USE OF A NON-COVALENT IMMOBILIZED COMPLEX

专利摘要:
use of an immobilized non-covalent complex and variant fc region of human igg1 isotype. the use of a non-covalent complex of neonatal fc receptor (fcrn) and beta-2-microglobulin (b2m) with the affinity chromatography ligand in general and, for example, for determination of the in vivo half life of a antibody by determining the ratio of retention times of the antibody to a reference antibody.
公开号:BR112014018005B1
申请号:R112014018005-9
申请日:2013-02-14
公开日:2021-06-29
发明作者:Roberto Falkenstein；Hubert Hertenberger；Petra Rueger；Tilman Schlothauer
申请人:F. Hoffmann-La Roche Ag；
IPC主号:

专利说明:

[001] It is reported herein the use of an affinity chromatography column comprising immobilized human neonatal Fc receptor as an affinity ligand and its use. BACKGROUND OF THE INVENTION
[002] An immunoglobulin generally comprises two of the so-called light chain (light chain) polypeptides and two of the so-called heavy (heavy chain) polypeptides. Each of the heavy and light chain polypeptides contains a variable domain (variable region) (generally the amino terminal part of the polypeptide chain) that comprises binding regions that are capable of interacting with an antigen. Each of the heavy and light chain polypeptides comprises a constant region (generally, the carboxyl terminal part). The heavy chain constant region mediates antibody binding (i) to cells that contain an Fc gamma receptor (FCYR), such as phagocytic cells, or (ii) to cells that contain the neonatal Fc receptor (FcRN) also known as the receptor Brambell. It also mediates binding to some factors, including classical complement system factors such as the (C1q) component.
[003] The neonatal Fc receptor (FcRn) is also known as the MHC class I relative receptor (for review, see Ward, E.S. and Ober, R.J., Advances in Immunology 103 (2009) 77-115). Studies have shown not only that this receptor serves to regulate IgG levels and distribution throughout adult life (Ghetie, V. et al, Nat. Biotechnol. 15 (1997) 637-640; Israel, EJ, Immunology 89 (1996) 573-578; Junghans, RP and Anderson, CL, Proc. Natl. Acad. Sci. USA 93 (1996) 5512-5516), but also that it has several other roles in various types of cells and tissues (see , for example, Akilesh, S. et al, Proc. Natl. Acad. Sci. USA 105 (2008) 967-972; Dickinson, BL et al, J. Clin. Invest. 104 (1999) 903-911; Kim, H. et al, Invest. Ophthalmol. Vis. Sci. 49 (2008) 2025-2029; Spiekermann, GM et al, J. Exp. Med. 196 (2002) 303-310 ; Zhu, X. et al, J. Immunol.166 (2001) 3266-3276). FcRn orthologs have been isolated from many species, including mice, rats, humans, sheep, cows, possums, pigs, and camels (Adamski, FM et al, Mol. Immunol. 37 (2000) 435-444; Ahouse, JJ et al. , J. Immunol. 151 (1993) 6076-6088; and Kacskovics, I. et al, J. Immunol. 164 (2000) 1889-1897; Kacskovics, I. et al, Dev. Comp. Immunol. 30 (2006) 1203-1215; Kandil, E. et al, J. Immunol. 154 (1995) 5907-5918; Mayer, B. et al, Immunology 107 (2002) 288-296; Schnulle, PM and Hurley, WL, Vet. Immunopathol.91 (2003) 227-231; Simister, NE and Mostov, KE, Nature 337 (1989) 184-187; Story, CM et al, J. Exp. Med. 180 (1994) 2377-2381), o which indicates that this receptor is present in essentially all species of mammals. The diverse functions of FcRn are dependent on its ability to select IgG away from lysosomal degradation within cells and release bound charge during exocytic events in the plasma membrane (Ober, RJ et al, Proc. Natl. Acad. Sci. USA 101 (2004 11076-11081; Ober, RJ et al, J. Immunol. 172 (2004) 2021-2029; Prabhat, P. et al, Proc. Natl. Acad. Sci. USA 104 (2007) 5889-5894). Furthermore, considering the potential for modulating IgG trafficking processes and behavior in vivo, the previous report on engineering antibodies to increase their half-life in mice (Ghetie et al, above) has expanded into an area of intense interest in the biopharmaceutical industry (Dall'Acqua, WF et al, J. Biol. Chem. 281 (2006) 23514-23524; Hinton, PR et al, J. Biol. Chem. 279 (2004) 6213-6216; Hinton, PR et al. al, J. Immunol. 176 (2006) 346-356; Shields, RL et al, J. Biol. Chem. 276 (2001) 6591-6604).
In WO 2005/047327, variants of neonatal Fc receptor binding polypeptides (FcRN), dimeric Fc binding proteins and related methods are reported. Polypeptide variants with altered effector function are reported in WO 2006/031370. In WO 2009/041643, a method of modifying the isoelectric point of antibodies by means of amino acid substitution in CDRs is reported. In WO 2010/048313, recombinant FcRn and its variants are reported for purification of Fc-containing fusion proteins. Magistrelli, G. et al report robust production of recombinant FcRn in mammalian cells, allowing targeted immobilization for IgG binding studies (J. Immunol. Meth. in print phase, available online 09.12.2011). BRIEF DESCRIPTION OF THE INVENTION
[005] One aspect as reported herein is the use of an immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) as an affinity chromatography ligand.
[006] It has been found that, with the method/use as reported herein, it is now possible to separate, isolate and characterize with respect to their in vivo properties antibody species with close relationship, i.e., with a single or a limited amount of different amino acid residues.
[007] In this way, the different species of antibodies, that is, an amino acid residue difference, isolated with the method as reported herein can be used to characterize/identify amino acid positions that influence the half-life relative to FcRn.
[008] Thus, with the method as reported herein, it is possible to separate different variants of a parental antibody and determine the specific ratio between these variants, which is not possible with currently known methods, as they only provide the sum of the modifications and not the individual species (ie, for a mixture of parent, variant 1, variant 2 and variant 1/2, mass spectrometry gives the total molecules that comprise variant 1, that is, variants that comprise a single variation (1) and also those that also comprise the second variation (1/2)).
[009] This can be achieved by the combination of (i) immobilization on a recombinantly produced human FcRn chromatography support in combination with recombinantly produced human beta 2 microglobulin and (ii) a linear pH gradient.
[010] It has been found that, for the given conditions, a wild-type IgG1 antibody has a retention time of about 42 to 49 minutes. In one embodiment, an Fc fusion protein antibody that comprises a wild-type Fc region of the IgG1 subclass has a retention time of about 45 minutes.
[011] An antibody that has modified Fc region with reduced FcRn binding has retention time that is shorter, while an antibody that has modified Fc region with improved FcRn binding has retention time that is longer. In one embodiment, the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase. In one embodiment, the solid phase is a chromatography material. In one embodiment, the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin.
[012] In one embodiment, use is in pH gradient affinity chromatography. In one embodiment, the pH gradient is from a first pH value to a second pH value, such that the first pH value is from about pH 3.5 to about pH 7.5 and the second value of pH is from about pH 6.0 to about pH 9.5. In one embodiment, the pH gradient is a gradient with increasing pH value or a gradient with decreasing pH value. In one embodiment, the first pH value is about pH 5.5 and the second pH value is about pH 8.8 or the first pH value is about pH 7.4 and the second pH value is about pH is about pH 6.0.
[013] In one embodiment, beta-2-microglobulin is of the same species as FcRn.
[014] In one embodiment, the use is for the determination of the in vivo half-life of an antibody by determining the ratio of the retention times of the antibody and a reference antibody.
[015] In one embodiment, the use is for the separation of antibodies or fusion polypeptides that comprise at least one Fc region.
[016] In one embodiment, the use is for the determination of methionine oxidation of an antibody.
[017] In one embodiment, the use is intended for determining the level of oligomerization of antibodies.
[018] In one embodiment, the use is for selecting a library of modified antibodies or modified fusion polypeptides from parental antibodies or parental fusion polypeptides, which comprise at least an FcRn binding portion of an Fc region to these modified antibodies or modified fusion polypeptides that have altered binding affinity for FcRn as compared to the parent antibody or parent fusion polypeptide.
[019] In one embodiment, the use is for the identification of antibodies or fusion polypeptides that comprise at least an FcRn binding portion of an Fc region that exhibit altered binding to the neonatal Fc receptor.
[020] In one embodiment, the antibody is a monospecific antibody or fusion polypeptide antibody fragment or a bispecific antibody or fusion polypeptide antibody fragment, or a trispecific antibody or fusion polypeptide antibody fragment, or tetraspecific antibody or antibody fragment of fusion polypeptide.
[021] In one embodiment, the use is intended to remove half-antibodies (half-antibodies) from IgG preparations.
[022] In one embodiment, the use is for the removal of antibody aggregates and antibody oligomers from IgG preparations.
[023] One aspect as reported herein is a human IgG1 isotype Fc region variant in which the amino acid at position 252 is changed from methionine to histidine and the amino acid at position 428 is changed from methionine to glutamic acid.
[024] One aspect as reported herein is a pre-determined in vivo half-live-half antibody selection method in which chromatography is performed and an antibody is selected that has retention time within a given retention time window with respect to a wild-type IgG1. BRIEF DESCRIPTION OF THE FIGURES
[025] Figure 1: Linearity of applied antibody and area under the chromatography curve using an FcRn column as reported herein.
[026] Figure 2: YTE mutant and wild type anti-IGF-1R antibody chromatogram on FcRn column as reported herein.
[027] Figure 3: FcRn chromatography (upper row A/B/C) of different antibody preparations containing different amounts of antibody by half as seen in SDS CE analysis (lower row A/B/C).
[028] Figure 4: FcRn chromatography of different antibody preparations containing different amounts of antibody monomer and aggregates.
[029] Figure 5: Influence of retention time in FcRn chromatography by the number of Fc regions in the chromatographed molecule.
[030] Figure 6: Impact on the oxidation of antibodies on the retention time of FcRn chromatography.
[031] Figure 7: Wild-type and HE mutant anti-Abeta antibody chromatogram on FcRn column as reported herein.
[032] Figure 8: Impact of oxidation of Met252 and Met428 on the interaction of FcRn. Applying a sample of an IgG1 antibody stored for two months at 40 °C (curve 2) to the FcRn column generates an early-eluting substance with a double peak that indicates oxidized IgG1 antibody, whereas applying an IgG1 antibody sample stored for two months at 25 °C (curve 1) and -80 °C (curve 3) to the FcRn column generates further elution, with virtually overlapping peaks. Chromatographic conditions: buffer A (20 mM MES, 150 mM NaCl, pH 5.5), buffer B (20 mM HEPES, 150 mM NaCl, pH 8.2), flow 0.5 ml/min, gradient between buffer A and buffer B: 60 minutes (standard).
[033] Figure 9: Surface plasma resonance (SPR) analysis of IgG1 antibody with stress. Sample application of IgG1 antibody stored for two months at 40°C to BIAcore for SPR analysis displays different sensorgrams for both Met252 and Met428 and wild type oxidized IgG1 antibody species.
[034] Figure 10: Impact of antibody aggregates on FcRn interaction. Chromatographic analysis of FcRn of anti-IL13R alpha antibody in the original sample, its isolated monomers and isolated aggregates. Chromatographic conditions: buffer A (20 mM MES, 150 mM NaCl, pH 5.5), buffer B (20 mM Tris-HCl, 150 mM NaCl, pH 8.8), flow 0.5 ml/ min, gradient between buffer A and buffer B: 50 minutes (standard).
[035] Figure 11: SPR analysis of anti-IL3R alpha antibody aggregates. Sensograms of anti-IL13R alpha antibody as reference standard (curve 1), in the original sample (3), of isolated anti-IL13R alpha antibody monomers (curve 2) and isolated anti-IL13R alpha antibody aggregates (curve 4).
[036] Figure 12: Impact of Fc mutations on pharmacokinetics in FcRn transgenic mice. Wild-type antibody or its triple mutant YTE was given as an injection of 10 mg/kg intravenous mixture alone to eight animals per group. Results are presented as mean ± standard deviation (SD), significant ANOVA analysis compared to wild-type antibody (+++, p<0.001). A: area under time curve and serum concentration from time 0 to 672 hours (AUC(0-672)). B: terminal half life. DETAILED DESCRIPTION OF THE INVENTION
[037] The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo.
[038] FcRn affinity chromatography can differentiate IgG samples by their peak area and retention time profile. It allows analysis of the interaction between FcRn and IgG in vitro and can provide insight into the structural and functional integrity of therapeutic IgG with respect to in vivo pharmacokinetics.
[039] In this way, FcRn affinity chromatography of mutant and wild-type IgGs can be used as semiquantitative prediction of in vivo pharmacokinetics. In addition, FcRn affinity chromatography can be used to monitor the FcRn-IgG interaction, for example, for characterization of IgG batches or for comparison studies.
[040] FcRn affinity liquid chromatography method with standardized pH gradient has been considered with conditions that closely resemble the mechanism of interaction between IgG and FcRn in vivo. Human FcRn was immobilized on the column as an affinity binder and a linear pH gradient was applied, for example, from pH 5.5 to 8.8.
[041] Analytical FcRn affinity chromatography, for example, allows the identification and characterization of IgG samples and variants by peak pattern and retention time profile. The method can differentiate (1) the same IgG with different Fab fragments, (2) oxidized IgG forms from non-oxidized IgG forms; (3) aggregates of monomers; and (4) antibodies with variations in the Fc region.
[042] Changes in the FcRn affinity chromatography profile of variant IgGs (Fc region variants) relative to wild-type IgG have been found to predict the in vivo pharmacokinetic profile. These results demonstrate that FcRn affinity chromatography is a useful new method of characterizing interactions between FcRn and IgG or integrity of IgG and at most an IgG as such. I. DEFINITIONS
[043] The expression “about” indicates a range of +/- 20% of the next numerical value. In one embodiment, the expression "about" indicates a range of +/- 10% of the next numerical value. In one embodiment, the expression "about" indicates a range of +/- 5% of the next numerical value.
[044] The term "alteration" indicates the substitution, addition or deletion of one or more amino acid residues in a parent antibody or fusion polypeptide comprising at least an FcRn binding portion of an Fc region to obtain a fusion polypeptide or modified antibody.
[045] The expression "amino acid substitution" indicates the replacement of at least one existing amino acid residue by another different amino acid residue (substitute amino acid residue). The substitute amino acid residue can be a "naturally occurring amino acid residue" selected from the group consisting of alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine ( asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine ( thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).
[046] The expression "insertion of amino acids" indicates the incorporation of at least one amino acid residue at a predetermined position in an amino acid sequence. In one embodiment, the insertion will be the insertion of one or two amino acid residues. The inserted amino acid residue(s) can be any naturally occurring or non-naturally occurring amino acid residue(s).
[047] The expression "amino acid deletion" indicates the removal of at least one amino acid residue at a predetermined position in an amino acid sequence.
[048] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) and antibody fragments, provided that display the FcRn binding property.
[049] The expression “buffer substance” indicates a substance that, when in solution, can level out changes in the pH value of the solution, for example, due to the addition or release of acidic or basic substances.
[050] The term "CH2 domain" indicates the part of an antibody heavy chain polypeptide that extends approximately from EU position 231 to EU position 340 (EU numbering system according to Kabat). In one embodiment, a CH2 domain has the amino acid sequence of SEQ ID NO. 1: APELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQ AND STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAK.
[051] The term "CH3 domain" indicates the part of an antibody heavy chain polypeptide that extends approximately from EU position 341 to EU position 446. In one embodiment, the CH3 domain has the amino acid sequence of SEQ ID NO. 2: GQPREPQ VYTLPPSRDE LTKNQVLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG.
[052] The term "class" of an antibody designates the type of constant domain or constant region of its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, y and μ, respectively.
[053] The term "Fc region of human origin" indicates the C-terminal region of an immunoglobulin heavy chain of human origin that contains at least a part of the hinge region, the CH2 domain and the CH3 domain. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain. In one embodiment, the Fc region has the amino acid sequence of SEQ ID NO. 10. The C-terminal lysine (Lys447) of the Fc region, however, may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, EA et al, Sequences of Proteins of Immunological Interest, fifth edition, Public Health Service, National Institutes of Health, Bethesda MD (1991), NIH Publication 91-3242.
[054] The term “FcRn” indicates the human neonatal Fc receptor. FcRn works to save IgG from the lysosomal degradation process, which results in reduced release and long half-life. FcRn is a heterodimeric protein consisting of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein (α-FcRn) and a 15 kDa β2 microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3 portion of the Fc domain of IgG. The interaction between IgG and FcRn is strictly pH dependent and occurs in 1:2 stoichiometry, with an IgG that binds to two FcRn molecules through its two heavy chains (Huber, AH et al, J. Mol. Biol. 230 (1993) 1077-1083). FcRn binding occurs in the endosome under acidic pH (pH < 6.5) and IgG is released on the neutral cell surface (pH about 7.4). The pH-sensitive nature of the interaction facilitates the FcRn-mediated protection of pinocytosed IgGs in cells against intracellular degradation through receptor binding within the acidic environment of endosomes. FcRn then facilitates the recycling of IgG to the cell surface and subsequent release into the blood stream upon exposure of the FcRn-IgG complex to the neutral pH environment outside the cell.
[055] The term "FcRn binding part of an Fc region" indicates the part of an antibody heavy chain polypeptide that extends from approximately EU position 243 to EU position 261, approximately from EU position 275 to EU position 293, from about EU position 302 to EU position 319, from about EU position 336 to EU position 348, from about EU position 367 to EU position 393 and about 408 and from about EU position 424 to EU position 440. In one realization, one or more of the following amino acid residues according to EU numbering of Kabat are F243, P244, P245 P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305, L306. T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, I336, S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380, W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439 and S440 changed (EU numbering).
[056] The term "full-length antibody" indicates an antibody that has a structure substantially similar to a native antibody structure or that has heavy chains that contain an Fc region as defined herein.
[057] The term "hinge region" indicates the part of an antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain, for example, from about position 216 to about position 230 according to the system of US numbering of Kabat. The hinge region is usually a dimeric molecule consisting of two polypeptides with identical amino acid sequence. The hinge region generally comprises about 25 amino acid residues and is flexible, allowing the antigen binding regions to move independently. The articulation region can be subdivided into three domains: the superior, intermediate, and inferior articulation domain (Roux et al, J. Immunol. 161 (1998) 4083).
[058] The terms "host cell", "host cell lineage" and "host cell culture" are used interchangeably and designate cells into which the exogenous nucleic acid has been introduced, including the offspring of those cells. Host cells include "transformers" and "transformed cells", which include the primary transformed cell and the offspring derived therefrom without regard to the number of passages. The offspring may not have nucleic acid content completely identical to a parent cell, but may contain Included in the present are mutant offspring that have the same biological function or activity as selected in the originally transformed cell.
[059] "Humanized" antibody means a chimeric antibody comprising amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human main-chain regions (FRs). In certain embodiments, a humanized antibody will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all of the HVRs (such as CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody can optionally comprise at least a portion of an antibody constant region derived from a human antibody. "Humanized form" of antibody, such as non-human antibody, means an antibody that has undergone humanization.
[060] The expression "hypervariable region" or "HVR", as used herein, indicates each of the regions of an antibody variable domain that are of hypervariable sequence and/or form structurally defined circuits ("hypervariable circuits"). Generally, antibodies with four native chains comprise six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs generally comprise amino acid residues from hypervariable circuits and/or "complementarity determining regions" (CDRs), where the latter have higher sequence variability and/or are involved in antigen recognition. Examples of hypervariable circuits occur at amino acid residues 2632 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3) (Chothia , C. and Lesk, AM, J. Mol. Biol. 196 (1987) 901-917). Examples of CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3 , 31-35B of H1, 50-65 of H2 and 95-102 of H3 (Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, Public Health Service, National Institutes of Health, Bethesda MD (1991), NIH Publication 91-3242). With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable circuits. CDRs also comprise “specificity-determining residues” or “SDRs”, which are residues that come into contact with antigen. SDRs are contained in regions of the CDRs called abbreviated CDRs or a-CDRs. Examples of α-CDRs (α-CDR-L1, α-CDR-L2, α-CDR-L3, α-CDR-H1, α-CDR-H2 and α-CDR-H3) occur at amino acid residues 3134 of L1 , 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2 and 95-102 of H3 (see Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619 -1633). Unless otherwise indicated, residues of HVR and other residues in the variable domain (such as FR residues) are numbered herein in accordance with Kabat et al, above.
[061] "Individual" or "patient" is mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs and horses), primates (such as humans and non-human primates such as monkeys), rabbits and rodents (such as rats, hamsters and mice) . In certain embodiments, the individual or patient is a human being.
[062] The term "monoclonal antibody" designates an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that comprise the population are identical and/or bind the same epitope, except for possible variant antibodies, by for example, which contain naturally occurring mutations or which arise during the production of a monoclonal antibody preparation, where such variants are generally present in smaller amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. In this way, the adjective “monoclonal” indicates the characteristic of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be considered to require the production of the antibody by any specific method. Monoclonal antibodies to be used in accordance with the present invention can be made, for example, by a number of methods, including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods. using transgenic animals that contain, in whole or in part, the human immunoglobulin loci, such methods and other examples of methods of making monoclonal antibodies are described herein.
[063] "Native antibodies" designates naturally occurring immunoglobulin molecules with variable structures. Native IgG antibodies, for example, are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are disulfide-linked. From the N-terminus to the C, each heavy chain has a variable region (VH), also called variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (CL). An antibody's light chain can be assigned to one of two types, called kappa (K) and lambda (A), based on the amino acid sequence of its constant domain.
[064] The term “non-naturally occurring amino acid residue” indicates an amino acid residue, other than the naturally occurring amino acid residues as listed above, that can be covalently linked to adjacent amino acid residues in a polypeptide chain. Examples of non-naturally occurring amino acid residues are norleucine, ornithine, norvaline and homoserine. Additional examples are listed in Ellman et al, Meth. Enzyme 202 (1991) 301-336. Examples of methods of synthesizing non-naturally occurring amino acid residues are reported, for example, in Noren et al, Science 244 (1989) 182 and Ellman et al, above.
[065] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a possible sequence that are identical to the amino acid residues in the polypeptide sequence of reference, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitution as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, using, for example, publicly available computer software such as BLAST software, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine appropriate sequence alignment parameters, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. For purposes of the present, however, percent amino acid sequence identity values are generated using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc. and the source code was deposited with user documentation at the US Copyright Office, Washington DC 20559, United States, where it was registered with on ° US Copyright Registry TUX510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program shall be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
[066] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the percent amino acid sequence identity of a given A amino acid sequence to, with or against a given B amino acid sequence (which may alternatively be expressed as a given amino acid sequence A that has or comprises a certain percent amino acid sequence identity to, with or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y where X is the amount of amino acid residues judged as identical matches by the ALIGN-2 sequence alignment program in the alignment of A and B in that program and Y is the total amount of amino acid residues in B. It will be appreciated that when the length of the sequence of amino acid A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity between A and B will not be equal to the percent identity. and amino acid sequences between B and A. Unless specifically indicated otherwise, all percent amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
[067] The term "pharmaceutical formulation" means a preparation that is in a form that allows the biological activity of an active ingredient contained therein to be effective and that does not contain additional components that are unacceptably toxic to a patient for whom the formulation would be administered.
[068] “Pharmaceutically acceptable carrier” means an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to the patient. Pharmaceutically acceptable carrier includes, but is not limited to, buffer, excipient, stabilizer or preservative.
[069] The expression "positive linear pH gradient" indicates a pH gradient that starts at a low pH value (ie more acidic) and ends at a higher pH value (ie less acidic, neutral or alkaline) . In one embodiment, the linear positive pH gradient starts at a pH value of about 5.5 and ends at a pH value of about 8.8.
[070] The expression “negative linear pH gradient” indicates a pH gradient that starts at a high pH value (ie neutral or alkaline) and ends at a lower pH value (ie neutral or acidic). In one embodiment, the linear negative pH gradient starts at a pH value of about 7.4 and ends at a pH value of about 6.0.
[071] As used herein, "treatment" (and its grammatical variations such as "treating" or "treating") indicates clinical intervention in an attempt to alter the natural course of the individual being treated and may be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease occurrence or recurrence, symptom relief, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, slowing of disease progress, improvement or reduction disease state and remission or better prognosis. In some embodiments, antibodies in accordance with the present invention are used to delay the development of a disease or to slow down the progress of a disease.
[072] The term "variable region" or "variable domain" indicates the domain of an antibody light or heavy chain that is involved in antibody binding to antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, for example, , Kindt, TJ et al, Kuby Immunology, sixth edition, WH Freeman & Co., New York (2007), p. 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a specific antigen can be isolated using a VH or VL domain of an antibody that binds the antigen to select a library of complementary VL or VH domains, respectively (see, for example, Portolano, S. et al, J. Immunol. 150 (1993) 880-887; Clackson, T. et al, Nature 352 (1991) 624-628).
[073] The terms "variant", "modified antibody" and "modified fusion polypeptide" indicate molecules that have an amino acid sequence that differs from the amino acid sequence of a parent molecule. Typically, these molecules have one or more alterations, insertions or deletions. In one embodiment, the modified antibody or modified fusion polypeptide comprises an amino acid sequence that comprises at least a portion of an Fc region that is not normally naturally occurring. These molecules have less than 100% sequence identity with the parent antibody or parent fusion polypeptide. In one embodiment, the variant antibody or variant fusion polypeptide has an amino acid sequence that has amino acid sequence identity of about 75% to less than 100% with the amino acid sequence of the parent antibody or parent fusion polypeptide, especially about 80% less than 100%, especially about 85% less than 100%, especially about 90% less than 100%, and especially about 95% less than 100%. In one embodiment, the parent antibody of the parent fusion polypeptide and the variant antibody or variant fusion polypeptide differ by one (a single), two, or three amino acid residues. II. COMPOSITIONS AND METHODS
[074] The human neonatal Fc receptor (FcRn) plays an important role in IgG catabolism. The in vitro FcRn binding properties/characteristics of an IgG indicate its in vivo pharmacokinetic properties. These in vitro methods would be of great importance during antibody development, as repeated in vivo studies can be avoided (animal experiments, reduced time and costs). Until now, such analyzes have generally been performed using plasma surface resonance (SPR) tests (Wang, W. et al, Drug Metab. Disp. 39 (2011) 1469-1477; Datta-Mannan, A. et al, Drug Metab Disp 40 (2012) 1545-1555; Vaughn, DE and Bjorkman, PJ, Biochemistry 36 (1997) 9374-9380; Raghavan, M. et al, Proc. Natl. Acad. Sci. USA 92 (1995) 11200 -11204; Martin, WL and Bjorkman, PJ, Biochemistry 38 (1999) 12639-12647). Asymmetric and calorimetric flow-field flow fractionation methods have also been described to determine the binding affinity of IgG to FcRn (Huber, AH et al, J. Mol. Biol. 230 (1993) 1077-1083; Pollastrini, J. et al, Anal. Biochem. 414 (2011) 88-98 ). In addition to being complex tests, several studies investigating the correlation between in vitro FcRn binding parameters determined by SPR and the serum half-life of antibodies in vivo have so far failed to demonstrate this correlation despite improved binding reaction conditions and appropriate modeling ( Gurbaxani, B. et al, Mol. Immunol. 43 (2006) 1462-1473; Gurbaxani, BM and Morrison, SL, Mol. Immunol. 43 (2006) 1379-1389; Gurbaxani, B., Clin. Immunol. 122 (2007) 121-124). Elaboration of the IgG1 Fc region to increase the affinity of IgG1 to FcRn at pH 6 and at neutral pH as measured by SPR technology did not result in increased pharmacokinetics in cynomolgus monkeys (Yeung, YA et al, J. Immunol. 182 ( 2009) 7663-7671). Only modest increases in FcRn affinity at pH 6 in the IgG1 N434A variant without simultaneous significant binding to FcRn at pH 7.4, however, resulted in increased pharmacokinetics in primates, demonstrating the importance of FcRn release at pH 7, 4 (see Yeung, YA, above).
[075] A combination of known methods can achieve analytical results comparable to those of FcRn affinity chromatography, but at the expense of greater complexity and effort.
[076] Current standard methods do not adequately reflect the physiological pH dependence of FcRn binding characteristics that require acidic pH for endosomal binding, but neutral pH for IgG release at the cell surface. The pH of the medium influences the self-association properties of the FcRn molecule. Current methods work under standard conditions at a given pH and therefore only detect a snapshot of the FcRn-IgG complex interaction, avoiding robust kinetic evaluation of the IgG-FcRn interaction. This may also be one of the reasons for the lack of correlation of FcRn affinity between in vitro FcRn analysis and in vivo pharmacokinetics found in several studies (see above).
[077] SPR analysis of the IgG-FcRn interaction provides a qualitative result that indicates expected or aberrant binding properties of a sample, but does not indicate the cause of aberrant binding nor a quantitative estimate of the amount of aberrantly bound antibody. Mass spectrometry also provides only qualitative information of an impaired IgG molecule integrity. On the other hand, FcRn affinity chromatography allows the analysis of the sample under appropriate physiological conditions with 2:1 predominant stoichiometry in a mixture of stoichiometries, including 1:2, 1:1 and 2:2 stoichiometry and a pH gradient that can be set to fine tune the separation of the different peaks found in a sample. The different peaks can be quantified by their area under the corresponding curve and the eluate corresponding to each peak is prone to secondary analysis, eg for functionality determinations, rechromatography or mass spectrometric analysis.
[078] Furthermore, in order to provide therapeutic regimens to treat the various diseases known today and also those to be discovered in the future, there is a need for specific antibodies as well as polypeptides that contain Fc part.
[079] To specifically define the FcRn binding characteristics of an antibody or a fusion polypeptide that contains Fc part, the residues involved in Fc part-mediated effector functions are modified and the resulting modified fusion polypeptides and antibodies need to be tested. If the required characteristics are not met, the same process is carried out again.
[080] In one embodiment, the Fc part is the fraction of an Fc region that mediates binding to FcRn.
[081] Thus, it would be advantageous to provide a method that predicts changes in the characteristic properties of a modified antibody based on a simple chromatographic method and that does not require in vivo studies to analyze changes in characteristics in the modified antibody.
[082] In some cases, antibodies with an extended half-life are desired. Drugs with a longer half-life in the circulation of patients in need of treatment need, for example, a lower dose or longer dosing intervals. These antibodies also have the advantage of greater exposure to a disease site, such as a tumor.
[083] One aspect as reported herein is the use of an immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin as an affinity chromatography ligand.
[084] It has been found that an affinity chromatography column comprising an immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin as affinity chromatography binder has unexpected stability. It can be used for at least more than one hundred chromatography cycles and up to about two hundred chromatography cycles (equilibrium - separation - regeneration) without loss of performance (selectivity and/or binding capacity).
[085] Also reported is an affinity chromatography column comprising a matrix and matrix-bound chromatographic functional groups, characterized in that the matrix-bound chromatographic functional group comprises a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2- microglobulin.
[086] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand for determining the in vivo half life of an antibody by means of determination of the ratio between the retention times of the antibody and a reference antibody. In one embodiment, the reference antibody is a full-length human IgG1 antibody.
[087] Also reported herein is a method of determining the in vivo half life of an antibody relative to a reference antibody by determining the ratio between the retention times determined on an FcRn affinity column as reported herein of the antibody and the reference antibody.
[088] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a linker for separation of antibodies or fusion polypeptides comprising at least one part Fc.
[089] Also reported herein is a method of separating antibodies or fusion polypeptides comprising at least one Fc part.
[090] In one embodiment, the separation is selected through purification, production and analysis.
[091] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand for separation of IgG1 subclass antibodies and IgG3 subclass antibodies.
[092] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand to determine the methionine oxidation of an antibody.
[093] A method of determining the impact on FcRn binding of oxidized methionine residues in the Fc part of an antibody using an affinity chromatography method reported herein is reported herein.
[094] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand to determine the level of oligomerization of an antibody.
[095] A method of determining the oligomerization level of an antibody using an affinity chromatography method as reported herein is reported herein.
[096] Generally, the starting point of the method reported herein is a parental antibody or a parental fusion polypeptide that is characterized by binding to FcRn.
[097] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as selection ligand for a library of modified antibodies or modified fusion polypeptides of parental antibodies or parental fusion polypeptides, which comprise at least an FcRn binding portion of an Fc region for such modified antibodies or modified fusion polypeptides which have altered binding affinity for FcRn compared to the parent antibody or fusion polypeptide parental.
[098] There is reported herein a method of selecting a library of modified antibodies or modified fusion polypeptides from parental antibodies or parental fusion polypeptides, which comprise at least an FcRn binding portion of an Fc region for such modified antibodies or modified fusion polypeptides which have altered binding affinity for FcRn as compared to the parent antibody or parent fusion polypeptide, the method comprising the following steps: a. applying the individual library members and the parental antibody or parental fusion polypeptide to an FcRn affinity chromatography column as reported herein; B. retrieving individual library members with a pH gradient and determining individual retention times; and c. selection of antibodies or fusion polypeptides that have altered binding affinity for FcRn as compared to the parent antibody or parent fusion polypeptide.
[099] There is reported herein a method of purifying antibodies or fusion polypeptides, comprising at least an FcRn binding part of an Fc region, from a mixture of polypeptides, wherein the method comprises applying the mixture to an FcRn affinity column as reported herein and eluting the antibodies or fusion polypeptide, which comprises at least an FcRn binding portion of a pH gradient Fc region, so as to purify the antibody or polypeptide of fusion. In one embodiment, the FcRn part of an Fc region is from a human Fc region, mouse Fc region, cynomolgus Fc region, rabbit Fc region, or hamster Fc region.
[0100] The terms "one" and "one" indicate one, two, three, four, five or six and up to 109.
[0101] In one embodiment, the production/reaction mixture or the crude or partially purified skin supernatant is applied to the FcRn affinity column at first pH value and the antibody or fusion polypeptide is recovered from the FcRn affinity column at second pH value.
[0102] In one embodiment, the first pH value is from about pH 3.5 to about pH 7.5. In one embodiment, the first pH value is from about pH 4 to about pH 7. In one embodiment, the first pH value is from about pH 4.5 to about pH 6.5. In one embodiment, the first pH value is from about pH 5 to about pH 6. In one embodiment, the first pH value is from about pH 5, about pH 5.5, or about pH 6.
[0103] In one embodiment, the first pH value is selected from about pH 3.5, about pH 3.6, about pH 3.7, about pH 3.8, about pH 3, 9, about pH 4.0, about pH 4.1, about pH 4.2, about pH 4.3, about pH 4.4, about pH 4.5, about pH 4.6 , about pH 4.7, about pH 4.8, about pH 4.9, about pH 5.0, about pH 5.1, about pH 5.2, about pH 5.3, about pH 5.4, about pH 5.5, about pH 5.6, about pH 5.7, about pH 5.8, about pH 5.9, about pH 6.0, about about pH 6.1, about pH 6.2, about pH 6.3, about pH 6.4, about pH 6.5, about pH 6.6, about pH 6.7, about pH 6.8, about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, about pH 7.4 and about pH 7.5.
[0104] In one embodiment, the second pH value is from about pH 8 to about pH 9.5. In one embodiment, the second pH value is from about pH 8.5 to about pH 9. In one embodiment, the second pH value is about pH 8. 8.
[0105] In one embodiment, the second pH value is selected from about pH 8.0, about pH 8.1, about pH 8.2, about pH 8.3, about pH 8, 4, about pH 8.5, about pH 8.6, about pH 8.7, about pH 8.8, about pH 8.9, about pH 9.0, about pH 9.1 , about pH 9.2, about pH 9.3, about pH 9.4 and about pH 9.5.
[0106] In one embodiment, each of the first given pH values is about pH 3.5, about pH 3.6, about pH 3.7, about pH 3.8, about pH 3.9 , about pH 4.0, about pH 4.1, about pH 4.2, about pH 4.3, about pH 4.4, about pH 4.5, about pH 4.6, about pH 4.7, about pH 4.8, about pH 4.9, about pH 5.0, about pH 5.1, about pH 5.2, about pH 5.3, about about pH 5.4, about pH 5.5, about pH 5.6, about pH 5.7, about pH 5.8, about pH 5.9, about pH 6.0, about pH 6.1, about pH 6.2, about pH 6.3, about pH 6.4, about pH 6.5, about pH 6.6, about pH 6.7, about pH 6.8, about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, about pH 7.4 and about pH 7 .5 is combined with each of the second given pH values of about pH 8.0, about pH 8.1, about pH 8.2, about pH 8.3, about pH 8.4, about about pH 8.5, about pH 8.6, about pH 8.7, about pH 8.8, about pH 8.9, about pH 9.0, about pH 9.1, about pH 9.2, about pH 9.3, about pH 9.4 and about pH 9.5.
[0107] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a linker to identify antibodies or fusion polypeptides comprising at least one part of FcRn binding from an Fc region (such as a constant domain of an immunoglobulin such as IgG1) that exhibit altered binding to the neonatal Fc receptor (FcRn).
[0108] A method of identifying antibodies or fusion polypeptides comprising at least an FcRn binding part of an Fc region (such as a constant domain of an immunoglobulin such as IgG1) that exhibit altered binding to the receptor is provided herein. Neonatal Fc (FcRn).
[0109] These modified fusion polypeptides or antibodies exhibit greater or lesser binding to FcRn compared to a fusion polypeptide or parent antibody or compared to a reference antibody or reference fusion protein and therefore have half-life in serum greater or lesser, respectively.
[0110] Fc region variants with higher affinity for FcRn (ie longer retention time on an FcRn column but still eluting before a pH 7.4 value as reported herein compared to an antibody) are predicted parent antibody or reference antibody) have longer serum half-lives compared to those with lower affinity for FcRn. Fc region variants with greater affinity for FcRn have application in methods of treating mammals, especially humans, where a long half-life of the administered fusion polypeptide or antibody is desired, such as in the treatment of a chronic disease or disorder. Fc region variants with lower affinity for FcRn have application in methods of treating mammals, especially humans, where a short half-life of the administered fusion polypeptide or antibody is desired, such as in in vivo diagnostic imaging.
[0111] It is very likely that Fc region variants with lower FcRn binding affinity are able to cross the placenta and therefore can be used in the treatment of diseases or disorders in pregnant women, especially in unborn children. In addition, reduced FcRn binding affinity for drugs intended for application/transport to the brain, kidney and/or liver may be desired.
[0112] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a linker to identify antibodies or fusion polypeptides that exhibit reduced transport across the epithelium of renal glomeruli from the vasculature.
[0113] In one embodiment, the antibody or fusion polypeptide comprising a modified Fc region as reported herein exhibits reduced transport across the epithelium of renal glomeruli from the vasculature.
[0114] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a linker to identify antibodies or fusion polypeptides that exhibit reduced transport across the blood brain barrier from the brain to the vascular space.
[0115] In one embodiment, the antibody or fusion polypeptide comprising a modified Fc region of human origin as reported herein exhibits reduced transport across the blood-brain barrier (BBB) from the brain to the vascular space.
[0116] Methods of making such fusion polypeptides and modified antibodies that comprise at least an FcRn binding part of an FcRn region and methods of using such fusion polypeptides and modified antibodies are reported herein.
[0117] In one embodiment, the antibody or polypeptide fusion reported herein comprises at least one binding site (such as at least one antigen binding site, at least one receptor binding site, or at least one site of linker binding). In one embodiment, the antibody or polypeptide fusion reported herein comprises at least two binding sites (such as at least two antigen binding sites, at least two receptor binding sites, at least two ligand binding sites, at least one antigen binding site and at least one receptor binding site or at least one antigen binding site and at least one ligand binding site, or at least one receptor binding site and at least one site of linker binding). In one embodiment, the antibody or polypeptide fusion reported herein comprises three binding sites (such as at least three antigen binding sites, at least three receptor binding sites, at least three ligand binding sites, or any mixture of at least three binding sites from the above). In one embodiment, the antibody or fusion polypeptides reported herein comprise four binding sites.
[0118] In an embodiment of all aspects reported herein, at least a part of an Fc region is at least a part of a Fc region of human origin. In one embodiment of all aspects reported herein, the FcRn is selected from human FcRn, cynomolgus FcRn, mouse FcRn, rat FcRn, sheep FcRn, dog FcRn and rabbit FcRn.
[0119] In a realization of all aspects reported herein, beta-2-microglobulin is of the same species as FcRn.
[0120] In an embodiment of all aspects reported herein, beta-2-microglobulin is of a different species than FcRn.
[0121] In one embodiment, the Fc regions or FcRn binding portions of an Fc region are derived from heavy chains of any isotype.
[0122] In one embodiment, the at least part of an Fc region comprises at least amino acid residues 282-340 of a CH2 domain of human origin (SEQ ID No. 1, numbering according to Kabat). In one embodiment, the at least part of an Fc region comprises a complete CH2 domain (about amino acid residues 231-340 of a human-derived antibody heavy chain polypeptide Fc region according to EU numbering under Kabat) . In one embodiment, the at least a portion of an Fc region comprises at least one CH2 domain and at least one of a hinge region (about amino acid residues 216-230 of a source antibody heavy chain polypeptide Fc region human according to EU numbering) or a CH3 domain (about amino acid residues 341-446 of a human source antibody heavy chain polypeptide Fc region according to EU numbering). In one embodiment, the at least a portion of the Fc region comprises a CH2 and CH3 domain of an antibody heavy chain of human origin. In one embodiment, the at least a portion of the Fc region comprises a hinge, CH2 domain and CH3 domain of an antibody heavy chain Fc region of human origin. Fc regions of human origin or FcRn binding parts of an Fc region of parts of human origin can be derived from heavy chains of any isotype, such as IgG1 (SEQ ID No. 3), IgG2 (SEQ ID No. 4), IgG3 (SEQ ID No. 5) and IgG4 (SEQ ID No. 6). In one embodiment, the human isotype is IgG1.
[0123] The Fc region of the parent antibody or comprised within the parent fusion polypeptide may be derived from different immunoglobulin molecules and/or different immunoglobulin isotypes. A parental antibody or parental fusion polypeptide, for example, can comprise a CH2 domain derived from an immunoglobulin of IgG1 isotype and a hinge region derived from an immunoglobulin of IgG3 isotype. Furthermore, for example, a parental antibody or parental fusion polypeptide can comprise a hinge region derived in part from the IgG1 immunoglobulin subtype and in part from the IgG3 immunoglobulin subtype, provided they are of human origin. A parental antibody or parental fusion polypeptide, for example, can comprise a chimeric hinge region derived in part from an IgG1 immunoglobulin isotype and in part from an IgG4 immunoglobulin isotype.
[0124] The parent antibody of the parent fusion polypeptide reported herein comprises at least one Fc region or one of its FcRn binding parts. In one embodiment, the parent antibody or parent polypeptide further comprises at least one binding domain (in one embodiment, selected from an antigen binding domain, receptor binding domain, or ligand binding domain). In one embodiment, the parent antibody or parent fusion polypeptide comprises at least one binding domain and at least one Fc region or one of its FcRn binding parts. In one embodiment, the parent antibody or parent fusion polypeptide comprises two binding domains and two Fc regions or two of its FcRn binding parts.
[0125] In one embodiment, the parental antibody or parental fusion polypeptide reported herein comprises at least one binding domain that specifically binds to a target that mediates a biological effect (in one embodiment, a ligand capable of binding to a cell surface receptor or to a cell surface receptor capable of binding a ligand) and mediates the transmission of a positive or negative signal to a cell along with at least one Fc region or one of its FcRn binding parts. In one embodiment, the measurement of the biological effect is carried out at a pH value of about pH 7.4. In one embodiment, the parent antibody or parent fusion polypeptide comprises at least one binding domain specific for an antigen directed towards reduction or elimination (in one embodiment, a cell surface antigen or soluble antigen) and at least one Fc region or a of its FcRn binding parts.
[0126] Antibodies that specifically bind to a target can be raised in mammals through various subcutaneous or intraperitoneal injections of the relevant antigen (such as purified antigen, cells or cell extracts that comprise such antigens or DNA encoding that antigen) and, optionally an adjuvant.
[0127] In one embodiment, the antibody is a monoclonal antibody.
[0128] In one embodiment, the fusion polypeptide reported herein comprises an antibody fragment (such as an scFv molecule, a minibody, a tetravalent minibody or a diabody) operably linked to an FcRn binding moiety. In one embodiment, the FcRn binding portion is a complete antibody heavy chain Fc region.
[0129] In one embodiment, the parent antibody is a bispecific antibody or the parent fusion polypeptide comprises a bispecific antibody or a bispecific antibody fragment.
[0130] In one embodiment, the parent antibody is a chimeric antibody.
[0131] In one embodiment, the parental fusion polypeptide comprises at least an FcRn binding portion of an Fc region. In one embodiment, the parent fusion polypeptide reported herein comprises one or more binding domains which, in turn, may each comprise a binding site. The parental fusion polypeptide can be bispecific (with one binding site that specifically binds to a first target and a second binding site that specifically binds to a second target) or multivalent (with two binding sites that specifically bind to the same target).
[0132] In an embodiment of all of the above aspects, the pH is a gradient from about pH 5.5 to about pH 8.8.
[0133] In one embodiment, the pH is gradient from about pH 5 to pH 6, about pH 6 to about pH 7 or about pH 7 to about pH 8.
[0134] Generally, the binding domain is fused to the C-terminus or N-terminus of at least an FcRn binding portion of an Fc region.
[0135] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand for selection of antibodies with binding to FcRn at pH 7 value .4 for (co)targeting in vivo. In one realization, co-direction is internalizing.
[0136] In one embodiment, therefore, the first pH is about pH 7.4. In one embodiment, the second pH is about pH 6.0.
[0137] Generally, the soluble extracellular domain of FcRn (SEQ ID NO 7 for human FcRn) with C-terminal His-Avi tag (SEQ ID NO 8) was co-expressed with β2-microglobulin (SEQ ID NO 9 for beta -2-human microglobulin) in mammalian cells. The non-covalent FcRn microglobulin complex was biotinylated and loaded onto streptavidin-derivatized sepharose.
[0138] In an embodiment of all aspects reported herein, the non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase.
[0139] A “solid phase” indicates a non-fluid substance and includes particles (including microparticles and spheres) made of materials such as polymer, metal (paramagnetic and ferromagnetic particles), glass and ceramic; gel substances such as silica, alumina and polymer gels; capillaries, which can be made of polymer, metal, glass and/or ceramics; zeolites and other porous substances; electrodes; microtiter plates; solid tapes; and spectrometer cuvettes, tubes or other sample containers. A solid-phase component of a test is distinguished from inert solid surfaces in that a “solid support” contains at least a portion on its surface that is intended to chemically interact with a molecule. A solid phase can be a stationary component, such as a chip, tube, tape, cuvette or microtiter plate, or it can be a non-stationary component, such as beads and microparticles. Microparticles can also be used as solid support for homogeneous test formats. Various microparticles that allow covalent or non-covalent binding of proteins and other substances can be used. Such particles include polymer particles such as polystyrene and (poly)methyl methacrylate; gold particles such as gold nanoparticles and gold colloids; and ceramic particles such as silica, glass and metal oxide particles. See, for example, Martin, C.R. et al, Analytical Chemistry-News & Features, May 1 (1998) 322A-327A, which is incorporated herein by reference. In one embodiment, the solid support is sepharose.
[0140] In one embodiment, the conjugation of the non-covalent complex to the solid phase is performed by chemical bonding through N-terminal groups and/or ε-amino (lysine), ε-amino groups of different lysines, functional groups carboxy, sulfhydryl, hydroxyl, and/or phenolics of the antibody backbone amino acid and/or sugar alcohol groups of the antibody carbohydrate structure.
[0141] In one embodiment, the non-covalent complex is conjugated to the solid phase via a specific binding pair. In one embodiment, the non-covalent complex is conjugated to biotin and immobilization to a solid support is accomplished by means of avidin or streptavidin immobilized on a solid support.
[0142] A specific binding pair (first component/second component), in one embodiment, is selected from streptavidin or avidin/biotin, antibody/antigen (see, for example, Hermanson, GT et al, Bioconjugate Techniques, Academic Press (1996)), lectin/polysaccharide, steroid/steroid binding protein, hormone/hormone receptor, enzyme/substrate, IgG/Protein A and/or G etc.
[0143] Recovery of antibody bound to the FcRn affinity column reported herein in the uses and methods reported herein is accomplished by linear gradient elution. In one embodiment, the linear gradient is a pH gradient or conductivity gradient.
[0144] In principle, any buffer substance can be used in the methods reported herein.
[0145] Fc residues critical for mouse Fc and mouse FcRn interaction have been identified through site-directed mutagenesis (see, for example, Dall'Acqua, WF et al, J. Immunol. 169 (2002), 5171 -5180). Residues I253, H310, H433, N434 and H435 (EU numbering according to Kabat) are involved in the interaction (Medesan, C. et al, Eur. J. Immunol. 26 (1996) 2533; Firan, M. et al, Int. Immunol. 13 (2001) 993; Kim, JK et al, Eur. J. Immunol. 24 (1994) 542). Residues I253, H310 and H435 were considered critical for human Fc interaction with murine FcRn (Kim, J.K. et al, Eur. J. Immunol. 29 (1999) 2819). Residues M252Y, S254T and T256E have been described by Dall'Acqua et al to increase FcRn binding through protein interaction studies (Dall'Acqua, WF et al, J.Biol. Chem. 281 (2006) 23514- 23524). Studies of the human Fc and human FcRn complex have demonstrated that residues I253, S254, H435 and Y436 are critical to the interaction (Firan, M. et al, Int. Immunol. 13 (2001) 993; Shields, RL et al, J Biol. Chem. 276 (2001) 6591-6604). In Yeung, Y.A. et al (J. Immunol. 182 (2009) 7667-7671), several mutants of residues 248 to 259, 301 to 317, 376 to 382 and 424 to 437 were reported and examined.
[0146] The retention time of different antibodies obtained with different elution buffers is shown in the table below. TABLE

[0147] The expression mutant YTE indicates the triple mutant M252Y/S254T/T256E.
[0148] In one embodiment, a pharmaceutically acceptable buffer substance is used, such as phosphoric acid or its salts, acetic acid or its salts, citric acid or its salts, morpholine, 2-(N-morpholino)ethanesulfonic acid (MES) or its salts, histidine or its salts, glycine or its salts, tris(hydroxymethyl)aminomethane (TRIS) or its salts, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or its salts.
[0149] In one embodiment, the buffer substance is selected from phosphoric acid or its salts, acetic acid or its salts, citric acid or its salts, histidine or its salts.
[0150] In one embodiment, the buffer substance has a concentration of 10 mM to 500 mM. In one embodiment, the buffer substance has a concentration of 10 mM to 300 mM. In one embodiment, the buffer substance has a concentration of 10 mM to 250 mM. In one embodiment, the buffer substance has a concentration of 10 mM to 100 mM. In one embodiment, the buffer substance has a concentration of 15 mM to 50 mM. In one embodiment, the buffer substance has a concentration of about 20 mM.
[0151] In one embodiment, the buffer substance in the first solution and the buffer substance in the second solution are the same buffer substance.
[0152] In one embodiment, the buffer substance in the first solution and the buffer substance in the second solution are different buffer substances.
[0153] In one embodiment, the first solution has a pH value from about pH 3.5 to about pH 7.5. In one embodiment, the first solution has a pH value of about pH 5 to about pH 6. In one embodiment, the first solution has a pH value of about pH 5. 5.
[0154] In one embodiment, the second solution has a pH value from about pH 7.0 to about pH 9.5. In one embodiment, the second solution has a pH value from about pH 8 to about pH 9. In one embodiment, the second solution has a pH value from about pH 8.2 to about pH 8.8.
[0155] An example of a first solution comprises 20 mM MES and 150 mM NaCl, adjusted to pH 5.5.
[0156] An example of a second solution comprises 20 mM TRIS and 150 mM NaCl, adjusted to pH 8.8.
[0157] An example of a second solution comprises 20 mM HEPES adjusted to pH 8.6.
[0158] An example of a second solution comprises 20 mM TRIS adjusted to pH 8.2.
[0159] In one embodiment, the buffered solution comprises an additional salt. In one embodiment, the additional salt is selected from sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, sodium citrate or potassium citrate. One embodiment comprises the buffered solution from 50 mM to 1000 mM of the additional salt. One embodiment comprises the 50 mM to 750 mM buffered solution of the additional salt. One embodiment comprises the 50 mM to 500 mM buffered solution of the additional salt. One embodiment comprises the 50 mM to 750 mM buffered solution of the additional salt. One embodiment comprises a buffered solution of about 50 mM to about 300 mM of the additional salt.
[0160] In one embodiment, the first and/or second solution comprises sodium chloride. In one embodiment, the first and/or second solution comprises about 50 mM to about 300 mM sodium chloride.
[0161] The type of salt and buffer substance has been found to influence retention time and resolution. An optimal salt concentration for antibody binding to FcRn (150 mM NaCl) can be determined. If the salt concentration is higher (300 mM), the binding to FcRn is reduced and a shorter retention time is obtained. The same is true for lower salt concentration (50 mM). 20 mM HEPES, pH 8.6, extended the retention time for all tested antibodies.
[0162] As can be seen in Figure 1, the amount of antibody applied exhibits a linear correlation with the area under the curve of the peak that eluted.
[0163]Eight antibodies were analyzed as complete antibodies and after splitting with the IDES enzyme. The split was controlled through SDS Page and SEC analytical. The Fc part and the Fab part of the antibody were separated by preparative SEC. In the Table below, the retention times of the complete antibody, the Fab part and the Fc part are given. TABLE

[0164] The expression mutant YTE indicates the triple mutant M252Y/S254T/T256E.
[0165] Generally, retention time of antibodies that have a wild-type Fc part (IgG1, IgG2 or IgG4) ranges from 45 to 49 minutes (tested with 35 therapeutic antibodies against 36 antigens, data not shown).
[0166] In the following table, the retention time is displayed in relation to the quantity of FcRn receiver immobilized per gram of column material. TABLE


[0167] The expression mutant YTE indicates the triple mutant M252Y/S254T/T256E.
[0168] The anti-Abeta antibody FAB fragment comprises a glycosylation site.
[0169] Thus, an aspect reported in the present is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand to detect the modification of FAB. In one embodiment, the modification is glycosylation or charge distribution.
[0170] Generally, retention time in the methods and uses reported at present depends on the slope of the pH gradient and the concentration of salt employed. Wild-type antibody is used as reference and weaker binding is indicated by shorter retention time (=early elution), while stronger binding is indicated by longer retention time (=after elution), but even before value of pH 7.4.
[0171] It was found that different mutants of the Fc part of IgG behave differently on the FcRn column, exhibiting modified retention times.
[0172] The anti-Abeta YTE antibody mutant, for example, exhibits longer retention time. The second peak of the anti-Abeta antibody YTE mutant is due to an additional glycosylation site introduced in the Fab part.
[0173] The anti-IGF-1R YTE antibody mutant, for example, exhibits longer retention time (see Figure 2). TABLE

[0174] The expression mutant YTE indicates the triple mutant M252Y/S254T/T256E.
[0175] It was found that, with the FcRn column as reported herein, it is possible to identify relevant amino acids for FcRn binding and evaluate the mutants in comparison to the unmodified wild-type antibody.
[0176] Aspects as reported herein are the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a linker to identify amino acids relevant to FcRn binding to assess mutants in comparison to unmodified wild-type antibody.
[0177] The results obtained with an anti-Her2 antibody are presented in the table below (see, for example, WO 2006/031370 as a reference example). TABLE


[0178] It was found that antibodies that exhibited further elution from the FcRn column, that is, which had longer retention time on the FcRn column, had a longer half-life in vivo. In vivo data are shown in the table below. TABLE

[0179] The expression mutant YTE indicates the triple mutant M252Y/S254T/T256E.
[0180] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand to determine the in vivo half life of an antibody.
[0181] The set of in vitro and in vivo experiments conducted with wild-type IgG and IgG variants with YTE mutations in the Fc part allowed to show semiquantitative correlation of the findings in FcRn affinity chromatography with those of in vivo pharmacokinetic studies with transgenic mice for human FcRn (Spiekerman, GM et al, J. Exp. Med. 196 (2002) 303-310; Dall'Acqua, WF et al, J. Biol. Chem. 281 (2006) 23514-23524). The YTE mutation generates significantly longer half-life and slower plasma release. The longer in vivo half life corresponded to longer retention time on FcRn chromatography. It has recently been shown that the extended half-life of an Fc-crafted trastuzumab variant has enhanced in vitro binding to FcRn as measured by flow cytometry (Petkova, SB et al, Int. Immunol. 18 (2006) 1759-1769 ). A variant of the anti-VEGF IgG1 antibody bevacizumab with eleven-fold increased FcRn affinity has been shown to have a five-fold extended half-life in human FcRn transgenic mice and three-fold longer half-life in cynomolgus monkeys (Zalevsky, J. et al, Nat. Biotechnol. 28 (2010) 157-159).
[0182] It has been found that analysis and removal of antibodies by half in IgG preparations can be achieved using an FcRn column as reported herein. An example of FcRn column chromatography is shown in Figure 3.
[0183] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand for removing antibodies by half of IgG preparations.
[0184] It has been found that oligomers and aggregates can be separated by means of FcRn chromatography as reported herein (see Figure 4).
[0185] One aspect reported herein is the use of a chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand for removal of antibody aggregates and antibody oligomers from preparations of IG G.
[0186] It was found that the retention time is influenced by the number of Fc parts comprised in the analyte molecule. This has been demonstrated using constructs that contain one or two Fc parts (see Figure 5).
[0187] It was found that the oxidation had an impact on the binding of FcRn and could be observed on the FcRn column (see Figure 6).
[0188] The antibody format has been shown to have no impact on binding to the FcRn column. This has been demonstrated for the knob-in-hole format and for several bispecific antibody formats. In this way, the FcRn column can be used for evaluating new antibody formats.
[0189] In one embodiment, the complex is monobiotinylated.
[0190] In one embodiment, chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand has stability of at least one hundred cycles in the methods and uses reported herein. A cycle is a pH gradient from the first pH value to the second pH value of the corresponding method or use, where, for material regeneration, no further change of conditions is required beyond the final conditions of the method or use. Thus, in one embodiment, a cycle is a pH gradient from a pH value of about pH 5.5 to a pH value of about pH 8.8.
[0191] An amino acid change from M to H at position 252 in combination with an amino acid change from M to E at position 428 has been found to result in reduced retention time (see Figure 7).
[0192] Antibody sample with Met252 and Met428 oxidation products illustrates the difference between the SPR method and FcRn affinity chromatography. Although SPR analysis of the samples detected a relative difference in sample binding of about 20% compared to the reference standard, it did not provide details of the heterogeneity of this sample. On the other hand, FcRn affinity chromatography of the same sample exhibited two distinct peaks, one at the retention time of the reference standard and the second peak significantly shifted to the left, indicating weaker interaction of the antibody in the stressed sample with the material from FcRn column at lower pH.
[0193] A chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand, as reported herein, can be used for isolation/separation of antibody fragments and thus provides alternative to conventional Protein A affinity chromatography. Furthermore, using the chromatography material reported herein, the separation can be carried out under more physiological conditions, such as pH value, compared to conventional Protein A affinity chromatography.
[0194] Chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand can be used for determination/separation/enrichment of antibody species that comprise modifications such as oxidation, variants of loading, glycosylation and deamidation. Chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand can be used depending on the selected pH gradient (initial/final pH value) for enrichment of certain species of antibodies .
[0195] Chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a ligand can be used for isolation/enrichment of antibody species by molecular weight variation/difference.
[0196] Chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a linker can be used for the isolation/enrichment of antibodies by the number of FcRn binding site on the molecule.
[0197] Chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a linker can be used for the isolation of amino acid modifications. Chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as a linker can be used for isolation/separation of bispecific antibody mismatches such as hole-hole dimers and halved antibodies. SPECIFIC PERFORMANCE 1. Use of immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) as affinity chromatography ligand in affinity chromatography with a positive linear pH gradient. 2. Use according to item 1, characterized in that it is in affinity chromatography with positive linear pH gradient to separate antibodies or fusion polypeptides comprising at least one Fc region. 3. Use according to any of items 1 to 2, characterized in that the neonatal Fc receptor and beta-2-microglobulin are, independently of each other, of human origin, or originated from mice, or originated from cynomolgus, or originated from rats or originated from rabbit. 4. Use according to any of items 1 to 3, characterized in that beta-2-microglobulin is of the same species as the neonatal Fc receptor. 5. Use according to any of items 1 to 4, characterized in that neonatal Fc receptor and beta-2-microglobulin are wild-type human neonatal Fc receptor and human wild-type beta-2-microglobulin each, independently between itself, with zero to ten amino acid residue modifications. 6. Use according to any of items 1 to 5, characterized in that the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase. 7. Use according to item 6, characterized in that the solid phase is a chromatography material. 8. Use according to either item 6 or 7, characterized in that the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 9. Use according to any of items 1 to 8, characterized in that the pH gradient is from a first pH value to a second pH value, so that the first pH value is from about pH 3.5 to about pH 7.5 and the second pH value is from about pH 6.0 to about pH 9.5. 10. Use according to any of items 1 to 9, characterized in that the first pH value is about pH 5.5 and the second pH value is about pH 8.8. 11. Use according to any of items 1 to 10, characterized in that the use is intended for the determination of the in vivo half-life of an antibody by means of determining the ratio of the retention times of the antibody and a reference antibody. 12. Use according to any of items 1 to 10, characterized in that the use is intended for the determination of the methionine oxidation of an antibody. 13. Use according to any of items 1 to 10, characterized in that the use is intended to determine the level of oligomerization of an antibody. 14. Use according to any of items 1 to 10, characterized in that the use is for selection of a library of modified antibodies or modified fusion polypeptides from parental antibodies or parental fusion polypeptides, which comprise at least a binding part of FcRn of an Fc region for those modified antibodies or modified fusion polypeptides that have altered binding affinity for FcRn compared to the parent antibody or parent fusion polypeptide. 15. Use according to any of items 1 to 10, characterized in that the use is for the identification of antibodies or fusion polypeptides that comprise at least an FcRn binding part of an Fc region that exhibit altered binding to the neonatal Fc receptor . 16. Use according to any of items 1 to 10, characterized in that the use is intended to remove antibodies by half of IgG preparations. 17. Use according to any of items 1 to 10, characterized in that the use is for the removal of antibody aggregates and antibody oligomers from IgG preparations. 18. Use according to any of items 1 to 17, characterized in that the antibody is a monospecific antibody or fusion polypeptide antibody fragment or a bispecific antibody or fusion polypeptide antibody fragment, or a trispecific antibody or antibody fragment of fusion polypeptide, or tetraspecific antibody or antibody fragment of fusion polypeptide. 19. Use of immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) as affinity chromatography binder in affinity chromatography with negative linear pH gradient. 20. Use according to item 19, characterized in that it is in affinity chromatography with a negative linear pH gradient to separate antibodies or fusion polypeptides which comprise at least one Fc region. 21. Use according to any of items 19 or 20, characterized in that the neonatal Fc receptor and beta-2-microglobulin are, independently of each other, of human origin, or mouse origin, or cynomolgus origin, or mouse origin or originated from rabbit. 22. Use according to any of items 19 to 21, characterized in that beta-2-microglobulin is of the same species as the neonatal Fc receptor. 23. Use according to any of items 19 to 22, characterized in that neonatal Fc receptor and beta-2-microglobulin are wild-type human neonatal Fc receptor and human wild-type beta-2-microglobulin each, independently between itself, with zero to ten amino acid residue modifications. 24. Use according to any of items 19 to 23, characterized in that the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase. 25. Use according to item 24, characterized in that the solid phase is a chromatography material. 26. Use according to either item 24 or 25, characterized in that the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin. 27. Use according to any of items 19 to 26, characterized in that the pH gradient is from a first pH value to a second pH value such that the first pH value is from about pH 7.0 to about pH 8.5 and the second pH value is from about pH 5.5 to about pH 6.9. 28. Use according to any of items 19 to 27, characterized in that the first pH value is about pH 7.4 and the second pH value is about pH 6.0. 29. Use according to any of items 19 to 28, characterized in that the use is intended for the determination of the in vivo half-life of an antibody by determining the ratio of the retention times of the antibody and a reference antibody. 30. Use according to any of items 19 to 28, characterized in that the use is for the determination of methionine oxidation of an antibody. 31. Use according to any of items 19 to 28, characterized in that the use is intended to determine the level of oligomerization of an antibody. 32. Use according to any of items 19 to 28, characterized in that the use is for the selection of a library of modified antibodies or modified fusion polypeptides from parental antibodies or parental fusion polypeptides, which comprise at least a binding part of FcRn of an Fc region for those modified antibodies or modified fusion polypeptides that have altered binding affinity for FcRn compared to the parent antibody or parent fusion polypeptide. 33. Use according to any of items 19 to 28, characterized in that the use is for the identification of antibodies or fusion polypeptides that comprise at least an FcRn binding part of an Fc region that exhibit altered binding to the neonatal Fc receptor . 34. Use according to any of items 19 to 28, characterized in that the use is for the removal of antibodies by half of IgG preparations. 35. Use according to any of items 19 to 28, characterized in that the use is for the removal of antibody aggregates and antibody oligomers from IgG preparations. 36. Use according to any of items 19 to 35, characterized in that the antibody is a monospecific antibody or fusion polypeptide antibody fragment or a bispecific antibody or fusion polypeptide antibody fragment, or a trispecific antibody or antibody fragment of fusion polypeptide, or tetraspecific antibody or antibody fragment of fusion polypeptide. 37. Use according to any of items 1 to 10, 18, 19 to 28 and 35, characterized in that the use is intended for the separation of IgG1 subclass antibodies from IgG3 subclass antibodies. 38. Human IgG1 isotype Fc region variant in which the amino acid at position 252 is changed from methionine to histidine and the amino acid at position 428 is changed from methionine to glutamic acid. 1. ANTIBODY FRAGMENTS
[0198] In certain embodiments, a fusion polypeptide provided herein comprises an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments, as well as other fragments described below. For analysis of certain antibody fragments, see Hudson, P.J. et al, Nat. Med. 9 (2003) 129-134. For analysis of scFv fragments, see, for example, Plueckthun, A., in: The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), p. 269-315; see also WO 93/16185, US 5,571,894 and US 5,587,458. For discussion of Fab and F(ab')2 fragments which comprise saved receptor binding epitope residues and have a longer half-life in vivo, see US 5,869,046.
[0199] Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, for example, EP 0.404,097; WO 1993/01161; Hudson, P.J. et al, Nat. Med. 9 (2003) 129-134; and Holliger, P. et al, Proc. Natl. Academic Sci. U.S.A. 90 (1993) 6444-6448. Triabodies and tetrabodies are also described in Hudson, P.J. et al, Nat. Med. 9 (2003) 129-134.
[0200] Single domain antibodies are antibody fragments that comprise, in whole or in part, the heavy chain variable domain or, in whole or in part, the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham MA; see, for example, U.S. Patent No. 6,248,516 B1).
[0201] Antibody fragments can be made by a variety of methods, including, but not limited to, proteolytic digestion of an intact antibody, as well as production by recombinant host cells (such as E. coli or phage), as described herein. 2. CHIMERIC AND HUMANIZED ANTIBODIES
[0202] In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in US 4,816,567; and Morrison, S.L. et al, Proc. Natl. Academic Sci. U.S.A. 81 (1984) 6851-6855). In one example, a chimeric antibody comprises a non-human variable region (such as a variable region derived from mouse, rat, hamster, rabbit or non-human primate such as monkey) and a human constant region. In a further example, a chimeric antibody is a “class-switched” antibody in which the class or subclass has been altered from the parent antibody. Chimeric antibodies include their antigen binding fragments.
[0203] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, such as CDRs (or parts thereof), are derived from a non-human antibody and FRs (or parts thereof) are derived from human antibody sequences. A humanized antibody will optionally also comprise at least a part of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (such as the antibody from which the HVR residues are derived), for example, to restore or increase the specificity or affinity of antibodies. .
[0204] Humanized antibodies and the methods of their elaboration are analyzed, for example, in Almagro, J.C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633 and are further described, for example, in Riechmann, I. et al, Nature 332 (1988) 323-329; Queen, C. et al, Proc. Natl. Academic Sci. U.S.A. 86 (1989) 10029-10033; US 5,821,337, US 7,527,791, US 6,982,321 and US 7,087,409; Kashmiri, S.V. et al, Methods 36 (2005) 25-34 (describing SDR grafting (a-CDR)); Padlan, E.A., Mol. Immunol. 28 (1991) 489-498 (describing "new surface formation"); Dall'Acqua, W.F. et al, Methods 36 (2005): 4360 (describing "RF shifting"); and Osbourn, J. et al, Methods 36 (2005) 6168 and Klimka, A. et al, Br. J. Cancer 83 (2000) 252-260 (describing the "guided selection" approach to FR switching).
[0205] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method (see, for example, Sims, MJ et al, J. Immunol. 151 ( 1993) 2296-2308); framework regions derived from the consensus sequence of human antibodies from a specific subset of light or heavy chain variable regions (see, for example, Carter, P. et al, Proc. Natl. Acad. Sci. USA 89 (1992), 4285-4289; and Presta, LG et al, J. Immunol. 151 (1993) 2623-2632); human mature main-chain regions (that have undergone somatic mutation) or human germ lineage main-chain regions (see, for example, Almagro, J.C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions derived from selection FR libraries (see, for example, Baca, M. et al, J. Biol. Chem. 272 (1997) 10678-10684; and Rosok, MJ et al, J. Biol. Chem 271 (1996) 22611-22618). 3. HUMAN ANTIBODIES
[0206] In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using a variety of methods known in the art. Human antibodies are generally described in van Dijk, M.A. and van de Winkel, J.G., Curr. Opinion Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opinion Immunol. 20 (2008) 450-459.
[0207] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. These animals typically contain, in whole or in part, human immunoglobulin loci, which replace endogenous immunoglobulin loci or are extrachromosomally present or are randomly integrated into the animal's chromosomes. In these transgenic mice, endogenous immunoglobulin loci were generally deactivated. For analysis of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125. See also, for example, US 6,075,181 and US 6,150,584 which describe XENOMOUSE® technology; US 5,770,429 which describes the HuMab® technology; US 7,041,870 which describes K-M MOUSE® technology; and US 2007/0061900, which describes VelociMouse® technology. Human variable regions of intact antibodies generated by such animals can be further modified, for example, by combining with a different human constant region.
[0208] Human antibodies can also be made by hybridoma-based methods. Human myeloma and human/mouse heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (see, for example, Kozbor, D., J. Immunol. 133 (1984) 3001-3005; Brodeur, BR et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987), pp. 51-63; and Boerner, P. et al, J. Immunol. 147 (1991) 86-95). Human antibodies generated by means of human B cell hybridoma technology are also described in Li, J. et al, Proc. Natl. Academic Sci. U.S.A. 103 (2006) 3557-3562. Additional methods include those described, for example, in US 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, J., Xiandai Mianyixue 26 (2006) 265-268 (which describes human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers, HP and Brandlein, S., Histology and Histopathology, 20 (2005) 927-937 and Vollmers, HP and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology, 27 (2005): 185-191.
[0209] Human antibodies can also be generated by isolating selected clone Fv variable domain sequences from human-derived phage display libraries. These variable domain sequences can then be combined with a desired human constant domain. Methods of selecting human antibodies from antibody libraries are described below. 4. LIBRARY DERIVED ANTIBODIES
[0210] Antibodies according to the present invention can be isolated by selecting combinatorial libraries in search of antibodies with the desired activity(ies). Several methods are known in the art, for example, to generate phage display libraries and to screen those libraries for antibodies that have the desired binding characteristics. Such methods are discussed, for example, in Hoogenboom, H.R. et al in Methods in Molecular Biology 178 (2002) 1-37 and further described, for example, in McCafferty, J. et al, Nature 348 (1990) 552-554; Clackson, T. et al, Nature 352 (1991) 624-628; Marks, J.D. et al, J. Mol. Biol. 222 (1992) 581-597; Marks, J.D. and Bradbury, A., Methods in Molecular Biology 248 (2003) 161-175; Sidhu, S.S. et al, J. Mol. Biol. 338 (2004) 299-310; Lee, C.V. et al, J. Mol. Biol. 340 (2004) 1073-1093; Fellouse, F.A., Proc. Natl. Academic Sci. U.S.A. 101 (2004) 12467-12472; and Lee, C.V. et al, J. Immunol. Methods 284 (2004) 119-132.
[0211] In certain phage display methods, VH and VL gene repertoires are cloned separately by means of polymerase chain reaction (PCR) and re-randomly combined into phage libraries, which can then be searched for phage of antigen binding as described in Winter, G. et al, Ann. Rev. Immunol. 12 (1994) 433-455. Phage typically displays antibody fragments, either in the form of single-stranded Fv fragments (scFv) or Fab fragments. Libraries from immunized sources provide antibodies with high affinity to the immunogen without the need for hybridoma construction. Alternatively, the native repertoire can be cloned (eg, from humans) to provide a single source of antibodies to a wide variety of self and non-self antigens without any immunization, as described by Griffiths, AD et al, EMBO J. , 12 (1993) 725-734. Finally, native libraries can also be constructed synthetically by cloning unrearranged V gene segments from stem cells, using PCR primers that contain random sequence to encode the highly variable CDR3 regions and perform rearrangement in vitro as described by Hoogenboom , HR and Winter, G., J. Mol. Biol. 227 (1992) 381-388. Patent publications describing human antibody phage libraries include, for example: US 5,750,373 , US 2005/0079574 , US 2005/0119455 , US 2005/0266000 , US 2007/0117126 , US 2007/0160598 , US 2007/0237764 , US 2007/0292936 and US 2009/0002360.
[0212] Antibodies or antibody fragments isolated from human antibody libraries are considered to be human antibodies or human antibody fragments herein. 5. MULTISPECIFIC ANTIBODIES
[0213] In certain embodiments, an antibody provided herein is a multispecific antibody, such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, bispecific antibodies can bind to two different epitopes of the same antigen. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express the antigen. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
[0214] Methods of elaborating multispecific antibodies include, but are not limited to, recombinant co-expression of two pairs of immunoglobulin light and heavy chains that have different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537- 540, WO 93/08829 and Traunecker, A. et al, EMBO J. 10 (1991) 3655-3659) and "knob-in-hole" engineering (see, for example, US 5,731,168 ). Multispecific antibodies can also be elaborated by performing electrostatic targeting effects to elaborate heterodimeric antibody Fc molecules (WO 2009/089004); cross-linking two or more antibodies or fragments (see, for example, US 4,676,980 and Brennan, M. et al, Science 229 (1985) 81-83); use of leucine zippers to produce bispecific antibodies (see, for example, Kostelny, SA et al, J. Immunol. 148 (1992) 1547-1553; use of "diabody" technology to make bispecific antibody fragments (see, by example, Holliger, P. et al, Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); use of single-stranded Fv (sFv) dimers (see, for example, Gruber, M. et al. , J. Immunol. 152 (1994) 5368-5374); and preparation of trispecific antibodies as described, for example, in Tutt, A. et al, J. Immunol. 147 (1991) 60-69).
[0215] Also included herein are antibodies elaborated with three or more functional antigen binding sites, including "Octopus antibodies" (see, for example, US 2006/0025576).
[0216] The antibody or fragment herein also includes a "Dual-Action Fab" or "DAF" which comprises an antigen binding site that binds to different antigens (see, for example, US 2008/0069820).
[0217] The antibody or fragment of the present also includes multispecific antibodies described in WO 2009/080251 , WO 2009/080252 , WO 2009/080253 , WO 2009/080254 , WO 2010/112193 , WO 2010/115589 , WO 2010/136172 , WO 2010/145792 and WO 2010/145793. 6. ANTIBODY VARIANTS
[0218] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. It may be desirable, for example, to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the antibody amino acid sequences. Any combination of deletion, insertion and substitution can be done to arrive at the final construct, as long as the final construct has the desired characteristics, such as antigen binding. A. SUBSTITUTION, INSERTION AND EXCLUSION VARIANTS
[0219] In certain embodiments, antibody variants that contain one or more amino acid substitutions are provided. Sites of interest for replacement mutagenesis include HVRs and FRs. Examples of changes are provided in Table 1 under the heading "Examples of substitutions" and as further described below with reference to amino acid side chain classes. Conservative substitutions are shown in Table 1 under the heading “preferred substitutions”. Amino acid substitutions can be introduced into an antibody of interest and products are selected to determine a desired activity, such as retained/enhanced antigen binding, reduced immunogenicity, or increased ADCC or CDC. TABLE

[0220] Amino acids can be grouped according to common side chain properties: 1. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; 2. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3. acids: Asp, Glu; 4. basic: His, Lys, Arg; 5. residues that influence chain orientation: Gly, Pro; 6. aromatics: Trp, Tyr, Phe.
[0221] Non-conservative substitutions imply replacing a member of one of these classes with another class.
[0222] One type of substitution variant involves the substitution of one or more hypervariable region (HVR) residues from a parent antibody (eg, human or humanized antibody). Generally, the resulting variant(s) selected for further study will contain modifications (such as improvements) in certain biological properties (such as increased affinity and reduced immunogenicity) with respect to the antibody parental and/or will possess certain biological properties substantially retained from the parental antibody. An example of a substitution variant is an affinity matured antibody, which may conveniently be generated using, for example, phage display based affinity maturation methods such as those described herein. Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and selected for a specific biological activity (such as binding affinity).
[0223] Changes (such as substitutions) in HVRs can be made, for example, to increase antibody affinity. These changes can be made at HVR “hot spots”, that is, residues encoded by codons that mutate at high frequency during the somatic maturation process (see, for example, Chowdhury, PS, Methods Mol. Biol. 207 (2008) ) 179-196) and/or SDRs (a-CDRs), wherein the resulting variant VH or VL is tested to determine binding affinity. Affinity maturation through the construction and reselection of secondary libraries has been described, for example, in Hoogenboom, H.R. et al in Methods in Molecular Biology 178 (2002) 1-37. In some affinity maturation embodiments, diversity is introduced into variable genes selected for maturation by any of a number of methods (such as error-proof PCR, strand alteration, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then selected to identify any antibody variants with the desired affinity. Another method of introducing diversity involves HVR-targeted approaches, in which several HVR residues (such as four to six residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning modeling or mutagenesis. CDR-H3 and CDR-L3 are particularly frequently targeted.
[0224] In certain embodiments, substitutions, insertions, or deletions may occur in up to one or more HVRs, provided such changes do not substantially reduce the antibody's ability to bind antigen. Conservative changes, for example (such as conservative substitutions as provided herein), that do not substantially reduce binding affinity can be made in HVRs. These changes may be outside the “hot spots” of HVR or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two or three amino acid substitutions.
[0225] A useful method of identifying certain residues or regions of the antibody that can be targeted for mutagenesis is termed "alanine scanning mutagenesis", as described by Cunningham, BC and Wells, JA, Science 244 (1989), 1081- 1085. In that method, a desired residue or group of residues (e.g., charged residues such as arg, asp, his, lys and glu) is identified and replaced with a neutral or negatively charged amino acid (such as alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions can be introduced at amino acid sites that demonstrate functional sensitivity to the initial substitutions. Alternatively or additionally, a crystal structure of the antigen-antibody complex identifies points of contact between the antibody and the antigen. These contact debris and neighboring debris can be directed or disposed of for possible replacement. Variants can be selected to determine if they contain the desired properties.
[0226] Amino acid sequence insertions include carboxyl and/or amino terminal fusions ranging in length from one residue to polypeptides that contain one hundred or more residues, as well as intrasequential insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusion at the N- or C-terminus of the antibody to an enzyme (for example, to ADEPT) or a polypeptide that increases the half-life of the antibody in serum. B. GLYCOSYLATION VARIANTS
[0227] In certain embodiments, an antibody provided herein is altered to increase or decrease the extent of antibody glycosylation. The addition or deletion of glycosylation sites to an antibody may conveniently be accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.
[0228] When the antibody comprises an Fc region, the carbohydrate bound to it can be altered. Native antibodies produced by mammalian cells typically comprise a branched biantennary oligosaccharide that is generally linked by an N-bond to Asn297 of the CH2 domain of the Fc region. See, for example, Wright, A. and Morrison, S.L., TIBTECH 15 (1997) 26-32. The oligosaccharide can include various carbohydrates, such as mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose linked to a GlcNAc on the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody in accordance with the present invention can be made to create antibody variants with certain enhanced properties.
[0229] In one embodiment, antibody variants are provided that have a carbohydrate structure that does not contain fucose linked (directly or indirectly) to an Fc region. The amount of fucose in that antibody can be, for example, from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297, relative to the sum of all glycostructures linked to Asn 297 (such as complex and hybrid high-mannose structures) as measured by MALDI-TOF mass spectrometry means as described, for example, in WO 2008/077546. Asn297 designates the asparagine residue located near position 297 in the Fc region (Fc region residue numbering EU); Asn297 can also be located, however, about three amino acids up or down in flow from position 297, that is, between positions 294 and 300, due to minor antibody sequence variations. These fucosylation variants may have enhanced ADCC function. See, for example, US 2003/0157108 and US 2004/0093621. Examples of publications relating to "defucosylated" or "fucose deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki, A. et al, J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al, Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines capable of producing defucosylated antibodies include CHO Lec13 cells deficient in protein fucosylation (Ripka, J. et al, Arch. Biochem. Biophys. 249 (1986) 533-545; US 2003/0157108; and WO 2004/056312, especially in Example 11) and knockout cell lines such as alpha-1,6-fucosyltransferase gene, FUT8, CHO knockout cells (see, for example, Yamane-Ohnuki, N. et al, Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y. et al, Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).
[0230] Antibody variants additionally have bisected oligosaccharides, such as in which a biantennary oligosaccharide linked to the Fc region of the antibody is bisected by GlcNAc. These antibody variants may have reduced fucosylation and/or enhanced ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878, US 6,602,684 and US 2005/0123546. Antibody variants with at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. These antibody variants may have enhanced CDC function. Such antibody variants are described, for example, in WO 1997/30087, WO 1998/58964 and WO 1999/22764. 7. REGION FC VARIANTS
[0231] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, so as to generate an Fc region variant. The Fc region variant may comprise a human Fc region sequence (such as human IgG1, IgG2, IgG3 or IgG4 Fc region) that comprises an amino acid modification (such as substitution) at one or more amino acid positions.
[0232] In certain embodiments, the present invention contemplates an antibody variant that possesses some but not all of the effector functions, making it a desirable candidate for applications in which the antibody half-life in vivo is important, but certain effector functions (such as as a complement and ADCC) are unnecessary or harmful. In vitro and/or in vivo cytotoxicity tests can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. Fc receptor (FcR) binding assays, for example, can be conducted to ensure that the antibody does not exhibit FcR binding (thus likely not to exhibit ADCC activity), but retains FcRn binding capacity. The primary cells for mediating ADCC, NK cells, express only FCYRIII, while monocytes express FCYRI, FCYRII and FCYRIII. FcR expression on hematopoietic cells is summarized in Table 3, p. 464, by Ravetch, J.V. and Kinet, J.P., Annu. Rev. Immunol. 9 (1991) 457-492. Non-limiting examples of in vitro tests to determine the ADCC activity of a molecule of interest are described in US 5,500,362 (see, for example, Hellstrom, I. et al, Proc. Natl. Acad. Sci. USA 83 (1986) ) 7059-7063; and Hellstsrom, I. et al, Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502); US 5,821,337 (see Bruggemann, M. et al, J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive test methods may be employed (see, for example, ACTI® non-radioactive cytotoxicity test for flow cytometry (CellTechnology, Inc., Mountain View CA; and CytoTox 96® non-radioactive cytotoxicity test) (Promega, Madison WI). Useful effector cells for these tests include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be determined in vivo, such as in an animal model such as that described in Clynes, R. et al, Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. C1q binding assays can also be conducted to confirm that the antibody is unable to bind C1q and, therefore, it does not show CDC activity See, for example, C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To determine complement activation, a CDC test can be performed (see, for example, Gazzano-Santoro , H. et al, J. Imm oneol. Methods 202 (1996) 163-171; Cragg, M.S. et al, Blood 101 (2003) 1045-1052; and Cragg, M.S. and M.J. Glennie, Blood 103 (2004) 2738-2743). Half-life/in vivo release and FcRn binding determinations can also be performed using methods known in the art (see, for example, Petkova, S.B. et al, Int. Immunol. 18 (2006) 1759-1769).
[0233] Antibodies with reduced effector function include those with substitution of one or more of the Fc region residues 238, 265, 269, 270, 297, 327 and 329 (US 6,737,056). These Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 for alanine (US 7,332,581 ).
[0234] Certain variants of antibodies with greater or lesser binding to FcRs are described (see, for example, US 6,737,056; WO 2004/056312; and Shields, RL et al, J. Biol. Chem. 276 (2001) 6591 -6604)
[0235] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that increase ADCC, such as substitutions at positions 298, 333 and/or 334 of the Fc region (EU residue numbering).
[0236] In some embodiments, changes are made in the Fc region that result in C1q binding and/or altered (i.e., increased or decreased) Complement Dependent Cytotoxicity (CDC) as described, for example, in US 6,194,551 , WO 99/51642 and Idusogie, EE et al, J. Immunol. 164 (2000) 4178-4184.
[0237] Antibodies with longer half-lives and enhanced binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer, RL et al, J. Immunol. 117 (1976) 587-593; and Kim, JK et al, J. Immunol. 24 (1994) 2429-2434), are described in US 2005/0014934. These antibodies comprise an Fc region with one or more substitutions that enhance the binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of the Fc region residues: 238, 252, 253, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, such as replacement of the Fc region residue 434 (US 7,371,826).
[0238] See also Duncan, A.R. and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821; and WO 94/29351 with reference to other examples of Fc region variants. 8. ANTIBODY VARIANTS MADE WITH CYSTEINE
[0239] In certain embodiments, it may be desirable to create cysteine-elaborated antibodies, such as "thioMAbs", in which one or more residues of an antibody are replaced by cysteine residues. In specific embodiments, substituted residues occur at accessible sites on the antibody. By replacing these residues with cysteine, reactive thiol groups are therefore positioned at accessible sites on the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or drug and linker moieties, to create an immunoconjugate, as described additionally in the present. In certain embodiments, any one or more of the following residues may be substituted for cysteine: V205 (Kabat numbering) light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Antibodies elaborated with cysteine can be generated as described, for example, in US 7,521,541. 9. ANTIBODY DERIVATIVES
[0240] In certain embodiments, an antibody provided herein may be further modified so that it contains additional non-proteinaceous moieties that are known in the art and readily available. Suitable portions for derivatizing the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3, 6-trioxane, ethylene maleic anhydride copolymer, polyamino acids (random homopolymers or copolymers) and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide and ethylene oxide copolymers, polyoxyethylated polyols (such as such as glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The amount of polymers bound to the antibody may vary and if more than one polymer is bound they may be identical or different molecules. Generally, the amount and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, if the antibody derivative will be used in therapy under conditions defined etc.
[0241] In another embodiment, conjugates of an antibody and non-proteinaceous portion that can be selectively heated through exposure to radiation are provided. In one embodiment, the non-proteinaceous portion is a carbon nanotube (Kam, N.W. et al, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 11600-11605). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm ordinary cells, but that heat the non-proteinaceous portion to a temperature at which cells near the non-proteinaceous portion of antibody are killed. III. RECOMBINANT COMPOSITIONS AND METHODS
[0242] Methods of producing monoclonal antibodies were first reported by Kohler and Milstein (Nature 256 (1975) 495-497). The production of recombinant antibodies with myeloma cells by the stable introduction of antibody-encoding nucleic acid (DNA) has then been reported (see Oi et al, Proc. Natl. Acad. Sci. U.S.A. 80 (1983) 63516355).
[0243] Antibody encoding nucleic acid (either for the full length antibody or for the variable domains) can be isolated and sequenced using standard procedures from an antibody-producing cell. After isolation, the encoding nucleic acid can be placed into one or more expression vectors. If only the variable domain encoding nucleic acid is isolated, the expression vector also comprises a nucleic acid encoding the light chain and/or heavy chain constant region, respectively (see, for example, US 5,658,570). The expression vector can be transfected into prokaryotic (E. coli) or eukaryotic (CHO, HEK, BHK and SP2/0) host cells that otherwise do not secrete antibodies.
[0244] If the encoding nucleic acid is derived from a display library, such as a phage display library, a yeast display library, or generally a cell surface display library, it can be cloned directly in the expression vector.
[0245] Antibodies can be produced using recombinant compositions and methods, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such nucleic acid may encode an amino acid sequence that comprises the VL and/or amino acid sequence that comprises the VH of the antibody (such as the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (such as expression vectors) that comprise such nucleic acid are provided. In a further embodiment, a host cell that comprises such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector that comprises a nucleic acid that encodes an amino acid sequence that comprises the VL of the antibody and an amino acid sequence that comprises the VH of the antibody. antibody; or (2) a first vector that comprises a nucleic acid that encodes an amino acid sequence that comprises the VL of the antibody and a second vector that comprises a nucleic acid that encodes an amino acid sequence that comprises the VH of the antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (such as a Y0, NS0, Sp20 cell). In one embodiment, a method of making an antibody as reported herein is provided, wherein the method comprises culturing a host cell that comprises a nucleic acid encoding the antibody, as provided above, under conditions appropriate for expression of the antibody. antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
[0246] For recombinant production of an antibody as reported herein, nucleic acid encoding an antibody, as described above, is isolated and inserted into one or more vectors for cloning and/or further expression in a host cell. Such nucleic acid can be easily isolated and sequenced using conventional procedures (using, for example, oligonucleotide probes that are capable of specifically binding to genes encoding the antibody light and heavy chains).
[0247] Appropriate host cells for cloning or expressing antibody-encoding vectors include eukaryotic or prokaryotic cells described herein. Antibodies can be produced, for example, in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199 and US 5,840,523 (see also Charlton, KA in Methods in Molecular Biology, Vol. 248, Lo, BKC ( ed.), Human Press, Totowa NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
[0248] In addition to prokaryotes, eukaryotic microbes such as yeasts or filamentous fungi are appropriate expression or cloning hosts for antibody-encoding vectors, including yeast and fungal strains whose glycosylation processes have been "humanized", which results in the production of an antibody with a fully or partially human glycosylation pattern. See Gerngross, T.U., Nat. Biotech. 22 (2004) 14091414; and Li, H. et al, Nat. Biotech. 24 (2006) 210-215.
[0249] Appropriate host cells for glycosylated antibody expression are also derived from multicellular organisms (vertebrates and invertebrates). Examples of invertebrate cells include insect and plant cells. Numerous bacilloviral strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
[0250] Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (which describe PLANTIBODIES® technology for producing antibodies in transgenic plants).
[0251] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted for suspension culture may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lineage (293 or 293 cells as described, for example, in Graham, F.L. et al, J. Gen. Virol. 36 (1977) 59-74); hamster baby kidney (BHK) cells; mouse sertoli cells (TM4 cells as described, for example, in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather, JP et al, Annals NY Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells , including CHO DHFR- cells (Urlaub, G. et al, Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Human Press, Totowa NJ (2004), pp. 255-268. III. IMMUNOCONJUGATES
[0252] The present invention also provides immunoconjugates comprising an antibody as reported herein conjugate to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (such as protein toxins, enzymatically active toxins of origin bacterial, fungal, plant or animal, or fragments thereof) or radioactive isotopes.
[0253] In one embodiment, an immunoconjugate is a drug-antibody conjugate (ADC) in which an antibody is conjugated to one or more drugs, including, but not limited to, maytansinoid (see US 5,208,020, US 5,416,064 and EP 0.425,235 B1); an auristatin such as monomethyl auristatin DE and DF drug moieties (MMAE and MMAF) (see US 5,635,483, US 5,780,588 and US 7,498,298); dolastatin; calicheamicin or its derivatives (see US 5,712,374, US 5,714,586, US 5,739,116, US 5,767,285, US 5,770,701, US 5,770,710, US 5,773,001 and US 5,877,296; Hinman, LM et al. al, Cancer Res. 53 (1993) 3336-3342; and Lode, HN et al, Cancer Res. 58 (1998) 2925-2928 ); an anthracycline such as daunomycin or doxorubicin (see Kratz, F. et al, Curr. Med. Chem. 13 (2006) 477-523; Jeffrey, SC et al, Bioorg. Med. Chem. Lett. 16 (2006) 358- 362; Torgov, MY et al, Bioconjug. Chem. 16 (2005), 717-721; Nagy, A. et al, Proc. Natl. Acad. Sci. USA 97 (2000) 829-834); Dubowchik, G.M. et al, Bioorg. & Med. Chem. Letters 12 (2002) 1529-1532; King, H.D. et al, J. Med. Chem. 45 (2002) 4336-4343; and U.S. Patent No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, teetaxel and ortataxel; trichothecene; and CC1065.
[0254] In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, active non-diphtheria toxin binding fragments, exotoxin chain A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diantin proteins, American Phytolaca proteins (PAPI, PAPII and PAP-S), Momordica inhibitor charantia, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin and the trichothecenes.
[0255] In another embodiment, an immunoconjugate comprises an antibody as described herein, conjugated to a radioactive atom to form a radioconjugate. A number of radioactive isotopes are available for the production of radioconjugates. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the conjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, such as TC99m or I123, or centrifuge mark for nuclear magnetic resonance (NMR) imaging (also known as resonance imaging magnetic, MRI), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0256] Conjugates of an antibody and cytotoxic agent can be made using a series of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), cyclohexane-1-carboxylate of succinimidyl-4-(N-maleimidomethyl) (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl HCl adipimidate), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), compounds bisazido (such as bis(p-azidobenzoyl) hexanediamine), bisdiazonium derivatives (such as bis(p-diazoniobenzoyl)-ethylenediamine), diisocyanates (such as 2,6-diisocyanate toluene) and fluorine compounds bisactive (such as 1,5-difluoro-2,4-dinitrobenzene). Ricin immunotoxin can be prepared, for example, as described in Vitetta, E.S. et al, Science 238 (1987) 1098-1104. Carbon-14-labelled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugating radionucleotide to the antibody. See WO 94/11026. The ligand can be a “divisible ligand” that enables the release of a cytotoxic drug into the cell. For example, an acid labile linker, peptidase sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker can be used (Chari, RV et al, Cancer Res. 52 (1992) 127-131; US 5,208,020 ).
[0257] Immunoconjugates or ADCs expressly contemplate herein, but without limitation, conjugates prepared with cross-linking reagents, which include, but are not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB and SVSB (succinimidyl-(4-vinyl sulfone) benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, United States). IV. DIAGNOSTIC AND DETECTION METHODS AND COMPOSITIONS
[0258] In certain embodiments, any of the antibodies provided herein is useful for detecting the presence of its antigen in biological samples. The term “detection”, as used herein, encompasses either qualitative or quantitative detection. In certain embodiments, biological samples comprise a cell or tissue.
[0259] In one embodiment, an antibody as reported herein is provided for use in a detection or diagnostic method. In a further aspect, a method of detecting the presence of antigen in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an antibody as described herein under permissive conditions for binding the antibody to its antigen and detecting whether a complex is formed between the antibody and the antigen. This method can be an in vitro or an in vivo method.
[0260] In certain embodiments, labeled antibodies as reported herein are provided. Marks include, but are not limited to, marks or portions that are directly detected (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive marks), as well as portions, such as enzymes or ligands, that are indirectly detected, for example, through enzymatic reaction or molecular interaction. Examples of labels include, but are not limited to, 32P, 14C, 125I, 3H and 131I radioisotopes, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansil, umbelliferone, luciferases such as wave luciferase -lum and bacterial luciferase (US 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, such as glucose oxidase , galactose oxidase and glucose 6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, tags centrifuges, bacteriophage marks, stable free radicals and the like. V. PHARMACEUTICAL FORMULATIONS
[0261] Pharmaceutical formulations of an antibody as described herein are prepared by mixing that antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (Ed. ) (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable vehicles are generally non-toxic to patients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenolic, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about ten residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (such as protein and Zn complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Examples of pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins such as rhuPH20 (HYLENEX®, Bayer International, Inc.). Certain examples of sHASEGPs and methods of use, including rhuPH20, are described in published U.S. Patent Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.
[0262] Examples of lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006/044908, the latter formulations including a histidine acetate buffer.
[0263] The present formulation may also contain more than one active ingredient as necessary for the specific indication being treated, preferably those with complementary activities that do not harm each other. These active ingredients are suitably present in combination, in amounts that are effective for the intended purpose.
[0264] Active ingredients can be captured in microcapsules prepared, for example, by means of agglutination methods or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively, in drug delivery systems colloidal (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These methods are described in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (Ed.) (1980).
[0265] Sustained-release preparations can be manufactured. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of molded articles such as films or microcapsules.
[0266] The formulations to be used for in vivo administration are generally sterile. Sterility can easily be achieved, for example, by means of filtration through sterile filter membranes. SAW. INDUSTRIALIZED ITEMS
[0267] In another aspect of the present invention, an industrialized article is provided that contains materials useful for the treatment, prevention and/or diagnosis of the disorders described above. The manufactured article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, ampoules, syringes, intravenous solution bags etc. Containers can be formed from a variety of materials, such as glass or plastic. The container contains a composition which, by itself or when combined with another composition, is effective for the treatment, prevention and/or diagnosis of the condition and may have a sterile access port (the container may be, for example, a bag of intravenous solution or ampoule containing a cap pierceable by a hypodermic injection needle). At least one active agent of the composition is an antibody according to the present invention. The label or package insert indicates that the composition is used to treat the chosen condition. Furthermore, the manufactured article may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody in accordance with the present invention; and (b) a second container with a composition contained therein, wherein the composition comprises an additional cytotoxic or other therapeutic agent. The manufactured article of this embodiment of the present invention may further comprise a package insert indicating that the compositions can be used for the treatment of a specific condition. Alternatively or additionally, the manufactured article may further comprise a second (or third) container which comprises a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
[0268] It is understood that any of the above industrialized articles may include an immunoconjugate according to the present invention in place of or in addition to an antibody as reported herein.
[0269] The following examples, figures and sequences are provided to aid in understanding the present invention, the true scope of which is defined in the appended claims. It is understood that modifications can be made to the procedures described without departing from the spirit of the present invention. EXAMPLES METHODS ELECTROSPRAY IONIZATION MASS SPECTROMETRY (ESI-MS)
[0270] Protein portions (50 μg) were deglycosylated by adding 0.5 μl of N-Glycanase Plus (Roche) and sodium phosphate buffer (0.1 M, pH 7.1) to obtain a final volume of 115 µl sample. The mixture was incubated at 37 °C for 18 hours. Then, for reduction and denaturation, 60 μl of 0.5 M TCEP (Pierce) in 4 M guanidine * HCl (Pierce) and 50 μl of 8 M guanidine * HCl were added. The mixture was incubated at 37 °C for thirty minutes. Samples were desalted by size exclusion chromatography (Sepharose G-25, isocratic, 40% acetonitrile with 2% formic acid). ESI mass spectra (+ve) were recorded on a Q-TOF instrument (maXis, Bruker) equipped with a nano ESI source (TriVersa NanoMate, Advion). The MS parameter definitions were as follows: Transfer: Funnel RF, 400 Vpp; ISCID energy, 0 eV; RF Multipole, 400 Vpp; Quadripole: ion energy, 4.0 eV; low mass, 600 m/z; source: dry gas, 8 l/min; dry gas temperature, 160 °C; collision cell: collision energy, 10 eV; RF collision: 2000 Vpp; ion cooler: RF Ion Cooler, 300 Vpp; transfer time: 120 μs; Pre Puls storage, 10 μs; scanning range m/z 600 to 2000. For data evaluation, a domestic development software (Mass Analyzer) was used. FCRN SURFACE PLASMA RESONANCE ANALYSIS (SPR)
[0271] The binding properties of wild-type and mutant antibody to FcRn were analyzed by means of surface plasma resonance (SPR) technology using a BIAcore T100 instrument (BIAcore AB, Uppsala, Sweden). This system is well established for studying molecular interactions. It allows continuous real-time monitoring of binder/analyte bindings and therefore the determination of kinetic parameters in various test setups. The SPR technology is based on measuring the refractive index near the surface of a gold-plated biosensor chip. Refractive index alterations indicate mass changes on the surface caused by the interaction of immobilized ligand with analyte injected in solution. If molecules bind to a ligand immobilized on the surface, the mass increases; in case of dissociation, the mass decreases. In the current test, the FcRn receptor was immobilized on a BIAcore CM5 biosensor chip (GE Healthcare Bioscience, Uppsala, Sweden) via amine coupling to a level of 400 response units (RU). The test was conducted at room temperature with PBS, 0.05% Tween 20, pH 6.0 (GE Healthcare Bioscience) as the conduction and dilution buffer. 200 nM of oxidized or native antibody samples were injected at a flow rate of 50 µl/min at room temperature. The association time was 180 s and the dissociation phase took 360 s. Chip surface regeneration was achieved by a short injection of HBS-P, pH 8.0. The evaluation of SPR data was performed by comparing the biological reaction signal height at 180 s after injection and at 300 s after injection. The corresponding parameters are the maximum RU level (180 s after injection) and later stability (300 s after the end of injection). EXAMPLE 1 FCRN AFFINITY COLUMN PREPARATION FCRN EXPRESSION IN HEK293 CELLS
[0272] FcRn was transiently expressed by transfection of HEK293 cells with two plasmids containing the coding sequence of FcRn and beta-2-microglobulin. Transfected cells were grown in shake flasks at 36.5 °C, 120 rpm (5 cm shaker amplitude), 80% humidity and 7% CO2. Cells were diluted every two to three days to a density of 3 to 4*105 cells/ml.
[0273] For transient expression, a 14 l stainless steel bioreactor started with a culture volume of 8 to 36.5 °C, pH 7.0 ± 0.2, 35% pO2 (gassing with N2 and air, flow total gas of 200 ml/min-1) and stirrer speed of 100 to 400 rpm. When cell density reached 20*105 cells/ml, 10 mg of plasmid DNA (equimolar amounts of the two plasmids) was diluted in 400 ml of Opti-MEM (Invitrogen). 20 ml of 293fectin (Invitrogen) was added to this mixture, which was then incubated for fifteen minutes at room temperature and then transferred to the fermentor. From the following day, the cells were supplied with nutrients in continuous mode: a feeding solution was added at the rate of 500 ml per day and glucose as needed to maintain the level at more than 2 g/l. The supernatant was collected seven days after transfection using a swinging overhead centrifuge with one liter buckets: 4000 rpm for 90 minutes. The supernatant (13 l) was cleaned by a Sartobran P filter (0.45 µm + 0.2 µm, Sartorius) and the FcRn beta-2-microglobulin complex was purified from it. NEONATAL CF RECEIVER BIOTINYLATION
[0274] A soluble extracellular domain of His-Avi tag FcRn that was co-expressed with β2-microglobulin in HEK293 cells was biotinylated after purification as follows: 1.2 mg to 12 mg of FcRn/β2-microglobulin in 5 ml of 20 mM of sodium citrate buffer, pH 5.5 containing 150 mM KCl, 250 µl PBS and a complete protease inhibitor pellet (Roche Diagnostics GmbH, Mannheim, Germany) were biotinylated using Avidity's biotinylation kit according to manufacturer's instructions (Bulk BIRA). Biotinylation reaction was carried out at room temperature for one night. The modified protein was dialyzed against 20 mM sodium phosphate buffer comprising 150 mM NaCl, pH 7.5, at 4°C overnight to remove excess biotin. COUPLING TO STREPTAVIDIN SEPHAROSE
[0275] One gram of streptavidin sepharose (GE Healthcare) was added to the biotinylated and dialyzed receptor (1.2 to 12 mg of FcRn/β2-microglobulin, for standard analytical application 3 mg were chosen) and incubated for two hours with shaking . The receptor derivatized sepharose was loaded onto a 1 ml column of XK (GE Healthcare). EXAMPLE 2 CHROMATOGRAPHY USING THE FCRN Affinity Column
[0276] The receptor-derivatized sepharose was loaded onto a 1 ml XK column (GE Healthcare) and the FcRn column was then equilibrated with 20 mM 2-(N-morpholino)-ethanesulfonic acid (MES) buffer containing 150 mM NaCl, pH 5.5. CONDITIONS Column dimensions: 50 mm x 5 mm Bed height: 5 cm Load: 50 μg sample Equilibrium buffer: 20 mM MES with 150 mM NaCl, adjusted to pH 5.5 Elution buffer: 20 mM Tris /HCl with 150 mM NaCl, adjusted to pH 8.8 Elution: 7.5 CV Equilibrium Buffer in 30 CV 100% Elution Buffer, 10 CV Elution Buffer
[0277] Fusion protein samples or antibodies containing 50 to 100 µg of protein were adjusted to pH 5.5 and applied to the FcRn column using ÃKTA 10 XT explorer or Dionex Summit (Dionex, Idstein, Germany). The 5 cm bed height column was then washed with 5-10 column volumes of 20 mM MES, 150 mM NaCl, pH 5.5 equilibration buffer. Affinity-linked Fc-containing proteins were eluted with a pH gradient to 20 mM Tris/HCl, 150 mM NaCl, pH 8.8, in 30 column volumes. For complete elution of modified antibodies, the pH increased in the gradient to pH 8.8. The experiments were carried out at room temperature. The elution profile was obtained by continuous measurement of absorbance at 280 nm. The time taken for an analytical peak, X, to reach the detector after sample injection was called the retention time. EXAMPLE 3 CORRELATION BETWEEN RETENTION TIME ON THE FCRN COLUMN AND IN VIVO HALF LIFE
[0278] The in vivo half-life was measured in human FcRn transgenic C57BL/6J mice after intravenous administration alone of 10 mg/kg (n = 8) and compared with retention time on the FcRn column (see table) . Antibodies that exhibited further elution from the FcRn column were found to have a longer half-life in FcRn transgenic mice. TABLE
EXAMPLE 4 PURIFICATION OF HUMAN FCRN, MOUSE FCRN AND CYNOMOLGUS FCRN
[0279] Clarified supernatants containing hexahis tagged proteins were loaded onto a Ni-NTA affinity chromatography resin (Qiagen, Hanbrechtikon, Switzerland) at 4 °C. After washing steps with 20 mM sodium phosphate buffer comprising 500 mM NaCl at pH 7.4 and containing 20 mM, respectively 100 mM imidazole, proteins were eluted at a flow rate of 2 ml/min using batch elution with the same buffer containing 300 mM imidazole on an ÃKTA Prime chromatography system (Amersham Pharmacia Biotech, Uppsala, Sweden). Fractions were pooled and further purified in sodium phosphate buffer containing 500 mM NaCl on size exclusion chromatography (Superdex® 200, GE Healthcare, Zurich, Switzerland). Purified proteins were quantified using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE) and analyzed by SDS PAGE on 4-12% NuPAGE Bis-Tris gels in MES buffer under denaturation and reduction conditions. EXAMPLE 5 AFINITY COLUMN CHROMATOGRAPHS OF MOUSE AND CYNOMOLGUS FCRN
[0280] In the following table, retention times of examples of human antibodies on affinity columns comprising FcRn from Cynomolgus monkeys are given. Data were obtained using the following conditions: Elution buffer: 20 mM TRIS/HCl, 150 nM NaCl, pH 8.5. Further description: see Example 2. The expression mutant YTE indicates the triple mutant M252Y/S254T/T256E.

[0281] In the table below, retention times of examples of human antibodies to murine FcRn are given. Data were obtained using the following conditions: 1.2 mg of receptor coupled on 1 ml of Sepharose. Elution buffer: 20 mM TRIS/HCl, 150 nM NaCl, pH 8.5. Further description: see Example 2. YTE mutants are not included in this table as they could not have been eluted unless the pH of the elution buffer had been adjusted to 9.5.

[0282] The Cynomolgus FcRn affinity column behaves similarly to a human FcRn affinity column with respect to binding of humanized antibodies. On the other hand, the binding of humanized antibodies to the murine FcRn column is stronger than to the human FcRn affinity column, as can be seen by posterior retention. EXAMPLE 6 ANTIBODY FRAGMENT GENERATION
[0283] The F(ab')2 fragment and the Fc region fragment were prepared by splitting the full length antibody diluted 1:1 with 100 mM Tris, pH 8.0, adding 1 μg of Cysteine protease IdeS per 50 μg of antibody and incubation for two hours at 37 °C. The resulting division products F(ab')2 and Fc were separated on a size exclusion chromatography (SEC) column (Superdex 200, GE Healthcare, Zurich, Switzerland) using an ÃKTA Explorer chromatography system (GE Healthcare, Uppsala , Sweden) and peak fractions were pooled. Molecular weight standards on the same column served to identify the two split products based on their retention times.
[0284] Retention times for full-length antibodies varied markedly. On the other hand, the retention times of the corresponding Fc parts of virtually all tested antibodies did not differ from each other (<1%).
[0285] By using plasmin to split the full-length antibodies, the same findings were obtained (data not shown). EXAMPLE 7 CORRELATION BETWEEN RETENTION TIME ON THE FCRN COLUMN AND THE OXIDATION STATE
[0286] The influence of antibody oxidation on retention time in FcRn affinity chromatography was studied. Oxidation of an IgG1 antibody (1 mg/ml) was observed by storing the antibody at 40°C for two months. Unmodified and oxidized antibody samples were analyzed by FcRn affinity chromatography and FcRn surface plasma resonance (SPR) technology. Antibody oxidation was characterized by peptide mapping and electrospray ionization mass spectrometry (ESI-MS).
[0287] ESI-MS data revealed that about 50% of IgG1 antibody heavy chains were oxidized in the solvent and exhibited residues of Met252 and Met428 upon storage in buffer (20 mM His/His*HCl, 240 mM tre- halose and 0.02% polysorbate 20) under accelerated conditions at 40 °C for two months. When samples stressed at 40 °C containing oxidized antibody and samples stored at -80 °C and at 25 °C were applied to an affinity column on which FcRn was previously immobilized, two main peaks may be separated (Figure 8) . The second peak in Figure 8 which consists of two virtually superimposed curves with longer retention time corresponded to the elution time of unmodified antibody in samples stored at 25 °C and -80 °C, while the initial elution peak represented antibody oxidized by Met252 and Met428. The presence of Met252 and oxidized Met428 was supported by the 16 Da mass change detected in the ESI-MS stressed sample. Analysis of the stressed antibody sample (40 °C) by SPR on the BIAcore showed the same reaction pattern in the sensorgram of the FcRn chromatography with two different antibody species, namely wild-type antibody and antibody oxidized by Met252 and Met428 (Figure 9). EXAMPLE 8 CORRELATION OF RETENTION TIME ON FCRN COLUMN WITH AGGREGATE FORMATION
[0288] The influence of antibody aggregate formation on FcRn interaction in affinity chromatography was evaluated for an anti-IL3R alpha antibody. Aggregate and antibody monomer fractions were isolated by size exclusion chromatography (SEC) and pooling of the corresponding peaks. The FcRn interaction of the fractions was analyzed using affinity chromatography and SPR technology.
[0289] Size exclusion chromatography (SEC) was used to isolate three anti-IL13R alpha antibody fractions that contain anti-IL13R alpha antibody monomers and multimeric aggregates for FcRn column chromatography. Based on the peak areas of the chromatogram, the anti-IL13R alpha antibody sample used for FcRn affinity chromatography contained 57% monomers and 43% anti-IL13R alpha antibody aggregates. Analysis of the unfractionated sample on FcRn affinity chromatography revealed two main peaks with different retention times (Figure 10). The smallest peak with the longest retention time corresponded to the aggregated fraction while the main part of the fastest elution peak corresponded to the monomer fraction.
[0290] In surface plasma resonance (SPR) analysis, an anti-IL13R alpha antibody reference standard was compared with two different pooled fractions enriched for aggregates (set 1) and for monomers (set 2) and with the set native. The exact composition of the native batch and pooled fractions is described in the Table below. TABLE
[0291] Composition of pooled native and enriched fractions of an anti-IL13R alpha antibody.

[0292] The monomer enriched pool sample sensorgram 2 was closer to the anti-IL13R alpha antibody reference standard followed by the aggregate poor sample sensorgram. On the other hand, aggregate enriched pool 1 was characterized by almost twice as high binding as FcRn in the SPR analysis (Figure 11). EXAMPLE 9 PHARMACOKINETIC STUDY IN HUMAN FCRN MICE
[0293] All procedures were conducted in accordance with the guidelines of the Association for the Determination and Certification of Care of Laboratory Animals (www.aaalac.org). The study was authorized by the Regional Council of Oberbayern, Germany.
[0294] Male and female C57BL/6J mice (background); with mouse FcRn deficiency, but hemizygous transgenics for human FcRn (huFcRn (276) -/tg (30, 31) were used throughout the pharmacokinetic study.
[0295] At the time of administration, the animals weighed from 17 to 25 g. The corresponding antibody was provided as an injection of an isolated intravenous mixture via the tail vein. Due to the limited blood volume of mice, it was necessary that three groups of four male and four female animals each covered nine sampling times, that is, three sampling times per animal. Blood samples were taken in group 1 at five minutes, 24 hours and 336 hours, in group 2 in two hours, 168 hours and 504 hours and in group 3 at eight hours, 48 hours and 672 hours after administration. Blood samples of about 100 μl were obtained by retrobulbar puncture and stored at room temperature for sixty minutes to allow clot formation. Serum samples of at least 40 μl were obtained by centrifugation at 9300 x g at 4 °C for three minutes and immediately frozen and stored at -20 °C until testing.
[0296] Serum concentrations of the therapeutic human antibodies in murine serum were determined by means of an antigen-captured enzyme-linked immunosorbent assay (ELISA) specific for the antigen binding region (Fab) of the administered antibody and its variants. All reagents or samples were incubated at room temperature on a shaker at 400 rpm. Each wash step included three cycles. Briefly, streptavidin-coated microtiter plates were coated with biotinylated antibody diluted in assay buffer. After washing with phosphate-buffered saline polysorbate (Tween 20), serum samples at various dilutions were added and incubated for one hour. After washing, bound human therapeutic antibodies were detected by subsequent incubation with human FCY-specific monoclonal antibody Fab fragments conjugated to digoxigenin that do not cross-react with mouse IgG. After washing, an anti-digoxigenin antibody conjugated to horseradish peroxidase (HRP) was added and incubated for one hour. After washing, ABTS (2,2'-azino-di[3-ethylbenzo]thiazoline sulfonate; Roche Diagnostics, Germany) was added as HRP substrate to form a colored reaction product. Absorbance of the resulting reaction product was read at 405 nm with reference wavelength at 490 nm. All serum samples and positive or negative control samples were analyzed in two copies and calibrated against reference standard.
[0297] The pharmacokinetic parameters were calculated through non-compartmental analysis using the WinNonlin® pharmacokinetic evaluation program (Pharsight, St. Louis MO, United States), version 5.2.1. Briefly, the area under the time/concentration curve AUC(0-672) was calculated using a linear trapezoidal ruler (with linear interpolation) from time 0 to infinity. The apparent terminal half-life (T1/2) was derived from the equation: T1/2 = ln2 / Àz. Total body release (CL) was calculated as dose/AUC. Statistically significant differences in pharmacokinetic parameters between wild-type antibody and its variants were determined by means of ANOVA analysis.
[0298] The pharmacokinetic study in C57BL/6J mice deficient in mouse FcRn but transgenic hemizygous for human FcRn (huFcRn (276) -/tg) demonstrated that the YTE mutation increased the antibody pharmacokinetics (Figure 12). At the level of statistical significance, the YTE mutant showed 1.74 times higher release of AUC(0-672), 1.95 times slower release and 2.2 times longer terminal half-life compared to wild-type antibody ( Table). TABLE
[0299] Pharmacokinetic parameters for wild-type antibody and its triple mutant YTE obtained by non-behavioral analysis of serum concentrations measured by ELISA after a single injection of 10 mg/kg intravenous mixture to human FcRn transgenic mice. Mean ± standard deviation, n = 8 per group, ANOVA analysis of significance compared to wild-type antibody (+++, p < 0.001). AUC(0-672), area under the time curve and serum concentration from time 0 to 672 hours.

权利要求:
Claims (11)
[0001]
1. USE OF AN IMMOBILIZED NON-COVALENT COMPLEX of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m), characterized by being as an affinity chromatography ligand in an affinity chromatography with a positive linear pH gradient, to separate antibodies or fusion polypeptides comprising at least one Fc region, wherein the immobilized non-covalent complex of a neonatal Fc receptor and beta-2-microglobulin is linked to a chromatography material and the non-covalent complex is conjugated to the solid phase by means of of a specific binding pair in which the pH gradient is from a first pH value to a second pH value, so that the first pH value is from pH 3.5 to pH 6.4 and the second pH value is pH is 7.4 to pH 9.5, and wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is mono-biotinylated and the solid phase is derivatized with streptavidin.
[0002]
2. USE according to claim 1, characterized in that neonatal Fc receptor and beta-2-microglobulin are wild-type human neonatal Fc receptor and human wild-type beta-2-microglobulin each, independently of each other, with 0 to 10 amino acid residue modifications.
[0003]
Use according to any one of claims 1 to 2, characterized in that the pH gradient is from a first pH value to a second pH value, so that the first pH value is pH 5.5 and the second pH value is pH 8.8.
[0004]
Use according to any one of claims 1 to 3, characterized in that it is for the determination of the in vivo half-life of an antibody by means of determining the ratio of the retention times of the antibody and a reference antibody.
[0005]
Use according to any one of claims 1 to 3, characterized in that it is for determining the methionine oxidation of an antibody.
[0006]
Use according to any one of claims 1 to 3, characterized in that it is for determining the level of oligomerization of an antibody.
[0007]
Use according to any one of claims 1 to 3, characterized in that it is for selecting a library of modified antibodies or modified fusion polypeptides from a parental antibody or a parental fusion polypeptide, which comprise at least a part of FcRn binding of an Fc region to such modified antibodies or modified fusion polypeptides that have altered binding affinity for FcRn compared to the parent antibody or parent fusion polypeptide.
[0008]
Use according to any one of claims 1 to 3, characterized in that it is for the identification of antibodies or fusion polypeptides that comprise at least an FcRn binding part of an Fc region that exhibit altered binding to the neonatal Fc receptor.
[0009]
Use according to any one of claims 1 to 3, characterized in that it is for removing half-antibodies (halfantibodies) from IgG preparations.
[0010]
Use according to any one of claims 1 to 3, characterized in that it is for removing aggregates of antibodies and antibody oligomers from IgG preparations.
[0011]
Use according to any one of claims 1 to 10, characterized in that the antibody is a monospecific antibody or a fusion polypeptide antibody fragment, or a bispecific antibody or a fusion polypeptide antibody fragment, or a trispecific antibody or a fusion polypeptide antibody fragment, or a tetraspecific antibody or a fusion polypeptide antibody fragment.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US4816567A|1983-04-08|1989-03-28|Genentech, Inc.|Recombinant immunoglobin preparations|

---

US4737456A|1985-05-09|1988-04-12|Syntex Inc.|Reducing interference in ligand-receptor binding assays|

---

US4676980A|1985-09-23|1987-06-30|The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services|Target specific cross-linked heteroantibodies|

---

US4900660A|1985-11-25|1990-02-13|University Of Florida|Streptococcal fc rc|

---

US6548640B1|1986-03-27|2003-04-15|Btg International Limited|Altered antibodies|

---

IL85035D0|1987-01-08|1988-06-30|Int Genetic Eng|Polynucleotide molecule,a chimeric antibody with specificity for human b cell surface antigen,a process for the preparation and methods utilizing the same|

---

AU600575B2|1987-03-18|1990-08-16|Sb2, Inc.|Altered antibodies|

---

US5770701A|1987-10-30|1998-06-23|American Cyanamid Company|Process for preparing targeted forms of methyltrithio antitumor agents|

---

US5606040A|1987-10-30|1997-02-25|American Cyanamid Company|Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methyl-trithio group|

---

ES2052027T5|1988-11-11|2005-04-16|Medical Research Council|IMMUNOGLOBULINE VARIABLE DOMAIN SEQUENCE CLONING.|

---

US5169936A|1989-04-14|1992-12-08|Biogen, Inc.|Protein purification on immobilized metal affinity resins effected by elution using a weak ligand|

---

DE3920358A1|1989-06-22|1991-01-17|Behringwerke Ag|BISPECIFIC AND OLIGO-SPECIFIC, MONO- AND OLIGOVALENT ANTI-BODY CONSTRUCTS, THEIR PRODUCTION AND USE|

---

CA2026147C|1989-10-25|2006-02-07|Ravi J. Chari|Cytotoxic agents comprising maytansinoids and their therapeutic use|

---

US5208020A|1989-10-25|1993-05-04|Immunogen Inc.|Cytotoxic agents comprising maytansinoids and their therapeutic use|

---

US5959177A|1989-10-27|1999-09-28|The Scripps Research Institute|Transgenic plants expressing assembled secretory antibodies|

---

US6075181A|1990-01-12|2000-06-13|Abgenix, Inc.|Human antibodies derived from immunized xenomice|

---

US6150584A|1990-01-12|2000-11-21|Abgenix, Inc.|Human antibodies derived from immunized xenomice|

---

US5770429A|1990-08-29|1998-06-23|Genpharm International, Inc.|Transgenic non-human animals capable of producing heterologous antibodies|

---

WO1992009690A2|1990-12-03|1992-06-11|Genentech, Inc.|Enrichment method for variant proteins with altered binding properties|

---

US5571894A|1991-02-05|1996-11-05|Ciba-Geigy Corporation|Recombinant antibodies specific for a growth factor receptor|

---

DE69233254T2|1991-06-14|2004-09-16|Genentech, Inc., South San Francisco|Humanized Heregulin antibody|

---

GB9114948D0|1991-07-11|1991-08-28|Pfizer Ltd|Process for preparing sertraline intermediates|

---

MX9204374A|1991-07-25|1993-03-01|Idec Pharma Corp|RECOMBINANT ANTIBODY AND METHOD FOR ITS PRODUCTION.|

---

US7018809B1|1991-09-19|2006-03-28|Genentech, Inc.|Expression of functional antibody fragments|

---

US5587458A|1991-10-07|1996-12-24|Aronex Pharmaceuticals, Inc.|Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof|

---

WO1993008829A1|1991-11-04|1993-05-13|The Regents Of The University Of California|Compositions that mediate killing of hiv-infected cells|

---

AT503496T|1992-02-06|2011-04-15|Novartis Vaccines & Diagnostic|BIOSYNTHETIC BINDEPROTEIN FOR TUMOR MARKERS|

---

DE69329503T2|1992-11-13|2001-05-03|Idec Pharma Corp|Therapeutic use of chimeric and labeled antibodies directed against a differentiation antigen, the expression of which is restricted to human B lymphocyte, for the treatment of B cell lymphoma|

---

US5635483A|1992-12-03|1997-06-03|Arizona Board Of Regents Acting On Behalf Of Arizona State University|Tumor inhibiting tetrapeptide bearing modified phenethyl amides|

---

US5780588A|1993-01-26|1998-07-14|Arizona Board Of Regents|Elucidation and synthesis of selected pentapeptides|

---

AU691811B2|1993-06-16|1998-05-28|Celltech Therapeutics Limited|Antibodies|

---

US5773001A|1994-06-03|1998-06-30|American Cyanamid Company|Conjugates of methyltrithio antitumor agents and intermediates for their synthesis|

---

US5789199A|1994-11-03|1998-08-04|Genentech, Inc.|Process for bacterial production of polypeptides|

---

US5840523A|1995-03-01|1998-11-24|Genetech, Inc.|Methods and compositions for secretion of heterologous polypeptides|

---

US5731168A|1995-03-01|1998-03-24|Genentech, Inc.|Method for making heteromultimeric polypeptides|

---

US5869046A|1995-04-14|1999-02-09|Genentech, Inc.|Altered polypeptides with increased half-life|

---

US5712374A|1995-06-07|1998-01-27|American Cyanamid Company|Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates|

---

US5714586A|1995-06-07|1998-02-03|American Cyanamid Company|Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates|

---

US6267958B1|1995-07-27|2001-07-31|Genentech, Inc.|Protein formulation|

---

GB9603256D0|1996-02-16|1996-04-17|Wellcome Found|Antibodies|

---

US6171586B1|1997-06-13|2001-01-09|Genentech, Inc.|Antibody formulation|

---

ES2244066T3|1997-06-24|2005-12-01|Genentech, Inc.|PROCEDURE AND COMPOSITIONS OF GALACTOSILATED GLICOPROTEINS.|

---

JP2001521909A|1997-10-31|2001-11-13|ジェネンテク・インコーポレイテッド|Methods and compositions comprising glycoprotein glycoforms|

---

US6610833B1|1997-11-24|2003-08-26|The Institute For Human Genetics And Biochemistry|Monoclonal human natural antibodies|

---

PT1034298E|1997-12-05|2012-02-03|Scripps Research Inst|Humanization of murine antibody|

---

US6194551B1|1998-04-02|2001-02-27|Genentech, Inc.|Polypeptide variants|

---

CA2323757C|1998-04-02|2011-08-02|Genentech, Inc.|Antibody variants and fragments thereof|

---

DK1071700T3|1998-04-20|2010-06-07|Glycart Biotechnology Ag|Glycosylation modification of antibodies to enhance antibody-dependent cellular cytotoxicity|

---

DK1082341T3|1998-06-01|2011-03-28|Agensys Inc|Useful tumor antigen in the diagnosis and treatment of prostate and colon cancer|

---

US6040498A|1998-08-11|2000-03-21|North Caroline State University|Genetically engineered duckweed|

---

HU0104865A3|1999-01-15|2004-07-28|Genentech Inc|Polypeptide variants with altered effector function|

---

US6737056B1|1999-01-15|2004-05-18|Genentech, Inc.|Polypeptide variants with altered effector function|

---

EP2270147B2|1999-04-09|2020-07-22|Kyowa Kirin Co., Ltd.|Method for controlling the activity of immunologically functional molecule|

---

PT1222292E|1999-10-04|2005-11-30|Medicago Inc|METHOD FOR REGULATING THE TRANSCRIPTION OF EXOGENEOUS GENES IN THE PRESENCE OF NITROGEN|

---

US7125978B1|1999-10-04|2006-10-24|Medicago Inc.|Promoter for regulating expression of foreign genes|

---

WO2001029246A1|1999-10-19|2001-04-26|Kyowa Hakko Kogyo Co., Ltd.|Process for producing polypeptide|

---

AU784983B2|1999-12-15|2006-08-17|Genentech Inc.|Shotgun scanning, a combinatorial method for mapping functional protein epitopes|

---

AU767394C|1999-12-29|2005-04-21|Immunogen, Inc.|Cytotoxic agents comprising modified doxorubicins and daunorubicins and their therapeutic use|

---

LT2857516T|2000-04-11|2017-09-11|Genentech, Inc.|Multivalent antibodies and uses therefor|

---

US6946292B2|2000-10-06|2005-09-20|Kyowa Hakko Kogyo Co., Ltd.|Cells producing antibody compositions with increased antibody dependent cytotoxic activity|

---

MXPA03002974A|2000-10-06|2004-05-05|Kyowa Hakko Kogyo Kk|Cells producing antibody compositions.|

---

US7064191B2|2000-10-06|2006-06-20|Kyowa Hakko Kogyo Co., Ltd.|Process for purifying antibody|

---

US6596541B2|2000-10-31|2003-07-22|Regeneron Pharmaceuticals, Inc.|Methods of modifying eukaryotic cells|

---

JP3523245B1|2000-11-30|2004-04-26|メダレックス，インコーポレーテッド|Transgenic chromosome-introduced rodents for the production of human antibodies|

---

KR20040054669A|2001-08-03|2004-06-25|글리카트 바이오테크놀로지 아게|Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity|

---

DE60232265D1|2001-10-25|2009-06-18|Genentech Inc|GLYCOPROTEIN COMPOSITIONS|

---

US20040093621A1|2001-12-25|2004-05-13|Kyowa Hakko Kogyo Co., Ltd|Antibody composition which specifically binds to CD20|

---

AT503829T|2002-04-09|2011-04-15|Kyowa Hakko Kirin Co Ltd|CELL WITH REDUCED OR DELETED ACTIVITY OF A PROTEIN INVOLVED IN GDP FUCOSET TRANSPORT|

---

US20050031613A1|2002-04-09|2005-02-10|Kazuyasu Nakamura|Therapeutic agent for patients having human FcgammaRIIIa|

---

JPWO2003085107A1|2002-04-09|2005-08-11|協和醗酵工業株式会社|Genome-modified cells|

---

WO2003084569A1|2002-04-09|2003-10-16|Kyowa Hakko Kogyo Co., Ltd.|Drug containing antibody composition|

---

EP1498490A4|2002-04-09|2006-11-29|Kyowa Hakko Kogyo Kk|Process for producing antibody composition|

---

WO2003085119A1|2002-04-09|2003-10-16|Kyowa Hakko Kogyo Co., Ltd.|METHOD OF ENHANCING ACTIVITY OF ANTIBODY COMPOSITION OF BINDING TO FcϜ RECEPTOR IIIa|

---

JP4753578B2|2002-06-03|2011-08-24|ジェネンテック，インコーポレイテッド|Synthetic antibody phage library|

---

US7361740B2|2002-10-15|2008-04-22|Pdl Biopharma, Inc.|Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis|

---

HU227217B1|2002-12-16|2010-11-29|Genentech Inc|Immunoglobulin variants and uses thereof|

---

AU2004205631A1|2003-01-16|2004-08-05|Genentech, Inc.|Synthetic antibody phage libraries|

---

US20060104968A1|2003-03-05|2006-05-18|Halozyme, Inc.|Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases|

---

US7871607B2|2003-03-05|2011-01-18|Halozyme, Inc.|Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases|

---

WO2005035586A1|2003-10-08|2005-04-21|Kyowa Hakko Kogyo Co., Ltd.|Fused protein composition|

---

WO2005035778A1|2003-10-09|2005-04-21|Kyowa Hakko Kogyo Co., Ltd.|PROCESS FOR PRODUCING ANTIBODY COMPOSITION BY USING RNA INHIBITING THE FUNCTION OF α1,6-FUCOSYLTRANSFERASE|

---

AU2004287643C1|2003-11-05|2012-05-31|Roche Glycart Ag|CD20 antibodies with increased FC receptor binding affinity and effector function|

---

PT2489364E|2003-11-06|2015-04-16|Seattle Genetics Inc|Monomethylvaline compounds conjugated to antibodies|

---

WO2005047327A2|2003-11-12|2005-05-26|Biogen Idec Ma Inc.|NEONATAL Fc RECEPTOR -BINDING POLYPEPTIDE VARIANTS, DIMERIC Fc BINDING PROTEINS AND METHODS RELATED THERETO|

---

CN101124245A|2003-11-12|2008-02-13|比奥根艾迪克Ma公司|Neonatal Fc receptor -binding polypeptide variants, dimeric Fc binding proteins and methods related thereto|

---

WO2005053742A1|2003-12-04|2005-06-16|Kyowa Hakko Kogyo Co., Ltd.|Medicine containing antibody composition|

---

ES2527292T3|2004-03-31|2015-01-22|Genentech, Inc.|Humanized anti-TGF-beta antibodies|

---

US7785903B2|2004-04-09|2010-08-31|Genentech, Inc.|Variable domain library and uses|

---

ES2403055T3|2004-04-13|2013-05-13|F. Hoffmann-La Roche Ag|Anti-P-selectin antibodies|

---

BRPI0515230A|2004-08-19|2008-07-15|Genentech Inc|isolated polypeptides, antibodies and nucleic acids, compositions, expression vector, isolated host cells, method of producing an antibody, manufactured articles, methods of treatment and alleviating dysfunction, methods of making and selecting a polypeptide, antibody cd20 binding agent, isolated anti-her2 antibody and uses of an antibody|

---

TWI309240B|2004-09-17|2009-05-01|Hoffmann La Roche|Anti-ox40l antibodies|

---

AU2005286607B2|2004-09-23|2011-01-27|Genentech, Inc.|Cysteine engineered antibodies and conjugates|

---

JO3000B1|2004-10-20|2016-09-05|Genentech Inc|Antibody Formulations.|

---

ES2577292T3|2005-11-07|2016-07-14|Genentech, Inc.|Binding polypeptides with diversified VH / VL hypervariable sequences and consensus|

---

WO2007064919A2|2005-12-02|2007-06-07|Genentech, Inc.|Binding polypeptides with restricted diversity sequences|

---

EP2016101A2|2006-05-09|2009-01-21|Genentech, Inc.|Binding polypeptides with optimized scaffolds|

---

EP1878739A1|2006-07-14|2008-01-16|LEK Pharmaceuticals D.D.|One step IMAC purification of proteins|

---

EP2471816A1|2006-08-30|2012-07-04|Genentech, Inc.|Multispecific antibodies|

---

US20080226635A1|2006-12-22|2008-09-18|Hans Koll|Antibodies against insulin-like growth factor I receptor and uses thereof|

---

US20090075378A1|2007-02-20|2009-03-19|Anaptysbio, Inc.|Somatic hypermutation systems|

---

US8198409B2|2007-05-21|2012-06-12|Nomadic Bioscience Co., Ltd.|Polypeptide, an affinity chromatography material, and a method for separating and/or purifying immunoglobulin|

---

CN100592373C|2007-05-25|2010-02-24|群康科技有限公司|Liquid crystal panel drive device and its drive method|

---

PL2202245T3|2007-09-26|2017-02-28|Chugai Seiyaku Kabushiki Kaisha|Method of modifying isoelectric point of antibody via amino acid substitution in cdr|

---

US20090162359A1|2007-12-21|2009-06-25|Christian Klein|Bivalent, bispecific antibodies|

---

US8227577B2|2007-12-21|2012-07-24|Hoffman-La Roche Inc.|Bivalent, bispecific antibodies|

---

US9266967B2|2007-12-21|2016-02-23|Hoffmann-La Roche, Inc.|Bivalent, bispecific antibodies|

---

US8242247B2|2007-12-21|2012-08-14|Hoffmann-La Roche Inc.|Bivalent, bispecific antibodies|

---

ES2563027T3|2008-01-07|2016-03-10|Amgen Inc.|Method for manufacturing antibody Fc heterodimer molecules using electrostatic conduction effects|

---

US20120021484A1|2008-10-22|2012-01-26|Biogen Idec Ma Inc.|Recombinant FcRn and Variants Thereof for Purification of Fc-Containing Fusion Proteins|

---

BRPI1014089A2|2009-04-02|2016-04-19|Roche Glycart Ag|multispecific antibodies comprising full length antibodies and single chain fab fragments|

---

EP2417156B1|2009-04-07|2015-02-11|Roche Glycart AG|Trivalent, bispecific antibodies|

---

EP2435473B1|2009-05-27|2013-10-02|F.Hoffmann-La Roche Ag|Tri- or tetraspecific antibodies|

---

US9676845B2|2009-06-16|2017-06-13|Hoffmann-La Roche, Inc.|Bispecific antigen binding proteins|

---

US8703132B2|2009-06-18|2014-04-22|Hoffmann-La Roche, Inc.|Bispecific, tetravalent antigen binding proteins|

---

WO2011106272A1|2010-02-23|2011-09-01|Merck Sharp & Dohme Corp.|Novel binding assays useful in identifying antibodies with altered half-lives|

---

CN105949313B|2011-03-29|2021-06-15|罗切格利卡特公司|Antibody Fc variants|

---

EP2734537A1|2011-07-20|2014-05-28|Zepteon, Incorporated|Polypeptide separation methods|

---

JP6288332B2|2017-03-06|2018-03-07|東洋紡株式会社|Novel glucose dehydrogenase|SG10201605397PA|2011-07-01|2016-08-30|Hoffmann La Roche|Method For Separation Of Monomeric Polypeptides From Aggregated Polypeptides|

---

KR20160134687A|2014-03-21|2016-11-23|에프. 호프만-라 로슈 아게|In vitro prediction of in vivo half-life of antibodies|

---

EP3162895B1|2014-06-27|2020-11-25|Tosoh Corporation|Improved fc-binding protein, method for producing said protein, antibody adsorbent using said protein, and method for separating antibody using said adsorbent|

---

SG11201701423RA|2014-09-03|2017-03-30|Boehringer Ingelheim Int|Compound targeting il-23a and tnf-alpha and uses thereof|

---

CA2960797A1|2014-11-06|2016-05-12|F. Hoffmann-La Roche Ag|Fc-region variants with modified fcrn-binding and methods of use|

---

EP3322436A4|2015-07-23|2019-03-27|Boehringer Ingelheim International GmbH|Compound targeting il-23a and b-cell activating factorand uses thereof|

---

EP3371202A1|2015-11-04|2018-09-12|Biogen MA Inc.|Conjugation methods for modifying or immobilizing proteins|

---

JP2018039735A|2016-09-05|2018-03-15|東ソー株式会社|Method for separating antibodies having differing complement dependent cytotoxic activities|

---

JP6976315B2|2016-09-19|2021-12-08|エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft|Affinity chromatography based on complement factors|

---

CN110088135A|2016-12-20|2019-08-02|豪夫迈·罗氏有限公司|The combination treatment of anti-CD20/ AntiCD3 McAb bispecific antibody and 4-1BBagonist|

---

MX2019011770A|2017-04-03|2020-01-09|F Hoffmann-La Roche Ag|Immunoconjugates of an anti-pd-1 antibody with a mutant il-2 or with il-15.|

---

EP3606963A1|2017-04-03|2020-02-12|H. Hoffnabb-La Roche Ag|Antibodies binding to steap-1|

---

JP2020512814A|2017-04-03|2020-04-30|エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト|Immunoconjugate|

---

JP6871415B2|2017-04-05|2021-05-12|エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト|Anti-LAG3 antibody|

---

AU2018250875A1|2017-04-13|2019-10-03|F. Hoffmann-La Roche Ag|An interleukin-2 immunoconjugate, a CD40 agonist, and optionally a PD-1 axis binding antagonist for use in methods of treating cancer|

---

CN110573626A|2017-04-28|2019-12-13|豪夫迈·罗氏有限公司|Antibody selection method|

---

JP6977536B2|2017-12-19|2021-12-08|東ソー株式会社|Method for evaluating the degree of antibody denaturation using gel filtration chromatography|

---

TW201930356A|2017-12-21|2019-08-01|瑞士商赫孚孟拉羅股份公司|Antibodies binding to HLA-A2/WT1|

---

EP3731864A1|2017-12-29|2020-11-04|F. Hoffmann-La Roche SA|Anti-vegf antibodies and methods of use|

---

TW201936641A|2018-02-09|2019-09-16|瑞士商赫孚孟拉羅股份公司|Antibodies binding to GPRC5D|

---

AU2019236372A1|2018-03-13|2020-07-30|F. Hoffmann-La Roche Ag|Therapeutic combination of 4-1 BB agonists with anti-CD20 antibodies|

---

KR20200135510A|2018-03-29|2020-12-02|제넨테크, 인크.|Regulation of lactation stimulating activity in mammalian cells|

---

AR115052A1|2018-04-18|2020-11-25|Hoffmann La Roche|MULTI-SPECIFIC ANTIBODIES AND THE USE OF THEM|

---

JP2020118664A|2018-06-20|2020-08-06|東ソー株式会社|Antibody separation method and disease inspection method|

---

MA50586A|2018-08-09|2020-09-16|Regeneron Pharma|METHODS FOR EVALUATING THE BINDING AFFINITY OF AN ANTIBODY VARIANT TO THE NEONATAL FC RECEPTOR|

---

EP3870331A1|2018-10-25|2021-09-01|F. Hoffmann-La Roche AG|Modification of antibody fcrn binding|

---

CN113195056A|2018-12-21|2021-07-30|豪夫迈·罗氏有限公司|Antibodies that bind to CD3|

---

PE20211294A1|2018-12-21|2021-07-20|Hoffmann La Roche|ANTIBODY JOINING VEGF AND IL-1BETA AND METHODS OF USE|

---

CN109725159B|2018-12-28|2021-10-08|江苏众红生物工程创药研究院有限公司|Human beta2Quantitative detection test paper card of microglobulin and clinical application|

---

AU2020253023A1|2019-03-29|2021-09-23|F. Hoffmann-La Roche Ag|Method for the generation of an FcRn expressing cell by targeted integration of multiple expression cassettes in a defined organization|

---

WO2021001289A1|2019-07-02|2021-01-07|F. Hoffmann-La Roche Ag|Immunoconjugates comprising a mutant interleukin-2 and an anti-cd8 antibody|

---

WO2022008688A1|2020-07-10|2022-01-13|F. Hoffmann-La Roche Ag|Antibodies which bind to cancer cells and target radionuclides to said cells|

---

AU2020313285A1|2019-07-12|2022-02-17|F. Hoffmann-La Roche Ag|Antibodies which bind to cancer cells and target radionuclides to said cells|

---

WO2021009146A1|2019-07-15|2021-01-21|F. Hoffmann-La Roche Ag|Antibodies binding to nkg2d|

---

CA3144524A1|2019-07-31|2021-02-04|F. Hoffmann-La Roche Ag|Antibodies binding to gprc5d|

---

CN114174338A|2019-07-31|2022-03-11|豪夫迈·罗氏有限公司|Antibodies that bind to GPRC5D|

---

WO2021055577A2|2019-09-18|2021-03-25|Genentech, Inc.|Anti-klk7 antibodies, anti-klk5 antibodies, multispecific anti-klk5/klk7 antibodies, and methods of use|

---

WO2021122875A1|2019-12-18|2021-06-24|F. Hoffmann-La Roche Ag|Antibodies binding to hla-a2/mage-a4|

---

WO2021123173A1|2019-12-20|2021-06-24|F. Hoffmann-La Roche Ag|Il-37 fusion proteins and uses thereof|

---

US20210309989A1|2020-03-26|2021-10-07|Genentech, Inc.|Modified mammalian cells|

---

WO2021198034A1|2020-03-30|2021-10-07|F. Hoffmann-La Roche Ag|Antibody that binds to vegf and pdgf-b and methods of use|

---

WO2021209402A2|2020-04-15|2021-10-21|F. Hoffmann-La Roche Ag|Immunoconjugates|

---

WO2021249990A2|2020-06-08|2021-12-16|Hoffmann-La Roche Inc.|Anti-hbv antibodies and methods of use|

---

WO2021255143A1|2020-06-19|2021-12-23|F. Hoffmann-La Roche Ag|Antibodies binding to cd3 and folr1|

---

WO2021255155A1|2020-06-19|2021-12-23|F. Hoffmann-La Roche Ag|Antibodies binding to cd3 and cd19|

---

WO2021255146A1|2020-06-19|2021-12-23|F. Hoffmann-La Roche Ag|Antibodies binding to cd3 and cea|

---

US20220010015A1|2020-06-19|2022-01-13|Hoffmann-La Roche Inc.|Antibodies binding to cd3|

---

US20220041672A1|2020-06-24|2022-02-10|Genentech, Inc.|Apoptosis resistant cell lines|

---

WO2022016037A1|2020-07-17|2022-01-20|Genentech, Inc.|Anti-notch2 antibodies and methods of use|

---

WO2022047222A2|2020-08-28|2022-03-03|Genentech, Inc.|Crispr/cas9 multiplex knockout of host cell proteins|

法律状态:
2018-07-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-12-24| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-06-01| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-06-29| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/02/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP12155630|2012-02-15|

---

EP12155630.2|2012-02-15|

---

PCT/EP2013/052932|WO2013120929A1|2012-02-15|2013-02-14|Fc-receptor based affinity chromatography|

---