing IL-1β (or its fragment) is first brought into contact with at least a first capture binding protein (eg antibody against IL-1β) in conditions that allow the formation of a first binding protein/IL-1β complex. If more than one capture binding protein is used, a first capture binding protein/IL-1β complex, comprising two or more capture proteins, is formed. In a sandwich assay, binding proteins, i.e., preferably the at least single capture binding protein, are used in amounts in molar excess of the predicted maximum amount of the IL-1β analyte (or its fragment) in the sample in test. For example, from approximately 5 μg to approximately 1 mg of antibody per ml of buffer (eg microparticle coating buffer) can be used.
[0391] Competitive inhibition immunoassays, which are often used to measure small analytes because only binding by a single antibody is required, comprise sequential and classical formats. In a sequential competitive inhibition immunoassay, a capture protein that binds to IL-1β is coated onto a well of a microtiter plate or other solid support. When the sample containing the IL-1β is added to the well, the IL-1β binds to the capture binding protein. After washing, a known amount of labeled IL-1β (eg with biotin or horseradish peroxidase (HRP)) is added to the well. A substrate for the enzyme label is needed to generate a signal. An example of a suitable substrate for HRP is 3,3',5,5'-tetramethylbenzidine (TMB). After washing, the signal generated by the labeled analyte is measured, being inversely proportional to the amount of IL-1β in the sample. In a classic competitive inhibition immunoassay, an IL-1β binding protein is coated onto a solid support (eg, microtiter plate well). However, unlike the sequential competitive inhibition immunoassay, the sample and labeled IL-1β are added to the well at the same time. Any IL-1β in the sample competes with the labeled IL-1β for binding to the capture binding protein. After washing, the signal generated by labeled IL-1β is inversely proportional to the amount of IL-1β in the sample.
[0392]Optionally, before the test sample is contacted with the at least single capture binding protein (for example, the first capture antibody), the at least single capture binding protein can bind to a solid support, which facilitates the separation of the first binding protein / IL-1β complex (or its fragment) from the test sample. The substrate to which the capture binding protein is bound can be any suitable solid support or solid phase that facilitates separation of the capture antibody-analyte complex from the sample.
[0393] Examples include well of a plate such as microtiter plate, test tube, porous gel (eg silica gel, agarose, dextran or gelatin), polymeric film (eg polyacrylamide), beads (eg , polystyrene or magnetic beads), filter/membrane strip (eg nitrocellulose or nylon), microparticles (eg latex particles), microparticles capable of magnetizing (eg microparticles with ferric oxide or oxide cores) of chromium and homo or heteropolymeric coatings and radii of approximately 1-10 microns). The substrate may comprise a suitable porous material with adequate surface affinity to bind antigen and sufficient porosity to allow access by detecting antibodies. A microporous material is generally preferred, although a gelatinous in a hydrated state may be used. Such porous substrates are preferably in the form of sheets, having a thickness of from approximately 0.01 to approximately 0.5 mm, preferably from approximately 0.1 mm. While pore size can vary widely, preferably the pore size is from approximately 0.025 to approximately 15 microns, more preferably from approximately 0.15 to approximately 15 microns. The surface of these substrates can be activated by chemical processes that cause an antibody to covalently bind to the substrate. The result is irreversible binding, usually by adsorption through hydrophobic forces, of the antigen or antibody to the substrate; alternatively, a chemical coupling agent or other means can be used to covalently bind the antibody to the substrate, so long as such binding does not interfere with the antibody's ability to bind the analyte. Alternatively, the antibody can be bound with microparticles, which have previously been coated with streptavidin (e.g., DYNAL® Magnetic Beads, Invitrogen, Carlsbad, California) or biotin (e.g., using streptavidin-coated Power-BindTM-SA-MP microparticles (Seradyn, Indianapolis, Indiana)) or species-specific monoclonal antibodies. If necessary, the substrate can be derivatized to allow for reactivity with various functional groups on the antibody. Such derivation requires the use of certain coupling agents, examples of which include, but are not limited to, maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. If desired, one or more capture reagents, such as antibodies (or fragments thereof), each specific to the analyte(s), can be affixed to solid phases in different physical or accessible locations (eg, such as in configuration of biochip (see, for example, US Patent No 6,225,047; PCT Publication No. WO 99/51773; US Patent No. 6,329,209; PCT Publication No. WO 00/56934; and US Patent No. 5,242,828). of capture is attached to a mass spectrometry probe such as the solid support, the amount of analyte bound to the probe can be detected by laser desorption/ionization mass spectrometry. Alternatively, a single column can be packed with different beads, which they are derived to the single or more capture reagents, thus capturing the analyte in a single place (see, antibody-derived bead-based technologies, eg Luminex xMAP technology (Austin, Texas)).
[0394] After the test sample under analysis for the analyte (or its fragment) is brought into contact with the at least single capture antibody (for example, the first capture antibody), the mixture is incubated to allow the formation of a first antibody (or multiple antibodies)-analyte (or fragment thereof) complex. Incubation can be carried out at a pH of approximately 4.5 to approximately 10.0 and a temperature of approximately 2 °C to approximately 45 °C and for a period of at least approximately one (1) minute to approximately eighteen (18) hours, preferably from approximately 1 to approximately 24 minutes, more preferably from approximately 4 to approximately 18 minutes. The immunoassay described herein can be conducted in a single step (meaning that the test sample, the at least single capture antibody, and the at least single detection antibody all added sequentially or simultaneously to a reaction vessel) or in more than one one step, such as two steps, three steps, etc.
[0395] After the formation of the complex (first or multiple) capture antibody/analyte (or its fragment), the complex is then contacted with the at least single detection antibody under conditions that allow the formation of a complex (first or multiple) capture antibody/analyte (or fragment thereof)/second detection antibody. Although indicated for clarity as the “second” antibody (eg second detection antibody), in fact, when multiple antibodies are used for capture and/or detection, the at least single detection antibody can be the second, third, the bedroom, etc. antibody used in the immunoassay. If the capture antibody/analyte complex (or its fragment) is brought into contact with more than one detection antibody, then a complex (first or multiple) capture antibody/analyte (or its fragment)/(multiple) antibody of detection is formed. As with the capture antibody (eg, the first capture antibody), when the at least one (eg, second and any subsequent) detection antibody is brought into contact with the capture antibody/analyte complex (or its fragment), an incubation period under conditions similar to those described above is required for the formation of the (first or multiple) capture antibody/analyte (or fragment thereof)/(second or multiple) detection antibody complex. Preferably, at least one detection antibody contains a detectable label. The detectable marker may be linked to the at least single detection antibody (eg the second detection antibody) before, simultaneously or after the formation of the (first or multiple) capture antibody/analyte (or fragment thereof)/(second) complex or multiple) detection antibody. Any detectable marker known in the prior art can be used (see discussion above, including references to Polak and Van Noorden (1997) and Haugland (1996)).
[0396] The detectable label can be linked to antibodies directly or through a coupling agent. An example of a coupling agent that can be used is EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, hydrochloride), commercially available from Sigma-Aldrich, St. Louis, Missouri. Other coupling agents that can be used are known in the state of the art. Methods for attaching a detectable label to an antibody are known in the art. Additionally, many detectable labels can be purchased or synthesized that already contain end groups that facilitate coupling of the detectable label to the antibody, such as CPSP-acridinium ester (i.e., 9-[N-tosyl-N-(3-carboxypropyl) ]-10-(3-sulfopropyl)acridinium carboxamide) or SPSP-acridinium ester (i.e., N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).
[0397] The (first or multiple) capture antibody/analyte (or fragment thereof)/(second or multiple) detection antibody complex can, but need not, be separated from the remainder of the sample prior to quantifying the marker. For example, if the at least single capture antibody (eg, the first capture antibody) is bound to a solid support, such as a cavity or bead, separation can be performed by removing the liquid (from the test sample) from the contact with solid support. Alternatively, if bound to a solid support, the at least single capture antibody (eg, the first capture antibody) may be simultaneously contacted with the sample containing the analyte and the at least single second detection antibody to form a first (multiple) antibody/analyte/second (multiple) antibody complex, followed by removal of the liquid (test sample) from contact with the solid support. If the at least single first capture antibody is not bound to a solid support, then the complex (first or multiple) capture antibody/analyte/(second or multiple) detection antibody need not be taken from the test sample for quantification of the highlighter.
[0398]After the formation of the capture antibody/analyte/labeled detection antibody complex (for example, the first capture antibody/analyte/second detection antibody complex), the amount of label in the complex is quantified using techniques known in the art of technique. For example, if an enzyme label is used, the labeled complex is reacted with a substrate for the label that provides a quantifiable reaction such as color development. If the label is radioactive, the label is quantified using appropriate means such as a scintillation counter. If the marker is fluorescent, the marker is quantified by stimulating it with a light of one color (which is known as the “excitation wavelength”) and by detecting another color (which is known as the “wavelength of emission”) which is emitted by the marker in response to stimulation. If the marker is chemiluminescent, the marker is quantified by detecting light emitted visually or using luminometers, x-ray film, high speed photographic film, CCD camera, camera, etc. Once the amount of the marker in the complex has been quantified, the concentration of analyte or its fragment in the test sample is determined by appropriate means, such as using a standard curve that has been generated with serial dilutions of the analyte or its fragment of known concentration. In addition to using serial dilutions of the analyte or its fragment, a standard curve can be generated by gravimetric techniques, by mass spectrometry or by others known in the state of the art.
[0399] In an assay with chemiluminescent microparticles using the ARCHITECT® analyzer, the pH of the conjugated diluent should be approximately 6.0 +/- 0.2, the microparticle coating buffer should be kept at approximately room temperature ( ie, from approximately 17 °C to approximately 27 °C), the pH of the microparticle coating buffer should be approximately 6.5 +/- 0.2 and the pH of the microparticle diluent should be approximately 7.8 +/- 0.2. The solids content is preferably less than about 0.2%, such as less than about 0.15%, less than about 0.14%, less than about 0.13%, less than about 0.12% or less to approximately 0.11%, such as approximately 0.10%.
[0400]FPIAs are based on competitive binding immunoassay principles. A fluorescence-labeled compound, when excited by linearly polarized light, will emit fluorescence with a degree of polarization inversely proportional to its rate of rotation. When a fluorescently labeled antibody-tracker complex is excited by linearly polarized light, the emitted light remains highly polarized because the fluorophore is prevented from rotating between the time the light is absorbed and the time the light is emitted. When a "tracker-free" compound (ie, a compound not bound to an antibody) is excited by linearly polarized light, its rotation is much faster than that of the corresponding tracker-antibody conjugate produced in a competitive binding immunoassay . FPIAs are advantageous over RIAs in that there are no radioactive substances requiring special handling and disposal. Additionally, FPIAs are homogeneous assays that can be performed quickly and easily.
[0401] In view of the foregoing, a method for determining the presence, amount or concentration of analyte (or its fragment) in a test sample is provided. The method comprises analyzing the test sample for an analyte (or its fragment) by an assay (i) employing (i') at least one antibody, an antibody fragment capable of binding an analyte, a capable antibody variant of binding an analyte, an antibody variant fragment capable of binding an analyte, and a DVD-Ig (or its fragment, variant or variant fragment) capable of binding an analyte; and (ii') at least a detectable marker and (ii) comprising comparing a signal generated by the detectable marker as a direct or indirect indication of the presence, amount or concentration of the analyte (or its fragment) in the test sample against a signal generated as a direct or indirect indication of the presence , quantity or concentration of the analyte (or its fragment) in a control or calibrator. The calibrator is optionally part of a series of calibrators, where each calibrator differs from the others by analyte concentration.
[0402] The method may comprise (i) contacting the test sample with at least a first specific binding partner for the analyte (or its fragment) selected from the group consisting of antibody, antibody fragment capable of binding to an analyte, antibody variant capable of binding an analyte, antibody variant fragment capable of binding an analyte and DVD-Ig (or its fragment, variant or variant fragment) capable of binding an analyte, so as to form a first specific binding partner/analyte complex (or its fragment), (ii) by first specific binding partner/analyte complex (or its fragment) in contact with at least one second specific binding partner for the analyte (or its fragment) selected from the group consisting of detectably labeled anti-analyte antibody, detectably labeled anti-analyte antibody fragment capable of binding to analyte, anti-analyte antibody variant detects detectably labeled anti-analyte antibody variant fragment capable of binding analyte capable of binding analyte or detectably labeled DVD-Ig (or its fragment, variant or variant fragment) so as to form a first partner complex of specific binding/analyte (or fragment thereof)/second specific binding partner and (iii) determining the presence, amount or concentration of analyte in the test sample, by detecting or measuring the overall signal by the detectable marker in the first binding partner complex specific/analyte (or fragment thereof)/second specific binding partner formed in sample (ii). A method in which at least one analyte-specific first binding partner (or fragment thereof) and/or at least one analyte-specific second binding partner (or fragment thereof) is a DVD-Ig (or fragment thereof, variant thereof) or variant fragment) as described herein may be preferred.
[0403] Alternatively, the method may comprise contacting the test sample with at least a first specific binding partner for an IL-1β analyte (or its fragment) selected from the group consisting of antibody, capable antibody fragment of binding to an analyte, antibody variant capable of binding to an analyte, antibody variant fragment capable of binding to an analyte and a DVD-Ig (or its fragment, variant or variant fragment) and simultaneously or in sequence, in any order, by contacting the test sample with at least one specific second binding partner, which is capable of competing with the analyte (or its fragment) for binding to the at least first specific binding partner and which is selected from the group consisting of a detectably labeled analyte, a detectably labeled analyte fragment capable of binding the first specific binding partner, an analyte variant detectably ma each capable of binding the first specific binding partner and a detectably labeled fragment of an analyte variant capable of binding the first specific binding partner. Any IL-1β (or fragment thereof) present in the test sample and the at least single second specific binding partner compete with each other to form a first specific binding partner/analyte complex (or fragment thereof) and a first binding partner complex specific/second specific binding partner, respectively. The method further comprises determining the presence, amount or concentration of analyte in the test sample by detecting or measuring the overall signal by the detectable label in the first specific binding partner/second specific binding partner complex formed in the sample (ii), wherein the The signal generated by the detectable label in the first specific binding partner/second specific binding partner complex is inversely proportional to the amount or concentration of analyte in the test sample.
[0404] The above methods may further comprise diagnosing, prognosticating or evaluating the effectiveness of a therapeutic/prophylactic treatment of a patient from whom the test sample was obtained. If the method further comprises evaluating the effectiveness of a therapeutic/prophylactic treatment of a patient from which the test sample was obtained, the method further comprises optionally modifying the therapeutic/prophylactic treatment of the patient, as necessary, to improve the effectiveness. The method can be adapted for use in an automatic system or semi-automatic system.
[0405] Regarding assay methods (and kits therefor), it may be possible to employ commercially available anti-analyte antibodies or methods for producing anti-analyte as described in the literature. Commercial suppliers of various antibodies include, but are not limited to, Santa Cruz Biotechnology Inc. (Santa Cruz, California), GenWay Biotech, Inc. (San Diego, California) and R&D Systems (RDS; Minneapolis, Minnesota).
[0406] In general, a predetermined level can be used as a benchmark against which to evaluate the results obtained when analyzing a test sample for an analyte or its fragment, for example, to detect disease or disease risk. In general, in such a comparison, the predetermined level is obtained by performing a given test a sufficient number of times and under appropriate conditions, such that a relationship or association of the presence, quantity or concentration of analyte with a given stage or outcome of a disease, disorder or condition or with certain clinical signs may be effected. Typically, the predetermined level is obtained with trials with reference individuals (or populations of individuals). The analyte measured can include its fragments, its degradation products and/or its enzymatic cleavage products.
[0407] Specifically, with respect to a predetermined level employed to monitor progression and/or treatment of a disease, the amount or concentration of analyte or its fragment may be "unchanged", "favorable" (or "favorably changed") or “unfavorably” (or “unfavorably altered”). "High" or "increased" refers to an amount or concentration in a test sample that is higher than a typical or normal level or range (eg, predetermined level) or that is higher than another level or reference range (eg, previous or baseline sample). The term "decreased" or "reduced" refers to an amount or concentration in a test sample that is lower than a typical or normal level or range (eg, predetermined level) or that is lower than another reference level or range (eg, previous or baseline sample). The term "altered" refers to an amount or concentration in a sample that is altered (increased or decreased) over a typical or normal level or range (eg, predetermined level) or over another reference level or range (eg , previous or basal sample).
[0408]The typical or normal level or range for an analyte is defined in accordance with standard practice. Since analyte levels in some circumstances will be very low, a supposed altered level or change may be considered to have occurred when there is any final change, compared to the typical or normal level or range, which cannot be explained by experimental error or sample variation. Therefore, the level measured in a given sample will be compared with the level or range of levels determined in similar samples from a supposedly normal individual. In this context, a “normal individual” is an individual with no detectable disease, for example, and a “normal” patient or population (sometimes called a “control”) is one that does not exhibit detectable disease, for example. Furthermore, given that an analyte is not routinely found at a high level in most of the human population, a "normal individual" can be considered one with no detectable substantially increased or elevated amount or concentration of analyte, and a "normal individual" patient or population ” (sometimes called “control”) is one that exhibits no substantially increased or high detectable amount or concentration of analyte. An “apparently normal individual” is one in whom the analyte has not yet been or is not currently being evaluated. The level of an analyte is said to be “high” when the analyte is normally undetectable (for example, the normal level is zero or within a range of around 25 to around 75 percentiles of normal people), but that is detected in a test sample, as well as when the analyte is present in the test sample at a higher level than normal. Therefore, inter alia, the present invention provides a method of screening an individual suffering from or at risk of suffering from a particular disease, disorder or condition. The assay method may involve assaying other markers and the like.
[0409] Therefore, the methods described herein can also be used to determine whether an individual has or is at risk of developing a particular disease, disorder, or condition. Specifically, such a method may comprise the steps of: (a) determining the concentration or amount of IL-1β (or its fragment) in a test sample from an individual (for example, using the methods described herein or methods known in the state of technique); and (b) compare the concentration or amount of IL-1β (or its fragment) determined in step (a) with a predetermined level, where if the concentration or amount of analyte determined in step (a) is favorable in relation to a predetermined level is then determined for the individual not to present or be at risk for the particular disease, disorder or condition. However, if the concentration or amount of IL-1β determined in step (a) is unfavorable to the predetermined level, then the individual has or is at risk for the particular disease, disorder or condition.
[0410]In addition, a method is provided to monitor the progression of disease in an individual. Ideally, the method comprises the steps of: (a) determining the concentration or amount of IL-1β in a test sample from an individual; (b) determine the concentration or amount of IL-1β in a subsequent test sample from the individual; and (c) compare the concentration or quantity of analyte determined in step (b) with the concentration or quantity of IL-1β determined in step (a), in which if the concentration or quantity determined in step (b) is unchanged or unfavorable when the concentration or amount of IL-1β determined in step (a), then it is determined for the individual that the disease has continued, progressed or worsened. By comparison, if the concentration or amount of IL-1β determined in step (b) is favorable when compared to the concentration or amount of IL-1β determined in step (a), then it is determined for the individual that the disease has discontinued, regressed or improved.
[0411] Optionally, the method further comprises comparing the concentration or amount of IL-1β analyte determined in step (b), for example, with a predetermined level. Additionally, optionally, the method comprises treating the subject with one or more pharmaceutical compositions for a period of time if the comparison shows that the concentration or amount of analyte determined in step (b), for example, is unfavorably changed from the level predetermined.
[0412] Still further, the methods can be employed to monitor treatment in an individual being treated with one or more pharmaceutical compositions. Specifically, such methods involve obtaining a first test sample from an individual, prior to being administered one or more pharmaceutical compositions. Thereafter, the concentration or amount of IL-1β in a first test sample from an individual is determined (for example, using the methods described herein or as known in the prior art). After the concentration or amount of IL-1β is determined, optionally, the concentration or amount of IL-1β is then compared to a predetermined level. If the concentration or amount of IL-1β determined in the first test sample is lower than the predetermined level, then the individual is not treated with one or more pharmaceutical compositions. However, if the concentration or amount of IL-1β determined in the first test sample is higher than the predetermined level, then the subject is treated with one or more pharmaceutical compositions for a period of time. The length of time the individual is treated with the single or more pharmaceutical compositions can be determined by the person skilled in the art (for example, the time period can be around seven (7) days to around two years, preferably around fourteen (14) days to around one (1) year).
[0413] During the treatment cycle with the single or more pharmaceutical compositions, a second and subsequent test samples are then obtained from the subject. The number of test samples and the time at which said test samples are obtained from the individual are not critical. For example, a second test sample could be obtained seven (7) days after the first administration to the subject of the single or more pharmaceutical compositions, a third test sample could be obtained two (2) weeks after the first administration to the subject of the single or more pharmaceutical compositions, a fourth test sample could be obtained three (3) weeks after the first administration to the subject of the single or more pharmaceutical compositions, a fifth test sample could be obtained four (4) weeks after the first administration to the subject of the single or more pharmaceutical compositions, etc.
[0414] After the second and subsequent test sample have been obtained from the individual, the concentration or amount of IL-1β analyte is determined in the second or subsequent test sample (for example, using the methods described herein or as known in the prior art). The concentration or amount of IL-1β determined in the second and subsequent test samples is then compared to the concentration or amount of analyte determined in the first test sample (for example, the test sample that was originally compared optionally to the predetermined level) . If the concentration or amount of IL-1β determined in step (c) is favorable, when compared to the concentration or amount of analyte determined in step (a), then it is determined for the individual that the disease has discontinued, regressed or improved, and the The individual must proceed with administration of the single or more pharmaceutical compositions from step (b). However, if the concentration or amount determined in step (c) does not change or is unfavorable, when compared to the concentration or amount of analyte determined in step (a), then it is determined for the individual that the disease has continued, progressed or worsened, and the subject must be treated with a higher concentration of the single or more pharmaceutical compositions administered to them in step (b) or the subject must be treated with one or more pharmaceutical compositions that are different from the single or more pharmaceutical compositions administered to them in step (b). Specifically, the subject may be treated with one or more pharmaceutical compositions that are different from the single or more pharmaceutical compositions that he or she has previously received to decrease or reduce said subject's analyte level.
[0415] In general, for trials in which repeat testing can be performed (for example, to monitor disease progression and/or response to treatment), a second or subsequent test sample is obtained within a period of time after the first test sample was obtained from the subject. Specifically, a second test sample from the subject may be taken minutes, hours, days, weeks, or years after the first test sample was taken from the subject. For example, the second test sample can be taken from the subject in a time period of approximately 1 minute, approximately 5 minutes, approximately 10 minutes, approximately 15 minutes, approximately 30 minutes, approximately 45 minutes, approximately 60 minutes, approximately 2 hours , approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 2 days, approximately 3 days, approximately 4 days , approximately 5 days, approximately 6 days, after approximately 7 days, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, approximately 11 weeks, approximately 12 weeks, approximately 13 weeks, approximately 14 weeks, approximately 15 weeks, approximately 16 weeks, approximately 17 weeks, approximately 18 weeks, approximately 19 weeks, approximately 20 weeks, approximately 21 weeks, approximately 22 weeks, approximately 23 weeks, approximately 24 weeks, approximately 25 weeks, approximately 26 weeks, approximately 27 weeks, approximately 28 weeks, approximately 29 weeks, approximately 30 weeks, approximately 31 weeks, approximately 32 weeks, approximately 33 weeks, approximately 34 weeks, approximately 35 weeks, approximately 36 weeks, approx approximately 37 weeks, approximately 38 weeks, approximately 39 weeks, approximately 40 weeks, approximately 41 weeks, approximately 42 weeks, approximately 43 weeks, approximately 44 weeks, approximately 45 weeks, approximately 46 weeks, approximately 47 weeks, approximately 48 weeks, approximately 49 weeks, approximately 50 weeks, approximately 51 weeks, approximately 52 weeks, approximately 1.5 years, approximately 2 years, approximately 2.5 years, approximately 3.0 years, approximately 3.5 years, approximately 4.0 years, approximately 4 .5 years, approximately 5.0 years, approximately 5.5 years, approximately 6.0 years, approximately 6.5 years, approximately 7.0 years, approximately 7.5 years, approximately 8.0 years, approximately 8, 5 years, approximately 9.0 years, approximately 9.5 years, or approximately 10.0 years after the subject's first test sample is obtained.
[0416] When employed to monitor disease progression, the above assay can be used to monitor disease progression in individuals suffering from acute conditions. Acute conditions, also known as critical care conditions, refer to life-threatening acute illnesses or other critical medical conditions involving, for example, the cardiovascular system or excretory system. Typically, critical care conditions refer to conditions that require acute medical intervention in a hospital setting (including, but not limited to, the emergency room, intensive care unit, trauma center or other emerging care setting) or administration by a paramedic or other medical personnel in the field. For critical care conditions, repeated monitoring is usually performed within a shorter timeframe, namely, minutes, hours or days (eg approximately 1 minute, approximately 5 minutes, approximately 10 minutes, approximately 15 minutes, approximately 30 minutes, approximately 45 minutes, approximately 60 minutes, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 2 days, approximately 3 days, approx. immally 4 days, approximately 5 days, approximately 6 days or approximately 7 days), and the initial trial is likewise generally carried out on a shorter time schedule, eg approximately minutes, hours or days from the onset of the disease or condition.
[0417] Assays can also be used to monitor disease progression in individuals suffering from chronic or non-acute conditions. Non-critical or non-acute care conditions refer to conditions other than life-threatening acute illness or other critical medical conditions involving the cardiovascular system and/or the excretory system. Typically, non-acute conditions include those of longer duration or chronic. For non-acute conditions, repeated monitoring is usually performed on a longer schedule, eg, hours, days, weeks, months or years (eg approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours , approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days , approximately 7 days, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks as, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, approximately 11 weeks, approximately 12 weeks, approximately 13 weeks, approximately 14 weeks, approximately 15 weeks, approximately 16 weeks, approximately 17 weeks approximately 18 weeks approximately 19 weeks approximately 20 weeks approximately 21 weeks approximately 22 weeks approximately 23 weeks approximately 24 weeks approximately 25 weeks approximately 26 weeks approximately 27 weeks approximately 28 weeks approximately 29 weeks, approximately 30 weeks, approximately 31 weeks, approximately 32 weeks, approximately 33 weeks, approximately 34 weeks, approximately 35 weeks, approximately 36 weeks, approximately 37 weeks, approximately 38 weeks, approximately 39 weeks, approximately 40 sec weeks, approximately 41 weeks, approximately 42 weeks, approximately 43 weeks, approximately 44 weeks, approximately 45 weeks, approximately 46 weeks, approximately 47 weeks, approximately 48 weeks, approximately 49 weeks, approximately 50 weeks, approximately 51 weeks, approximately 52 weeks, approximately 1.5 years, approximately 2 years, approximately 2.5 years, approximately 3.0 years, approximately 3.5 years, approximately 4.0 years, approximately 4.5 years, approximately 5.0 years, approximately 5.5 , years, approximately 6.0 years, approximately 6.5 years, approximately 7.0 years, approximately 7.5 years, approximately 8.0 years, approximately 8.5 years, approximately 9.0 years, approximately 9.5 years or approximately 10.0 years), and the initial trial, likewise, is generally carried out within a longer timeframe, for example approximately hours, days, months or years from the onset of the disease or condition. to.
[0418] Additionally, the above assays can be performed using a first test sample, obtained from an individual, wherein the first test sample is obtained from a source such as urine, serum or plasma. Optionally, the above tests can then be repeated using a second test sample obtained from the individual, where the second test sample is obtained from another source. For example, if the first test sample was obtained from urine, the second test sample could be obtained from serum or plasma. The results obtained from the tests using the first test sample and the second test sample can be compared. Comparison can be used to assess the status of an individual's disease or condition.
[0419] In addition, the present invention is also related to methods of determining whether an individual predisposed to or suffering from a particular disease, disorder or condition will benefit from treatment. In particular, the invention relates to diagnostic methods and products for a companion analyte. Thus, the method of "monitoring the treatment of disease in an individual" as described herein may ideally still encompass selecting or identifying candidates for therapy.
[0420] Consequently, in special embodiments, the invention also provides a method for determining whether an individual presenting or at risk for a particular disease, disorder or condition is a candidate for therapy. In general, an individual is someone who has some symptom of a particular disease, disorder or condition or who has actually been diagnosed or who is at risk for a particular disease, disorder or disease and/or who demonstrates an unfavorable concentration or amount of analyte or its fragment, as described herein.
[0421] The method optionally comprises an assay, as described herein, in which IL-1β is evaluated before and after treatment of an individual with one or more pharmaceutical compositions (for example, especially with a pharmaceutical product related to a mechanism of action involving the analyte), with immunosuppressive therapy or by immunoabsorption therapy, or when the analyte is evaluated after such treatment and its concentration or quantity is compared against a predetermined level. An unfavorable concentration or amount of IL-1β observed after treatment confirms that the individual will not benefit from further receiving or continuing treatment, whereas a favorable concentration or amount of analyte observed after treatment confirms that the individual will benefit from receiving more or continuing treatment. Confirmation helps manage clinical trials and provide better patient care. Needless to say, while certain embodiments of the present invention are advantageous when employed to assess a particular disease, disorder, or condition, as discussed herein, assays and kits can be employed to assess analyte in other diseases, disorders, and conditions. The assay method may involve assaying other markers and the like.
[0422] The test method can also be used to identify a compound that ameliorates a particular disease, disorder or condition. For example, an analyte expressing cell can be contacted with a candidate compound. The level of analyte expression in the cell in contact with the compound can be compared to that of a control cell using the assay method described herein. II. Kits
[0423] The present invention also provides a kit to analyze a test sample for the presence, amount or concentration of an analyte (or its fragment) in a test sample. The kit comprises at least one component for analyzing the test sample for IL-1β (or its fragment) and instructions for analyzing the test sample for the analyte (or its fragment). The at least single component for analyzing the test sample for analyte (or its fragment) may include a composition containing an anti-IL-1β binding protein, such as monoclonal antibody or DVD-Ig (or its fragment, variant or fragment of variant) as described herein, which is optionally immobilized on a solid phase.
[0424] The kit may include at least one component to analyze the test sample for an IL-1β analyte by immunoassay, for example, immunoassays with chemiluminescent microparticles and instructions for analyzing the test sample for the IL- analyte 1β by immunoassay, e.g. immunoassay with chemiluminescent microparticles. For example, the kit can include at least one specific binding partner for IL-1/3, such as anti-IL-1/3 monoclonal/polyclonal antibody (or its fragment capable of binding to the IL-1/3 analyte, its variant capable of binding bind to the analyte or a fragment of a variant capable of binding to the analyte) or an anti-IL-1β DVD-Ig (or its fragment, variant or variant fragment), any of these may be detectably labeled. Alternatively or additionally, the kit may include detectably labeled IL-1β analyte (or its fragment that can bind to an anti-analyte monoclonal/polyclonal antibody or an anti-analyte DVD-Ig) (or its fragment, variant or fragment of variant)), capable of competing with any analyte, in the tested sample, for binding to an anti-analyte monoclonal/polyclonal antibody (or its fragment capable of binding to the analyte, its variant capable of binding to the analyte or fragment of a variant capable of binding to analyte) or an anti-analyte DVD-Ig (or its fragment, variant or variant fragment), which may be immobilized on a solid support. The kit can include a calibrator or control, eg isolated or purified analyte. The kit may comprise at least one container (eg tube, microtiter plates, which may already be coated with a first specific binding partner, for example) for conducting the assay, and/or a buffer, such as a buffer. assay or wash buffer, both of which may be supplied as a stock solution, a substrate solution for the detectable marker (eg enzyme marker) or stop solution. Preferably, the kit includes all components, i.e. reagents, standards, buffers, diluents, etc., which are necessary for carrying out the assay. Instructions may be in printed form or computer-readable form, such as on disc, CD, DVD, or the like.
[0425] Any binding protein such as anti-IL-1β binding protein or anti-analyte or tracer DVD-Ig can incorporate a detectable marker as described herein such as fluorophore, radioactive moiety, enzyme, biotin/avidin marker, chromophore , chemiluminescent label or the like, or the kit can include reagents to perform detectable labeling. Antibodies, calibrators and/or controls can be supplied in separate containers or can be pre-distributed in an appropriate assay format, for example, in microtiter plates.
[0426] Optionally, the kit includes quality control components (eg, sensitivity panels, calibrators and positive controls). The preparation of quality control reagents is well known in the art and is described in insert inserts of a variety of immunodiagnostic products. Sensitivity panel members are optionally used to establish assay performance characteristics and optionally further useful indicators of the integrity of the immunoassay kit reagents and assay standardization.
[0427] The kit may optionally include other reagents necessary to conduct a diagnostic assay or to facilitate quality control assessments, such as buffers, salts, enzymes, enzyme cofactors, enzyme substrates, detection reagents and the like. Other components, such as buffers and solutions for the isolation and/or treatment of a test sample (eg, pre-treatment reagents) may also be included in the kit. The kit can additionally include one or more other controls. One or more of the kit components may be in lyophilized form, in which case the kit may further comprise reagents suitable for reconstitution of the lyophilized components.
[0428]The various kit components are optionally supplied in suitable containers as needed, eg a microtiter plate. The kit may further include containers for holding or storing a sample (eg, urine sample container or cartridge). When appropriate, the kit may also contain reaction vessels, mixing vessels and other components that facilitate the preparation of reagents or the test sample. The kit can also include one or more instruments to aid in obtaining a test sample, such as a syringe, pipette, forceps, measuring spoon or the like.
[0429] If the detectable label is at least one acridinium compound, the kit can comprise at least one acridinium-9-carboxamide, at least one aryl ester of acridinium-9-carboxylate or any combination thereof. If the detectable label is at least one acridinium compound, the kit may also comprise a source of hydrogen peroxide, such as buffer, solution and/or at least one basic solution. If desired, the kit can contain a solid phase, such as magnetic particle, microsphere, test tube, microtiter plate, cuvette, membrane, scaffold molecule, film, filter paper, disk or chip. III. Adaptation of kit and method
[0430] The kit (or its components), as well as the method for determining the presence, amount or concentration of an analyte in a test sample by an assay, such as an immunoassay as described herein, can be adapted for use in a variety of automatic and semi-automatic systems (including those in which the solid phase comprises a microparticle), as described in, for example, US Patent Nos. 5,089,424 and 5,006,309 and as commercially available, for example, from Abbott Laboratories (Abbott Park, Illinois) as ARCHITECT®.
[0431]Some of the differences between an automated or semi-automatic system, when compared to a non-automatic system (eg, ELISA), include the substrate to which the specific binding partner (eg, monoclonal/polyclonal anti-analyte antibody (or its fragment, its variant or fragment of its variant) or anti-analyte DVD-Ig (or its fragment, its variant or fragment of its variant) is fixed; which, in any way; may affect the sandwich-like formation and the reactivity of the analyte) and the extent and timing of capture, detection and/or any additional washing steps. A non-automatic format, such as an ELISA, may require a relatively longer incubation time with the sample and capture reagent (eg about 2 hours), whereas an automatic or semi-automatic format (eg ARCHITECT® , Abbott Laboratories) may have a relatively shorter incubation time (eg approximately 18 minutes for ARCHITECT®). Likewise, while a non-automatic format, such as an ELISA, can incubate a detection antibody, such as a conjugated reagent, for a relatively longer incubation time (eg, about 2 hours), an automatic or semi-automatic format can (eg ARCHITECT®) may have a relatively shorter incubation time (eg approximately 4 minutes for ARCHITECT®).
[0432]Other platforms available from Abbott Laboratories include, but are not limited to, AxSYM®, IMx® (see, for example, U.S. Patent No. 5,294,404), PRISM®, EIA (sphere) and Quantum™ II, in addition to other platforms. Additionally, the tests, kits and kit components can be used in other formats, for example, in electrochemical test systems or other manuals or at service points. The present invention applies, for example, to the Abbott Point of Care electrochemical immunoassay system (i-STAT®, Abbott Laboratories) which performs sandwich-type immunoassays. Immunosensors and their methods of manufacturing and operating in single-use testing devices are described, for example, in US Patent No. 5,063,081, US Publication No. 2003/0170881, US Publication No. 2004/0018577, US Publication No. 2005/0054078 and US Publication No. 2006/0160164.
[0433]Specifically, with respect to adapting an analyte assay to the I-STAT® system, the following configuration is preferred. A microfabricated silicon chip is produced with a pair of gold electrodes for amperometric work and a reference silver-silver chloride electrode. One of the working electrodes, polystyrene beads (0.2 mm in diameter) with immobilized anti-analyte, a monoclonal/polyclonal antibody (or its fragment, its variant or its variant fragment) or anti-analyte DVD-Ig (or its fragment, its variant or fragment of its variant) are adhered to a standardized polyvinyl alcohol polymeric coating on the electrode. This chip is mounted on an I-STAT® cartridge with a flowable format suitable for immunoassay. On the part of the cartridge compartment wall in which the sample is retained, there is a layer comprising a specific binding partner for an analyte, such as anti-analyte, monoclonal/polyclonal antibody (or its fragment, its variant or its variant fragment capable of binding to analyte) or anti-analyte DVD-Ig (or its fragment, its variant or fragment of its variant capable of binding to the analyte), which may be detectably landmarks. Inside the cartridge fluid pouch is an aqueous reagent that includes p-aminophenol phosphate.
[0434]In operation, a sample suspected of containing an analyte is added to the test cartridge holding compartment, and the cartridge is inserted into the I-STAT® reader. After the specific binding partner for an analyte has been dissolved in the sample, a pumping element within the cartridge forces the sample into a tube containing the chip. There, it undergoes oscillation to promote the formation of the sandwich. In the penultimate step of the assay, the liquid is forced out of the bag and into the tube to wash the sample from the chip into a waste compartment. In the final step of the assay, the alkaline phosphatase tag reacts with the p-aminophenol phosphate to cleave the phosphate group and allow the released p-aminophenol to undergo electrochemical oxidation at the working electrode. Based on the measured current, the reader is able to calculate the amount of analyte in the sample through an embedded algorithm and a factory-determined calibration curve.
[0435]In addition, it is evident that the methods and kits described herein necessarily encompass other reagents and methods for performing the immunoassay. For example, various buffers are included, such as are known in the prior art and/or which can be readily prepared or optimized to be used, for example, for washing, as a conjugate diluent, microparticle diluent and/or as a calibrate diluent . An exemplary conjugate diluent is the ARCHITECT® Conjugate Diluent, employed in certain kits (Abbott Laboratories, Abbott Park, Illinois) and containing 2-(N-morpholino)ethanesulfonic acid (MES), a salt, a protein blocker, a antimicrobial agent and a detergent. An exemplary calibrator diluent is the ARCHITECT® human calibrator diluent employed in certain kits (Abbott Laboratories, Abbott Park, Illinois), which comprises a buffer containing MES, another salt, a protein blocker, and an antimicrobial agent. Additionally, as described in US Patent Application Serial No. 12/650 241 (US Publication No. 2010/0167301; see also PCT Publication NoWO 2010/078443), signal generation can be improved, for example, in a cartridge format I-Stat, with a nucleic acid sequence linked to the signaling antibody as a signal enhancer.
[0436] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the invention described herein are obvious and that they can be made using suitable equivalents without departing from the scope of the invention or the modalities disclosed in this patent application.
[0437] Having now been described in detail, the present invention will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to limit the invention. Examples Example 1: Generation of affinity-matured humanized antibodies against IL-1β from clone E26
[0438] Table 6 provides the VH and VL amino acid sequences of the mouse humanized antibody E26 (GlaxoSmithKline, PCT Publication No. WO 95/01997). The amino acid residues of individual CDRs of each VH and VL sequence are indicated in bold. Table 6. Amino acid sequences of VH and VL regions of humanized antibody E26


[0439] Affinity-matured humanized E26 antibodies were obtained as follows. A light chain library was constructed to contain limited mutagenesis at the following residues: CDRL1: 30, 31, 32; CDRL2: 50, 53, 55, 56; CDRL3: 92, 93, 94, 96 and 97 (Kabat numbering). Two heavy chain libraries were produced to contain limited mutagenesis in CDRH1 and CDRH2 contained at residues 31, 33, 50, 52a, 55, 56, 57, 58 and 60 (Kabat numbering) or in CDRH3 at residues 95, 96 , 97, 98, 99, 100, 100a, 100b and 102. The heavy chain libraries also contained binary diversities at residues 23(A/S), 24(A/S), 62(T/S), 84(P /A), 88(G/A), 91(F/Y) and 108(P/L) to allow scaffold germline formation during selection in libraries. All three libraries were selected separately to decrease cynomolgus (cyno) IL-1β concentrations. All mutant CDR sequences were then combined into a single library containing mutations in the VH CDRs only, and another library having mutations in all six CDRs. These two combined libraries were subjected to more stringent selection conditions with human IL-1/3 and cyno before individual antibodies were identified and expressed in IgG protein forms for further characterization.
[0440] Table 7 provides a list of the VH region amino acid sequences of affinity matured antibodies against IL-1β, derived from humanized E26. The amino acid residues of individual CDRs of each VH sequence are indicated in bold. Table 7. Affinity Matured E26 Variant VH Amino Acid Sequences















[0441] Table 8 provides a list of the amino acid sequences of VL regions of affinity matured humanized antibodies against IL-1β, derived from E26. The amino acid residues of individual CDRs of each VL sequence are indicated in bold. The N-terminal mutation from D (Asp) to G (Gly) seen in some affinity-matured VL sequences in Table 8 below was most likely the result of unintended mutagenesis, which occurred during the polymerase chain reaction (PCR), carried out during the construction of the library. The N-terminal G residue could be removed without consequence when these regions were used in the construction of IgG molecules.





[0442] The sequences of the individual CDRs of the VH and VL regions of the affinity matured humanized E26 IL-1β antibodies in the above tables can be aligned to provide CDR consensus sequences such as those shown in Table 9. Table 9. Sequence of consensus for affinity matured VH and VL sequences






Exemplo 2: Caracterização funcional de anticorpos contra IL-1β Exemplo 2.1: Protocolo de ensaio imunoenzimático de adsorção para IL-1β[0443] The sequences in Table 10 were converted to IgG for further characterization. Clone E26.13 was mutated in the J region of the heavy and light chain variable region, named E26.13 JM VH and E26.13 JM VL, respectively, to remove framework mutations that did not belong to the germline. Amino acid residues of individual CDRs are indicated in bold. Table 10. Affinity Matured E26 Variant VH and VL Amino Acid Sequences














Example 2: Functional characterization of antibodies against IL-1β Example 2.1: Immunoenzyme adsorption assay protocol for IL-1β
[0444] In order to determine whether anti-IL-1β mAbs bind to human IL-1β, ELISA plates (Nunc, MaxiSorp, Rochester, New York) were incubated overnight at 4°C with anti-Fc antibody human diluted in Pierce Coat buffer at 2 µg/ml (Jackson Immunoresearch, West Grove, Pennsylvania). The plates were washed five times in wash buffer (PBS containing 0.05% Tween 20) and blocked for 1 hour at 25 oC with 200 μl per well of Superblock blocking buffer (Thermo scientific, No 37515). The blocking buffer was removed by lightly tapping the plates, and 2 μg/ml of each antibody in PBS containing 10% Superblock, 0.5% Tween 20 was added to the wells at 100 μl/well, which were incubated at 25 oC for 1 hour. The wells were washed five times in 1XPBST, and 1 μg/ml biotinylated antigen was titrated in serial 1:6 dilutions (to a μg to pg range in PBS containing 10% Superblock, 0.05% Tween 20). Each antigen dilution was then added to the plates and incubated for 1 hour at 25 oC. Wells were washed five times in 1XPBST and incubated for 1 hour at 25°C with polyHRP streptavidin (KPL No 474-3000, Gaithersburg, Maryland). Wells were washed in 1XPBST, and 100 µl of ULTRA-TMB ELISA (Pierce, Rockford, Illinois) was added per well. After color development, the reaction was stopped with 1N HCl, the absorbance being read at 450 nM. The results are shown in Table 11, where the numerical value indicates the binding of anti-IL-1/3 antibodies to human IL-1/3. Table 11. Antibody binding to human IL-1β by ELISA
Example 2.2: Neutralizing Potency of Antibodies against IL-1β
[0445] In order to examine the functional activity of the anti-human IL-1/3 antibodies in the invention, the antibodies were used in an assay with MRC-5 that measures the antibody's ability to inhibit the activity of IL-1/3. The MRC-5 cell line is a human lung fibroblast cell line that produces IL-8 in response to human IL-1β in a dose-dependent manner. This cell line also produces IL-8 in response to cynomolgus IL-1β (IL-1β cyno). MRC-5 cells were originally obtained from ATCC and subcultured in complete MEM with 10% FBS and maintained at 37°C in a 5% CO2 incubator. In order to determine the neutralizing potency of an antibody against IL-1β, antibodies (50 μl) were added to a 96-well plate (final concentration range from 1E-7 to 1E-15 M), pre-incubated with 50 μl of human IL-1β or cyno (final concentration 50 pg/ml) for 1 hour at 37°C, CO2. 5%. Antigen-antibody complexes (100 μL) were then added to MRC-5 cells (pre-seeded for 24 hours at a concentration of 1E5/ml at 100 μl cells/well). Assay plates were incubated overnight at 37°C in 5% CO 2 incubator. The potency of the antibodies was determined by their ability to inhibit IL-8 production. Human IL-8 production is measured by a chemiluminescence-based assay. Table 12 summarizes antibody potencies to human IL-1/3 and cyno. Table 12. Neutralizing potency of antibodies against IL-1β
ND: Not determined. Example 2.3: Measuring the affinity of antibodies against IL-1β by surface plasmon resonance
[0446] The BIACORE assay (Biacore, Inc, Piscataway, New Jersey) determines antibody affinity with constant kinetic measurements for on-rate and off-rate. Antibody binding to purified recombinant human IL-1β and cynomolgus IL-1β was determined by surface plasmon resonance measurements with a Biacore® 3000 apparatus (Biacore® AB, Uppsala, Sweden), with HBS-EP running (10 mM HEPES [H 7.4], 150 mM NaCl, 3 mM EDTA and 0.005% P20 surfactant) at 25°C. All chemicals were obtained from Biacore® AB (Uppsala, Sweden) or a different source as described herein. Approximately 5000 RU of goat anti-mouse IgG polyclonal antibody specific fragment, (FCY), (Pierce Biotechnology Inc, Rockford, Illinois), diluted in 10 mM sodium acetate (pH 4.5), was directly immobilized through a research grade CM5 biosensor chip using a standard amine coupling kit per manufacturer's instructions and procedures at 25 μg/ml. Unreacted clusters on the surface of the biosensor were blocked with ethanolamine. The modified carboxymethyl dextran surface in flow cell 2 and 4 was used as the reaction surface. Unmodified carboxymethyl dextran without goat anti-mouse Ig, in flow cell 1 and 3, was used as the reference surface. For kinetic analysis, rate equations derived from the 1:1 Langmuir binding model were simultaneously fitted to the association and dissociation phases of all eight injections (using global fit analysis) using the Biaevaluation 4.0 software .1. Purified antibodies were diluted in saline with HEPES buffer for capture on reaction surfaces with goat anti-human IgG. Antibodies to be captured as a ligand (25 μg/ml) were injected onto reaction matrices at a flow rate of 5 μl/minute. The constants for the association and dissociation rates, kon (unit: M-1s-1) and koff (unit: s-1) were determined at a continuous flow rate of 25 μl/min. Rate constants were derived by kinetic binding measurements made at ten different antigen concentrations, ranging from 10 - 200 nM. The equilibrium dissociation constant (unit: M) of the reaction between antibodies and the target antigen was then calculated from the kinetic constants for the rate by the following formula: KD = koff/kon. The binding is recorded as a function of time, and the kinetic constants for the rate are calculated. In this trial, rates for association as fast as 106 M-1s-1 and rates for dissociation as slow as 10-6 s-1 can be measured. Table 13 shows the affinity measurements for anti-human IL-1/3 antibodies. Table 13. Affinity of antibodies by human IL-1β and cyno by Biacore
ND: Not determined. Example 3: Generation of IL-1α/β DVD-Ig™ molecules against IL-1α/β Example 3.1: Construction of artificial DVD-Ig DNA against IL-1α/β
[0447] An anti-IL-1α antibody variable domain ("X3"; see, PCT Publication No. WO 95/14780) was combined with multiple variable domains of antibody against IL-1β in DVD-Ig format (Wu et al. ., Nature Biotechnol., 25: 1290-1297 (2007);PCT Publication No. WO 2007/024715 A2), with PCR amplification, overlaying DNA sequences with intervening linker. X3 was also mutated in the J region of the heavy and light variable region, called X3 JM VH and X3 JM VL, respectively, to remove mutations in the framework that did not belong to the germline. PCR amplified products were subcloned into expression vectors suitable for transient expression in HEK293 cells, and open reading frame regions were confirmed by sequencing prior to DVD-Ig expression. Example 3.2: Expression and production of DVD-Ig binding proteins to IL-1α/β
[0448]After DNA sequence confirmation, all constructed DVD-Ig DNAs were expanded in E. coli and purified with Qiagen Hispeed Maxi Prep (Catalogue No. 12662, QIAGEN). DVD-Ig DNA was transfected into log phase 293E cells (0.5 x 106/ml, >95% viability), mixing PEI and DNA in a 2:1 ratio with 0.2 μg/ml strand DNA heavy and 0.3 µg/ml of light chain DNA. The DNA:PEI complex was formed at room temperature in a CT hood for fifteen minutes, before being added to the 293E cells. Twenty-four hours later, 0.5% TN1 was added to 293E cells. On the fifth day, the supernatant was collected to measure the human IgG1 titer. Cell supernatant was collected on the seventh day and filtered through a 0.2 µM PES filter. The supernatant was purified by Protein A affinity chromatography on Sepharose according to the manufacturer's instructions. Purified DVD-Igs were collected by column elution with 0.1 M glycine (pH 2.99), and dialyzed immediately into 15 mM histidine buffer (pH 6.0). Binding proteins were quantified by A280 and analyzed by mass spectrometry and SEC. Example 3.3: IL-1α/β-Binding DVD-Igs Constructed Sequences
[0449] Amino acid sequences of heavy and light chains of DVD-Ig proteins capable of binding to human IL-1β and human IL-1α were determined. The amino acid sequences of the heavy chain variable (VH), light chain variable (VL), light chain constant (CL) and heavy chain constant (CH) regions of IL-1α/β DVD-Ig binding proteins are shown in Table 14, below. In Table 14, the amino acid sequences for the VL regions E26.13 and E26.35 are designated SEQ ID NO:238 and SEQ ID NO:239, respectively, in place of SEQ ID NO:205 and SEQ ID NO:209, previously shown in Table 10, taking into account the inclusion of an arginine (R) residue at the C-terminus. This C-terminal arginine residue is understood by those skilled in the field of antibody construction as the amino acid residue at the junction of the VL and CL kappa regions of an IgG molecule and is sometimes included in the CL region or, as in Table 14 below, in the VL region. Table 14. Variable and constant region sequences of DVD-Ig proteins that bind to IL-1α/β





























[0450] Linker sequences are indicated by underlined residues. Example 4: Functional characterization of DVD-Ig against IL-1α/β Example 4.1: Immunoenzyme adsorption assay protocol for IL-1α/β
[0451] Binding to IL-1β and IL-1α by IL-1β/α binding DVD-Igs proteins was assessed by ELISA (assay described above, Example 2.1). The results are shown in Table 15. Table 15. DVD-Ig IL-1α/β binding proteins to human IL-1α or IL-1β by ELISA
Example 4.2: IL-1α/β Bioassay and Neutralization Assay
[0452]MRC5 cells were seeded in plates at 1.5 - 2 x 104 cells per well in a volume of 100 µL and incubated overnight at 37°C, 5% CO 2 . A 20 μg/ml working stock of DVD-Ig (4X concentrate) was prepared in MEM complete medium. An eight-point serial dilution was performed (5 μg/ml - 0.0003 μg/ml) with complete MEM in Marsh dilution plates. Sixty-five μL/well of each antibody dilution was added, in four replicates, to a 96-well V-bottom plate (Costar No. 3894) in addition to 65 μL of a 200 pg/mL solution of IL-1α or IL-1β or 65 μL of a mixed solution, containing a 50 pg/mL solution of both IL-1α and IL-1β. Control wells received 65 μL of IL-1α or IL-1β at 200 pg/mL or IL-1α/β mixed at 50 pg/mL (4x concentrate) plus 65 μL of MEM medium, and control media wells received 130 µL of medium. After 1 hour of incubation, 100 µl of the DVD-Ig/Ag mixture was added to the MRC5 cells. All well volumes were equal to 200 µL. All reagents on the plate were then concentrated 1X. After 16 - 20 hours of incubation, the well contents (150 µL) were transferred to a 96-well round bottom plate (Costar No 3799) and placed in a freezer at -20°C. Supernatants were tested for hIL-8 levels with a human IL-8 ELISA kit (R&D Systems, Minneapolis, Minnesota) or MSD hIL-8 (Chemiluminescence Kit). Neutralizing potency was determined by the percentage inhibition calculated in relation to the control value of IL-1α, IL-1β or isolated IL-1α/β (Table 16). Table 16. Potency of DVD-Ig molecules against IL-1α/β on human IL-1e and IL-1β and IL-1α and IL-1β from Cynomolgus
ND: Not determined. Example 4.3: Measurement of affinity of DVD-Ig molecules for IL-1α/β
[0453] The binding of DVD-Igs against IL-1α/β to purified human IL-1β and IL-1α and to cynomolgus IL-1β and IL-1α was determined by surface plasmon resonance as described in Example 2.3 and the results are shown in Table 17. Table 17. Measurement of affinity of DVD-Ig molecules against IL-1α/β
Incorporation by reference
[0454] The present invention incorporates, in its entirety, well-known techniques in the field of molecular biology and drug delivery by reference in this patent application. These techniques include, among others, those described in the following publications: Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Ausubel, F.M. et al. eds., Short Protocols In Molecular Biology (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X); Controlled Drug Bioavailability Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Giegé et al., chapter 1, In Crystallization of Nucleic Acids and Proteins, A Practical Approach, 2nd ed., (Ducruix and Giegé, eds.) (Oxford University Press, New York, 1999) p. 1-16; Goodson, J.M., Chapter 6, In Medical Applications of Controlled Release, Vol. II, Applications and Evaluation, (Langer and Wise, eds.) (CRC Press, Inc., Boca Raton, 1984), p. 115-138; Hammerling et al., eds., "Monoclonal Antibodies and T-Cell Hybridomas", In Research Monographs in Immunology, vol. 3 (J.L. Turk, General Editor) (Elsevier, New York, 1981), p. 563587; Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987); Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Kontermann and Dübel, eds., Antibody Engineering (2001) Springer-Verlag, New York, 790 pp. (ISBN 3-540-41354-5); Kriegler, Gene Transfer and Expression , A Laboratory Manual, Stockton Press, NY (1990); Lu and Weiner eds., Cloning and Expression Vectors for Gene Function Analysis (2001) BioTechniques Press. Westborough, Mass. 298 pp. (ISBN 1-881299-21-X) Goodson, JM, Medical Applications of Controlled Release, (Langer and Wise, eds.) (CRC Press, Boca Raton, 1974); Old and Primrose, Principles of Gene Manipulation: An Introduction to Genetic Engineering (3rd Ed. 1985) Blackwell Scientific Publications, Boston; Studies in Microbiology, V.2:409 pp. (ISBN 0-63201318-4); Sambrook, J. et al., Molecular Cloning: A Laboratory Manual (2nd Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3 (ISBN 0-87969-309-6); Sustained and Controlled Release Drug Delivery Systems, (J.R. Robinson, ed.) (Marcel Dekker, Inc., New York, 1978); Winnacker, E.L. From Genes To Clones: Introduction To Gene Technology (1987) VCH Publishers, N.Y. (translated by Horst Ibelgaufts), 634 pp. (ISBN 0-89573-614-4).
[0455] The contents of all cited references (including references in literature, patents, patent applications, and Internet sites) that are referenced throughout this patent application are hereby expressly incorporated in their entirety by reference in this application of patent, as are the references cited therein. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology and cell biology, which are well known in the art. Equivalents
[0456] The invention can be embodied in other specific forms without deviating from its spirit or its essential characteristics. The foregoing embodiments are therefore considered to be illustrative in all respects, not limitations to the invention described in this patent application. Therefore, the scope of the invention is indicated by the appended claims rather than the foregoing description, and all changes which fall within the meaning and range of equivalence of the claims are therefore intended to be covered by this application. of patent.

权利要求:
Claims (7)
[0001]
1. A binding protein CHARACTERIZED by the fact that it comprises first and second polypeptide chains, wherein said first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a first heavy chain variable domain; VD2 is a second heavy chain variable domain; C is a heavy chain constant domain; X1 is a linker with the proviso that it is not CH1; X2 is an Fc region; n is independently 0 or 1; and wherein the second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a first light chain variable domain; VD2 is a second variable domain of the light chain; C is a constant domain of the light chain; X1 is a linker with the proviso that it is not CH1; X2 does not comprise an Fc region; n is independently 0 or 1; and wherein said first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 212; wherein said second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 215; and wherein the binding protein binds to human IL-1β and human IL-1a.
[0002]
2. The binding protein according to claim 1, CHARACTERIZED by the fact that: VD1-(X1)n-VD2 in the first polypeptide chain is SEQ ID NO: 212 and -C-(X2)n in the first polypeptide chain is SEQ ID NO: 214; and VD1-(X1)n-VD2 in the second polypeptide chain is SEQ ID NO: 215 and -C-(X2)n in the second polypeptide chain is SEQ ID NO: 5.
[0003]
3. The binding protein according to claim 1, CHARACTERIZED by the fact that the heavy chain constant region is an IgG1 heavy chain constant region.
[0004]
4. The binding protein according to claim 3, CHARACTERIZED by the fact that the IgG1 heavy chain constant region comprises mutations in the hinge region L234A and L235A.
[0005]
5. Binding protein according to any one of claims 1 to 4, CHARACTERIZED by the fact that the binding protein comprises two first polypeptide chains and two second polypeptide chains.
[0006]
6. Dual variable domain immunoglobulin binding protein (DVD-Ig) E26.13-SS-X3, CHARACTERIZED by the fact that the variable and constant region sequences are defined as follows: Protein region Sequence identifier Variable heavy chain (VH) SEQ ID NO: 212 Constant Heavy Chain (CH) SEQ ID NO: 214 Variable Light Chain (VL) SEQ ID NO: 215 Constant Light Chain (CL) SEQ ID NO: 5
[0007]
7. Pharmaceutical composition CHARACTERIZED by the fact that it comprises a binding protein, as defined in any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

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法律状态:
2018-07-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-09| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NAO 10196/2001, QUE MODIFICOU A LEI NAO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUAANCIA PRA VIA DA ANVISA. CONSIDERANDO A APROVAA AO DOS TERMOS DO PARECER NAO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NAO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDAANCIAS CABA-VEIS. |

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2020-10-06| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-11-03| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-05-18| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-08-10| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 13/05/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

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US33491710P| true| 2010-05-14|2010-05-14|

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US61/334,917|2010-05-14|

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US201061425701P| true| 2010-12-21|2010-12-21|

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US61/425,701|2010-12-21|

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PCT/US2011/036444|WO2011143562A2|2010-05-14|2011-05-13|Il-1 binding proteins|BR122014011544A| BR122014011544A2|2010-05-14|2011-05-13|il-1 binding proteins|