bispecific antibody molecules and their method of production, as well as pharmaceutical composition

专利摘要:
bispecific antibody molecules and their method of production, as well as pharmaceutical composition and nucleic acid molecule. the present invention relates to bispecific antibody molecules consisting of fab fragment comprising first binding site for first antigen, single chain fv fragment comprising second binding site for second antigen, and immunoglobulin ch2 domain, wherein the fab fragment and the single-chain fv fragment are linked via the ch2 domain, wherein at least one amino acid residue of the ch2 domain which is capable of mediating binding to fc receptors is absent or mutated, and wherein additionally one or more residue(s) ) amino acid(s) of sequence positions 226, 228 and 229, is(are) absent or mutated, as well as to the method of production of said bispecific antibody molecules. said bispecific antibody molecules can be used in the treatment of proliferative diseases such as tumors. the present invention further relates to tetrameric antibody molecule, pharmaceutical composition, nucleic acid molecule and the method of producing an antibody molecule.
公开号:BR112014015018A2
申请号:R112014015018-4
申请日:2012-11-12
公开日:2020-10-27
发明作者:Gundram Jung；Michael Durben；Ludger Grosse-Hovest
申请人:Synimmune Gmbh；
IPC主号:

专利说明:

[001] [001] This application claims the priority right of provisional United States patent application No. 61 / 577,327 filed with the US Patent and Trademark Office on December 19, 2011, the entire content of which is incorporated here, to this application for patent, for all purposes. INVENTION AREA!
[002] [002] The present invention relates to a bispecific antibody molecule, as well as a method for producing it, its use and a nucleic acid molecule encoding the bispecific antibody molecule. The invention in particular provides an antibody molecule that is able to mediate immune cell activation restricted to target cells. BACKGROUND
[003] [003] Monoclonal antibodies against the T cell receptor complex (antigen-specific TOCR / CD3 have the ability to effectively activate T cells. However, this activation requires that the antibody be - through its Fc portion - multimerized on the surface of cells expressing Fc receptor, which often also provides accessory signals for T cell activation (Davis, L., Vida, R. and Lipsky, PE, Regulation of human T Iymphocyte mitogenesis by antibodies to CD3, J. Immunol. [1986] 137: 3758-3767).
[004] [004] Bispecific antibodies, which recognize both an antigen on target cells (for example, FLT3 or CD19 on leukemia cells, the CSPGA4 antigen on melanoma cells or EGFR
[1988] [1988] 9: 257-260; Jung, G., Brandl, M., Eisner, W., Fraunberger, P., Reifenberger, G., Schlegel, U., Wiestler, OD, Reulen, HJ, Wilkins, W. Local immunotherapy of glioma patients with a combination of 2 bispecific antibody fragments and resting autologous Iymphocytes: evidence for in situ T-cell activation and therapeutic efficacy, Int J Cancer. [2001] 91: 225-30), and in addition to focusing activated cells on the target cell (Staerz, UD, Kanagawa, O., and Bevan, MJ, Hybrid antibodies can target sites for attack by T cells, Nature [ 1985] 314: 628-631; Perez, P., Hoffman, RW, Shaw, S., Bluestone, JA, and Segal, DM Specific targeting of cytotoxic T cells by anti-T3 linked to anti-target cell antibody, Nature [ 1985] 316: 354-356; Jung, G., Hon- sik, CJ, Reisfeld, RA and Muller-Eberhard, HJ Activation of human peripheral blood mononuclear cells by anti-T3: killing of tumor tar- get cells coated with anti-target-anti-T3 conjugates, Proc. Natl. Acad. Sci. USA, 83: 4479-4483, 1986). As a consequence, tumor cell lysis mediated by T cells occurs. Agonist antibodies to a co-stimulating T cell molecule such as CD28, reinforce anti-CD3 mediated T cell activation. The referred co-stimulatory antibodies are particularly effective if they are also provided in a bispecific format (Grosse-Hovest, L., Hartlapp, |., Marwan, W., Brem, G., Rammensee, HG, and Jung, G ., A recombinant bispecific single-chain antibody induces targeted, supra-agonistic CD28-stimulation and tumor cell killing, Eur. J. Immunol. [2003] 33: 1334-1340). In any case, we consider this an absolute requirement for therapeutic applications of bispecific antibodies.
[1988] [1988] 9: 257-260; Jung, G., Freimann, U., Von Marshall, Z., Reisfeld, R. A., and Wilmanns, W., Target cell-induced T cell activation with bi- and trispecific antibody fragments, Eur. J. Immunol. [1991] 21: 2431-2435). Binding to said Fc receptors would result in activation of T cells in vivo, which occurs, regardless of binding to a target antigen, at any location where cells expressing Fc receptors can be found, for example, within the entire hematopoietic, lymphatic and reticuloendothelial system. According to experience, the activation of referred T cells results in systemic activation of T cells, accompanied by a cytokine release syndrome, an adverse reaction feared during the therapeutic use of antibodies or T cells activating cytokines (Rosenberg, SA, Lotze, MT, Yang, JC, Aebersold, PM, Linehan, WM, Seipp, CA, and White, DE, Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients, Ann Surg. [1989] 210: 474-484; Tibben, JG, Boerman, OC, Massuger, LF, Schijf, CP, Claessens, RA, and Corstens, FH, Pharmacokinetics, biodistribution and bio-logical effects of intravenously administered bispecific monoclonal anti-body OC / TR F (ab ') 2 in ovarian carcinoma patients, Int. J. Cancer
[1996] [1996] 66: 477-483; Kroesen, B. J., Buter, J., Sleijfer, D. T., Janssen, R. A., van der Graaf, W. T., The, T.H., of, L.L. and Mulder, N. H., Phase | study of intravenously applied bispecific antibody in renal cell cancer patients receiving subcutaneous interleukin 2, Br. J. Cancer
[1994] [1994] 70: 652-661). Therefore, the objective in formatting bispecific CD3 antibodies needs to be to avoid systemic Fc-mediated T cell activation, thereby enabling restricted activation to target cells, which is exclusively dependent on binding of the bisected specific antibody to the target portion. corresponding target antigen (Jung, G., & Eberhard, HJ, An in-vitro model for tumor immunotherapy with antibody heteroconjugates, Immunol. Today [1988] 9: 257- 260; Jung, G., Freimann, U., Von Marshall, Z., Reisfeld, RA, and Wilmanns, W. Target cell-induced T cell activation with bi- and trispecific antibody fragments, Eur. J. Inmunol. [1991] 21: 2431-2435). From the above, it appears that when selecting the target antigen, care must be taken to express it as restricted to malignant cells as possible. In this way, activation by non-malignant cells and an accompanying release of cytokines can be kept as low as possible.
[005] [005] Similar considerations apply if bispecific antibodies are constructed that contain binding of agonistic effector antibodies to trigger receptors on immune cells other than T cells, such as CD16 expressed on NK cells. In any case, the Fc-mediated binding of antibodies to Fc receptors should be avoided according to the reasoning outlined above for T cells.
[006] [006] The bispecific antibody that has gone further in clinical development today is Blinatumomab (Micromet, Inc., Rockville, MD), a bispecific single chain antibody with specificity for CD19xCD3 and a remarkable therapeutic activity against cells from leukemia and lymphoma (Bargou, R., et al., Tumor regression in cancer patients by very low doses of a T cell-engaging antibody, Science [2008] 321: 974-977; Topp, MS, and others., Targeted therapy with the T-cell-engaging antibody blinatumomab of chemotherapy- refractory minimal residual disease in B-lineage acute Iymphoblastic leukemia patients results in high response rate and prolonged leukemi- a-free survival, J. Clin. Oncol. [2011] 29 : 2493-2498).
[007] [007] As the single chain format does not contain any domain from the Fc part, this antibody is restricted to target cells within the meaning explained above, that is, it only activates T cells in the presence of target cells expressing CD19 (Brischwein, K., et al., Strictly tar-get cell-dependent activation of T cells by bispecific single-chain anti-body constructs of the BIiTE class, J. Inmunother. [2007] 30: 798-807).
[008] [008] However, CD19 is also expressed on normal B cells so that, despite being restricted to target cells, after therapeutic application, a systemic release of cytokines occurs, causing significant cytotoxicity at daily doses of around 100 ug ( Bargou, R., et al., Tumor regression in cancer patients by very low doses of a T cell-engaging antibody, Science [2008] 321: 974-977; Topp, MS, et al., Targeted therapy with the T- cell-engaging anti-body blinatumomab of chemotherapy-refractory minimal residual dyseosis in B-lineage acute Iymphoblastic leukemia patients results in high response rate and prolonged leukemia-free survival, J. Clin. Oncol.
[2011] [2011] 29: 2493-2498).
[009] [009] Furthermore, the single chain format has the following disadvantages: (i) the molecular weight of about 50 kDa is relatively low and is associated with a short serum half-life, (ii) antibodies of this format easily aggregate and (iii) are difficult to produce in conventional fermentation processes (Grosse-Hovest, L., Hartlapp, |. Marwan, W., Brem, G., Rammensee, HG, and Jung, G., A recombi- nant bispecific single-chain antibody induces targeted, supra-agonistic CD28-stimulation and tumor cell killing, Eur. J. Immunol. [2003] 33: 1334-1340; Grosse-Hovest, L., et al., Cloned transgenic farm ani - mals produce a bispecific antibody for T cell-mediated tumor cell killing, Proc. Natl. Acad. Sci. USA [2004] 101: 6858-6863).
[0010] It is, therefore, an object of the present invention to provide a bispecific antibody molecule that overcomes at least some of the above difficulties and that can be generally used in therapy, among others for activation of immune cells strictly restricted to the target cells as described above. SUMMARY OF THE INVENTION
[0011] [0011] In a first aspect the present invention provides a recombinant bispecific antibody molecule. The recombinant bispecific antibody molecule consists of a Fab fragment, a single chain Fv fragment and an immunoglobulin CH2 domain. The Fab fragment includes a first binding site for a first antigen. The single-stranded Fv fragment includes a second binding site for a second antigen. The Fab fragment and the single chain Fv fragment are linked to each other via the CH2 domain. In typical embodiments, the cysteine residues forming heavy interchain disulphide bonds (C226 and C229 in immunoglobulins - human IgG) are exchanged.
[0012] [0012] In a second aspect the invention provides a tetrameric antibody molecule. The tetrameric antibody molecule includes a dimer of the antibody molecule according to the first aspect. The dimer is generally defined by a bond between cysteine residues of two antibody molecules of the first aspect, namely, between cysteines in the hinge region. The cysteine residues referred to are typically preserved amino acids (C226 and C229 in immunoglobulins - human IgG).
[0013] [0013] In a third aspect the invention provides a recombinant bispecific antibody molecule. The recombinant bispecific antibody molecule includes a Fab fragment that includes a first binding site for a first antigen, a single chain Fv fragment that includes a second binding site for a second antigen, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain. The Fab fragment and the single chain Fv fragment are linked via the CH2 domain / CH3 domain. At least one CH2 domain amino acid residue that has the ability to mediate binding to Fc receptors is missing or mutated. In typical embodiments of this aspect at least one of the cysteine residues forming inter-chain disulfide bonds (C226 and C229 in antibodies - human IgG) are exchanged. In some embodiments, the mentioned molecules may contain additional modifications in the CH3 region that prevent diffusion with homotypic CH3 domains.
[0014] [0014] In a fourth aspect the invention provides a tetrameric antibody molecule. The tetrameric antibody molecule consists of a dimer of the recombinant bispecific antibody molecule according to the third aspect. The dimer is usually defined by a link between preserved cysteines in the articulation region (C226 and C229 in antibodies - human IgG).
[0015] [0015] In a fifth aspect the invention provides an additional recombinant bispecific antibody molecule. This antibody molecule includes a Fab fragment including a first binding site for a first antigen, a single chain Fv fragment including a second binding site for a second antigen, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain. . The Fab fragment and the single chain Fv fragment are linked to each other via the CH2 domain and the CH3 domain. At least one cysteine residue from this antibody molecule that is capable of forming a disulfide bridge for dimerization is missing or mutated.
[0016] [0016] In a sixth aspect the invention provides a nucleic acid molecule. The nucleic acid molecule encodes an antibody molecule according to any one of the first, second, third, fourth or fifth aspect.
[0017] [0017] In a seventh aspect the invention provides a pharmaceutical composition. The pharmaceutical composition includes an antibody molecule according to one of the first, second, third, fourth or fifth aspect.
[0018] [0018] In an eighth aspect the invention provides a method of treating a disease. The method includes using an antibody molecule according to one of the first, second, third, fourth or fifth aspects. Usually the antibody molecule is administered to a patient who needs it.
[0019] [0019] In a ninth aspect the invention provides a host cell that includes a nucleic acid molecule according to the sixth aspect.
[0020] [0020] In a tenth aspect, the invention provides a method of producing an antibody molecule according to one of the first, second, third, fourth and fifth aspects. The method includes expression of a nucleic acid encoding the antibody molecule under conditions that allow expression of the nucleic acid molecule.
[0021] [0021] These aspects of the invention will be more fully understood in view of the description, drawings and non-limiting examples that follow. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] [0022] Fig. 1 schematically represents modalities of bispecific antibody molecules according to the invention.
[0023] [0023] Fig. 1A represents a bivalent molecule with a Fab fragment, a CH2 domain and a single chain Fv fragment. The antibody molecule has a backbone in which the CH2 domain is linked through its N-terminus to the heavy chain CH1 and VH domains of a Fab fragment and through its C-terminus to a Fv chain fragment (bsFc-1/2 format).
[0024] [0024] Fig. 1B represents a divalent antibody molecule with a backbone in which the CH2 domain is linked to the light chain of a Fab fragment, that is, in which the backbone includes a VL domain and a CL domain, a articulation region, a CH2 domain and a single chain Fv fragment.
[0025] [0025] Fig. 1C shows a divalent antibody molecule in which the main chain includes a VL domain and a CH1 domain, a hinge region, a CH2 domain and a single chain Fv fragment. A second lighter chain includes a VH domain and a CL domain. In the antibody molecule of Fig. 1C the Fab fragment is therefore not a “classic (naturally occurring) Fab” fragment in which the light and heavy chain variable domains are fused to their respective constant domain (CL or CH1, respectively) but a “hybrid” Fab fragment in which the variable domain is merged with the domain of the “opposite chain”, that is, the VH domain is merged with the CL domain and the VL domain is merged with the CH1 domain .
[0026] [0026] Fig. 1D represents a divalent antibody molecule with a backbone in which the CH2 domain is linked to a CL domain and a VH domain. A second, lower weight chain includes a VL domain and a CH1 domain. The antibody molecule of Fig. 1D therefore includes a "hybrid Fab fragment" (which includes the first binding site) since it is also present in the molecule of Fig.1C.
[0027] [0027] Fig. 1E represents a bivalent antibody molecule with a development as in Fig. 1A, in which amino acids in the CH2 domain and / or in the articulation region have been modified (indicated by “X” as represented in Fig. 10, bsFc format “º-1/2). Likewise, the mentioned modifications can be inserted in the molecules represented in 1B to 1D. In the molecules represented in Figs. 1A to 1E the cysteine residues that form interchain disulfide bonds (C226 and C229 in human IgG antibodies) are exchanged to prevent the formation of dimers (e).
[0028] [0028] Fig. 1F represents, as an illustrative modality, a tetravalent molecule that is a dimer of the unit represented in Fig. 1A. A similar molecule can also be constructed in the Fab configurations shown in Figs. 18 to 1D with and without
[0029] [0029] Fig. 1G represents, as an illustrative embodiment, a tetravalent molecule that is a dimer of a unit that includes a Fab fragment, a CH2 domain, a CH3 domain and a single chain Fv fragment. Amino acids in the CH2 domain and in the hinge region were modified (X); summarized in Fig. 1P. The two main chains of the antibody include a VH domain and a CH1 domain, a hinge region, a CH2 domain, a CH3 domain and a single chain Fv fragment (bsFc “* - 1 format). Similar molecules can also be constructed in the Fab configurations shown in Figs. 1A to 1E. In all of these molecules, dimers are defined by means of preserved cysteines in the articulation region (C226 and C229 in human IgG antibodies).
[0030] [0030] Fig. 1H represents a tetravalent molecule, which is a dimer of a unit that includes with a Fab fragment, a CH2 domain, a CH3 domain and a single chain Fv fragment. Within the Fab fragment the two main chains of the antibody include a VH domain and a CL domain.
[0031] [0031] Fig. 11 shows a tetravalent antibody with a general development as shown in Fig. 1G. In contrast to the modality of Fig. 1G, only one of the two main chains of this antibody includes amino acids in the CH2 domain and in the articulation region that have been modified (indicated by “X”).
[0032] [0032] Fig. 1J represents a tetravalent molecule in which the two main chains include a VL domain and a CL domain, a hinge region, a CH2 domain, a CH3 domain and a single chain Fv fragment.
[0033] [0033] Fig. 1K represents a tetravalent molecule with two structurally different Fab fragments. The first main chain of the antibody includes a VL domain and a CL domain, a pivot region, a CH2 domain, a CH3 domain and a single-chain Fv fragment. The second main strand of the antibody includes a VH domain and a CH1 domain, a hinge region, a CH2 domain, a CH3 domain and a single chain Fv fragment.
[0034] [0034] Fig. 1L represents a tetravalent molecule, which is a dimer of a unit that includes a Fab fragment, a CH2 domain, a CH3 domain and a single chain Fv fragment. Within the Fab fragment, the two main chains of the antibody include a VL domain and a CH1 domain.
[0035] [0035] Fig. 1M represents an additional tetravalent molecule with two structurally different Fab fragments. The first backbone of the antibody includes a VL domain and a CH1 domain, a hinge region, a CH2 domain, a CH3 domain and a single chain Fv fragment. The second main strand of the antibody includes a VH domain and a CL domain, a hinge region, a CH2 domain, a CH3 domain and a single chain Fv fragment.
[0036] [0036] Fig. 1N represents, as an illustrative embodiment, a bivalent molecule with a Fab fragment, a CH2 domain and a CH3 domain and a single chain Fv fragment. The antibody molecule has a backbone in which the CH2 domain is linked through its N-terminus to the heavy chain CH1 and VH domains of a Fab fragment and through its C-terminus to a CH3 domain which is read - cattle through its C-terminus to a single chain Fv fragment. A similar molecule can also be constructed in the Fab configurations shown in Figs. 1A to 1D and may contain modifications of Fc in the articulation region and CH2 (“X”) as shown in Figs. 1EÉ and 10. In addition, they may contain modifications in the CH3 domain that prevent dimerization of this domain and may influence the binding to the neonatal Fc receptor (FCcRn). Examples of waste
[0037] [0037] Additional illustrative modalities not shown in Figs. 1A to 1N include molecules where, in relation to the modalities represented, the C-terminal single-chain Fv part may be in a VL-VH orientation instead of the represented VH-VL orientation, meaning that the VL domain is merged with the respective constant domain.
[0038] [0038] Fig. 10 lists illustrative modifications that can be made to the divalent antibody variants shown in Figs.1A to 1D and Fig. 1N in order to obtain deficient Fc derivatives as exemplified in Fig. 1E. Modifications are identical to those shown in Fig. 1P with the exception of preserved cysteines (C226 and C229 in human IgG antibodies). The numbering of amino acids is in accordance with the numbering Kabat [European Union index (EU-Index)]. wt = human IgG1 wild-type sequence; [1 = no caute; Glycan = knockout of 1 with elimination of portions of saccharides [) 1297; [12-5 additional knockout variants in continuation of [D1; - = the amino acid has been eliminated.
[0039] [0039] Fig. 1P lists illustrative modifications that can be used to obtain a tetravalent molecule as shown in Fig. 1F to IM. The amino acid numbering is in accordance with the Kabat numbering [European Union index]. wt = human IgG1 wild sequence; / [11 = knockout; Glycan = knockout del) 1 with elimination of portions of saccharides [) [297; 12-5 additional rating variants in continuation of [) 1; - = the amino acid has been deleted.
[0040] [0040] Figs. 2A to 2C represent a schematic representation of the cloning procedure for the production of an optimized heavy chain (main chain) for the antibodies represented in Fig. 1, or as bivalent or tetravalent bispecific antibodies with Fc parts attenuated by modified ADCC. i) The original vector, based on the pcDNA3 plasmid backbone (Invitrogen; CMV promoter and bovine growth hormone termination signal are eliminated), is represented. This plasmid contains the Ig heavy chain of the human El isotype with regulatory elements of the immunoglobulin heavy chain locus. ii) The exchange of a VDJ (heavy chain variable domain) or VJ (light chain variable domain) element through the Aatll and Clal restriction endonuclease site is indicated. iii) Simple exchange (through restriction sites) is shown
[0041] [0041] In Fig. 2B and 2C) the regions adjacent to the inserted VDJ - CH1 and scFv elements, respectively, are shown in detail.
[0042] [0042] Figs. 2D-F represents a schematic representation of the cloning procedure for the production of the human monospecific antibody light chain. i) The parental vector, based on the pCR-Script plasmid backbone (Stratagene; lacZ promoter and termination signal are eliminated) contains the VJ region and the C region of the human I-gene as well as regulatory elements of the light chain locus immunoglobulin. ii) Exchange of a VJ element (variable domain of the light chain) or a VDJ element (variable domain of the heavy chain) through the restriction endonucleases Xho! and Spel.
[0043] [0043] In Figs. 2E and 2F the regions adjacent to the inserted VJ and CL elements are shown in detail.
[0044] [0044] Boxes represent exons; circles represent reinforcing elements and thin lines represent UT regions and intron sequences. L, and L ', leader sequences encoded by two different exons (also shown in Figures 2B and 2E); V, variable regions; D, region of diversity; J, joining regions; CH1, CH2, CH3, CL, exons of heavy and light constant chains, respectively, H, articulation region, scFv, single chain Fv fragment; X = amino acid changes. Notl, Aatll, Clal, Mlul, BspEl, Spel, Xhol, Kpnl, Xhol, Spel, Pmil, BsmBI, Sall, restriction endonucleases used for cloning; Amp " And Neo" represent the coding regions for resistance to Ampicillin and Neomycin respectively.
[0045] [0045] Cleavage sites for secretory signal peptides are indicated by |; and exon-intron limits by [, 1. |
[0046] [0046] Fig. 3A illustrates activation of T cells restricted to target cells (incorporation of * H-thymidine) by two bispecific antibodies of different format according to the invention, having specificity for FLT3 X CD3. The antibodies are used on cells that do not include (empty symbols) and that include (filled symbols) FLT3 / CD19 positive REH cells. 0, e: bivalent antibody molecule as shown in Fig. 1A with the sequence "Glycan" as shown in Figs. 1E and Fig. 10 Fab fragment (bsFc * º- 1/2 format) with FLT3 binding site, scFv fragment with CD3 binding site. 5, =: tetravalent antibody molecule as shown in Fig. 1G with the sequence [1 as shown in Fig. 1P, (bsFc “º-1 format). Fab fragment, with FLT3 binding site, scFv fragment with CD3 binding site. *: intact monospecific anti-CD3 antibody without target cells. In the absence of target cells, intact monospecific anti-CD3 antibodies effectively activate T cells in an Fc / FcR-dependent manner whereas bispecific antibodies are ineffective. This demonstrates that the bispecific shape of the invention lacks Fc / FcR binding as well as entirely. Fig. 3B illustrates the activation of T cells restricted to target cells (TNF release) by different bispecific bispecific antibodies according to the invention, used on cells that do not include (empty symbols) and that include ( filled symbols) FLT3 / CD19 positive REH cells. 0, e: bivalent antibody molecule as shown in Fig. 1A with the sequence "Glycan" as shown in Fig. 1E and Fig 10, Fab fragment with FLT3 binding site, scFv fragment with CD3 binding site; 0.4: bivalent antibody molecule as depicted in 1E with the "Glycan" sequence as depicted in Fig 10, Fab fragment with CD19 binding site, scFv fragment with TCR binding site; V, YV: bivalent antibody molecule as shown in Fig. 1E with the sequence “Glycan” as shown in Fig 10, Fab fragment with CSPGA binding site, scFv fragment with CD3 binding site. Chondroitin sulfate proteoglycan CSPG4 is a target antigen of melanoma cells and is not expressed on REH cells.
[0047] [0047] Fig. 4 represents the specific lysis of REH cells expressing FLT3 / CD19 (A) and SKMel63 cells expressing CSPG (B), respectively, by bispecific antibodies according to the invention and by positive T killer cells. for CD8 activated in a 4-hour chromium release test. e: FLT3 X CD3, bsFc “º-1/2 format as shown in Fig. 1E; =: FLT3 X CD3, bsFc “º * -1 format as shown in Fig. 1G; V: CSPG4 X CD3, bsFc “-1/2 format as shown in Fig. 1E; +: CD19 X TCR, for-
[0048] [0048] Fig. 5 shows a comparison of FLT3 X CD3 antibodies of identical specificity in three different formats: bispecific single chain format (bs-scFv), bsFc format “º-1/2 as represented in Fig. 1E , and bsFc “* - 1 format as shown in Fig. 1G. A: determination of aggregation (percentage values) by means of gel filtration. Aggregates are migrating close to the empty volume and are 43%, 0%, 2% for bs-scFv, bsFcko-1/2, bsFcko-1, respectively. It is concluded that the formation of aggregates is considerably more pronounced if the antibody is expressed as bs-scFv instead of bsFcko-1/2 or bsFcko-1. B: production rate after transfection of antibody genes into production and purification cells using affinity chromatography. As can be seen, the formation of aggregates is significantly reduced for the two formats of Fc “º according to the invention, and the production rates are substantially higher than with the bispecific single chain format (bs-scFv).
[0049] [0049] Fig. 6A shows the illustrative light chain sequences that can be included in an antibody of the invention. The respective peptide chains correspond to the mature protein without the corresponding leader peptide sequence. The strings contain a variable N-terminal domain represented in bold and a constant C-terminal domain represented in italics. The complementarity determining regions (CDRs) of the variable domain are underlined.
[0050] [0050] Fig. 6B represents the sequences of illustrative backbone chains, which in the present case can also be treated as heavy chains which can be included and are an antibody of the invention. This particular backbone for the bsFc-1/2 format (Figs 1E) includes a VH domain, a CH1 domain, a hinge region, a modified CH2 domain, a VL domain and a VH domain of an scFv fragment. In sequence example 21) (SEQ ID NO: 26) the main chain contains a CH3 domain as shown in Fig. 1G to 1M example (bsFc Ҽ-1 format).
[0051] [0051] The VH domains are represented in bold, the CH1 domain in regular text, and the articulation regions, CH2 and CH3 in regular, underlined text. The main chain additionally includes a VL domain, which is represented in bold and italic text, and a VH (bold) domain of an scFv fragment. The VH and VL domains are linked to each other through a chain, which is represented in italic text and underlined. Complementarity-determining residues (CDRs) from the respective VL and VH regions are underlined. The CH2 domain and the scFv fragment are linked to each other through a small linker (GQPSG), which is represented in italics. DETAILED DESCRIPTION
[0052] [0052] The present invention relates to a recombinant bispecific antibody molecule. This antibody molecule is composed of elements that are also found in native immunoglobulins, that is, naturally occurring, namely, heavy chain domains and immunoglobulin light chains.
[0053] [0053] The term "antibody" generally refers to a proteinaceous binding molecule with functions similar to immunoglobulins. Typical examples of an antibody are immunoglobulins, as well as derivatives or functional fragments of them that still retain the specificity of binding. Techniques for the production of antibodies are well known in the art. The term "antibody" also includes immunoglobulins (Ig's) of different classes (i.e., IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2 etc.). Illustrative examples of an antibody are Fab, F (ab ') fragments, Fv fragments, single chain Fv fragments (scFy), diabodies or domain antibodies (Holt LJ et al., Trends Biotechnol. 21 (11), 2003, 484-490).
[0054] [0054] An antibody molecule according to the invention can carry one or more domains that have a sequence of at least about 60%, at least about 70%, at least about 75%, at least about 80% at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98 % or at least about 99% sequence identity with a naturally occurring domain corresponding to an immunoglobulin M, an immunoglobulin G, an immunoglobulin A, an immunoglobulin D or an immunoglobulin E. in this respect, the term "about" or "approximately" as used herein, in this patent application, means within a 20% deviation, such as within a 10% deviation or within 5% of a given value or reach.
[0055] Therefore, the main chain (longest polypeptide chain) of an antibody molecule of the invention may include, including consisting of domains with the identity of the above sequences with a corresponding domain of an immuno heavy chain
[0056] [0056] "Percentage (%) of sequence identity" with respect to the amino acid sequences disclosed here, in this patent application, is defined as the percentage of amino acid residues in a candidate sequence that are identical in a paired way with the amino acid residues in a reference sequence, that is, an antibody molecule of the present disclosure, after aligning the sequences and introducing gaps, if necessary, in order to obtain the maximum percentage of sequence identity, and does not contain - considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining the identity percentage of amino acid sequences can be obtained in various ways that are within the knowledge of the art, for example, using software publicly available computing resources such as BLAST, ALIGN, or Megalign (DNASTAR) software. People skilled in the art can determine appropriate parameters for measure alignment, including any algorithms needed to obtain maximum alignment over the entire length of the sequences being compared. The same is true for nucleotide sequences disclosed here, in this patent application.
[0057] [0057] The term "variables" refers to the portions of the immunoglobulin domains that show variability in their sequence and that are involved in determining the specificity and binding affinity of a particular antibody (that is, the one or more "variable domains"). Variability is not evenly distributed across all variable domains of antibodies; it is concentrated in subdomains of each of the variable regions of heavy and light chains. These subdomains are called "hypervariable regions", "H-VR," or "HV," or "complementarity-determining regions" (C-DRs). The most conserved (ie, non-hypervariable) portions of the variable domains they are called "framework" (FR) regions ("structure" regions). Each of the naturally occurring heavy and light chain variable domains includes four FR regions, largely adopting a B-sheet configuration, connected by three regions hypervariables, which form loops connecting, and in some cases forming part of, the B-sheet structure.The hypervariable regions in each chain are kept together in close proximity by the FR and, with the hypervariable regions of the other chain, contribute to the formation of the antigenic binding site (see Kabat et al., see below). Generally, naturally occurring immunoglobulins include six CDRs (see below); three in VH (H1, H2, H3), and three in VL ( L1, L2, L3) .In immunoglobulins that occur naturally Mostly, H3 and L3 have the greatest diversity of the six CDRs, and H3 in particular is believed to play a unique role in conferring fine specificity to immunoglobulins. The constant domains are not directly involved in antigen binding, but have several effector functions, such as, for example, antibody-dependent cell cytotoxicity and complement activation.
[0058] The corresponding mu heavy chain, gamma heavy chain, alpha heavy chain, delta heavy chain, epsilon heavy chain, lamina light chain or immunoglobulin light chain can be of any species, such as a mammal species, including a species of rodent, an amphibian, for example, from the subclass Lissamphibia which includes, for example, frogs, frogs, salamanders or newts or a species of invertebrate. Examples of mammals include, but are not limited to, a rat, a mouse, a rabbit, a guinea pig, a squirrel, a hamster, a hedgehog, a platypus, an American pika, an armadillo, a dog, a lemur, a goat, a pig, a cow, an opossum, a horse, a bat, a marmot, an orangutan, a rhesus monkey, a paunchy monkey, a monkey, a chimpanzee, a tamarin (saguinus oedipus) , a marmoset or a human.
[0059] [0059] The term "immunoglobulin" refers to a glycoprotein that includes
[0060] [0060] Each immunoglobulin light chain includes an N-terminal variable (V) (VL) domain and a constant (C) (CL) domain. Each heavy chain includes an N-terminal V domain (VH), three or four C domains (CHs), and a joint region. An antibody molecule according to the invention likewise contains these domains and regions (although a bispecific antibody molecule binding site is formed only by a single chain Fv fragment).
[0061] [0061] An immunoglobulin, when used here, in this patent application, is typically a tetrameric glycosylated protein composed of two light chains (L) of approximately 25 kDa each and two heavy chains (H) of approximately 50 kDa each. Two types of light chain, called lamda and capa, can be found in immunoglobulins. Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be assigned to five main classes: A, D / E GeM, and several of these can be further divided into subclasses (iso-types), for example, I9G1 , IgG2, I9G3, IgG4, IgA1, and I9gA2. An IgM immunoglobulin consists of 5 of the basic heterotetramer unit together with an additional polypeptide called a J-block, and contains 10 antigenic binding sites, whereas IgA immunoglobulins contain 2 to 5 of the 4 basic chain units. which can polymerize to form multipurpose assemblies in combination with the J chain. In the case of IgGs, the 4-chain unit generally has about 150,000 daltons.
[0062] [0062] The term "amino acid" or "amino acid residue" refers to an o- or B-amino carboxylic acid.
[0063] [0063] When used in connection with a protein or a peptide, the term "amino acid" or "amino acid residue" typically refers to an α-amino carboxylic acid having its definition recognized in the art such as an amino acid selected from among group consisting of: L-alanine (Ala or A); L-arginine (Arg or R); L-asparagine (Asn or N); L-aspartic acid (Asp or D); L-cysteine (Cys or C); L-glutamine (GEm or OQ); L-glutamic acid (Glu or E); glycine (Gly or G); L-histidine (His or H); L-isoleucine (ILE or |): L-leucine (Leu or L); L-lysine (Lys or K); L-methionine (Met or M); L-phenylalanine (Phe or F); L- proline (Pro or P); L-serine (Ser or S); L-threonine (Thr or T); L-tryptophan (Trp or W); L-tyrosine (Tyr or Y); and L-valine (Val or V), although modified, synthetic, or rare amino acids such as, for example, taurine, ornithine, selenocysteine, homocystine, hydroxyproline, thioproline, iodo-tyrosine, 3-nitro-tyrosine, ornithine , citrullina, canavanina,
[0064] [0064] The term "epitope", also known as the "antigenic determinant", refers to the portion of an antigen to which an antibody or T cell receptor specifically binds, thereby forming a complex. Therefore, the term "epitope" includes any determinant molecule or protein capable of specific binding to an immunoglobulin or a T cell receptor. The one or more binding sites (parapet) of an antibody molecule described here, in this patent application, you can specifically link to / interact with conformational or continuous epitopes, which are unique to the target structure. Epitopic determinants generally consist of chemically active surface groups of molecules such as amino acids or sugar side chains and generally have three specific dimensional structural characteristics as well as specific charge characteristics. In some embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar, phosphoryl, or sulfonyl side chains, and, in certain embodiments, may have three specific dimensional structural characteristics, and / or characteristics of specific loads. With respect to polypeptide antigens, a conformational or discontinuous epitope is characterized by the presence of two or more distinct amino acid residues, separated in the primary sequence, but joining a consistent structure on the surface of the molecule when the polypeptide folds into the native protein / in the native antigen (Sela, M., Science (1969) 166, 1365-1374; Laver, WG, et al. Cell (1990) 61, 553-556). The two or more distinguishing amino acid residues that contribute to the epitope may be present on separate sections of one or more polypeptide chains.
[0065] [0065] An antibody or antibody molecule / antibody fragment is said to bind specifically to an antigen when it recognizes its target antigen in a complex mixture of proteins and / or macromolecules. Antibodies are said to "bind to the same epitope" if antibodies cross-compete so that only one antibody can bind to the epitope at any given point in time, that is, one antibody prevents binding or the modulating effect of the other .
[0066] [0066] The term "specific" in this context, or "specifically recognizing", also used as "targeted to", means according to this invention that the antibody molecule is specifically capable of interacting with and / or bind to at least two, for example, at least three or at least four amino acids from an epitope but do not essentially bind to another epitope or another antigen. The connection mentioned can be exemplified by the specificity of a "key and lock principle". Specific binding is believed to be effected for specific reasons in the amino acid sequence of the antibody binding region, and the antibody and epitope or antigen bind to each other as a result of their primary, secondary or tertiary structure as well as as a result of minor modifications to said structure. The specific interaction of the epitope / antigen interaction site with its specific epitope / antigen can also result in a simple connection of that site to the antigen. Furthermore, the specific interaction of the antigenic interaction site with its specific epitope / antigen may alternatively result in the initiation of a signal, such as, for example, due to the induction of a modification of the antigen's conformation or an oligomerization of the antigen.
[0067] [0067] Typically, the bond is considered specific when the bonding affinity is greater than 10º M. In particular, the bond is considered specific when the bonding affinity is about
[0068] [0068] In some embodiments an antigen to which an antibody according to the invention binds is an antigen that is included in the extracellular matrix or is a cell surface antigen. In some modalities an antigen to which an antibody according to the invention binds is a tumor-associated antigen. It is understood that an antigen associated with a similar tumor may be included in the extracellular matrix or may be a cell surface antigen.
[0069] [0069] The term "extracellular matrix" refers to the tissue region of a multicellular animal, including a human that is found in the intercellular space, that is, between the cells of the respective tissue. The extracellular matrix is largely a network of proteins such as fibrillar and non-fibrillar collagens or elastin, glycoproteins such as laminin or fibronectin, proteoglycans such as chondroitin sulphate or keratane and polysaccharide sulphate such as hyaluronic acid. The extracellular matrix serves among others in the segregation of different tissues from each other or in the regulation of intercellular communication. In some embodiments, a tumor-associated antigen can be expressed partially or exclusively in the extracellular matrix of a tumor.
[0070] [0070] The term "cell surface antigen" as used here, in this patent application, refers to a molecule that is presented on the surface of a cell. Typically a similar molecule is located on or over the cell's plasma membrane in such a way that at least part of this molecule remains accessible from the environment, that is, from outside the cell.
[0071] [0071] Examples of a tumor associated antigen that is included in the extracellular matrix include, but are not limited to, a proteoglycan such as Chondroitin Sulfate Proteoglycan 4 associated with Melanoma (CSPG4) or CD44v6, including a mucin such as Muc-1 or a membrane-bound enzyme such as carbonic anhydrase IX (CAIX). Examples for similar antigens are Tenascin and fibroblast activation protein (FAP).
[0072] [0072] The term "isolated antibody molecule" as used herein, in this patent application, refers to an antibody molecule that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are matters that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody molecule is purified to more than 95% by weight of the antibody as determined by the Lowry method, such as more than 99% by weight. In some embodiments, the antibody molecule is purified to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a rotating cup sequencer. In some embodiments, the antibody is purified to homogeneity as assessed by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver dye. An isolated antibody molecule in some embodiments may be present within recombinant cells with one or more components of the antibody's natural environment not being present. Typically, an isolated antibody is prepared by at least one purification step.
[0073] [0073] The terms "V4" and "V," are used here, in this patent application, to refer to the heavy chain variable domain and the light chain variable domain respectively of an immunoglobulin. An immunoglobulin light or heavy chain variable region consists of a framework region interrupted by three hypervariable regions. Thus, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region includes amino acid residues from a "Complementarity-Determining Region" or "CDR". There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Accordingly, "CDRs" as used herein, in this patent application, refer to all three heavy chain CDRs (CDRH1, CDRH2 and CD-RH3), or to all three light chain CDRs (CDRL1, CDRL2 and C-DRL3) or both to all heavy chain and all light chain CDRs, if appropriate. Three CDRs make up the binding character of a light chain variable region and three make up the binding character of a heavy chain variable region. CDRs determine the antigenic specificity of an immunoglobulin molecule and are separated by amino acid sequences that include framework or scaffolding regions. The exact CDR limits and extensions related to the definition are subject to different classification and numbering systems. The structure and protein fold of the antibody may mean that other residues are considered part of the antigenic binding region and would be understood as such by a skilled person. CDRs provide the majority of contact residues for binding immunoglobulin to the antigen or epitope.
[0074] [0074] CDR3 is typically the largest source of molecular diversity within the antibody binding site. H3, for example, can be as short as two amino acid residues or greater than 26 amino acids. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of the structure of antibodies, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988. A person skilled in the art will recognize that each subunit structure, for example, a CH, VH, CL, VL, CDR, FR structure, includes active fragments, for example, the portion of the subunit. VH, VL, or CDR binds to the antigen, that is, the antigen binding fragment, or, for example, the portion of the CH subunit that binds to and / or activates, for example, an Fc receptor and / or complement . CDRs typically refer to Kabat CDRs, as described in Sequences of Proteins of immunological Interest, US Department of Health and Human Services (1991), eds. Kabat and others. Another pattern for characterizing the antigenic binding site is referring to hypervariable loops as described by Chothia. See, for example, Chothia, and others. (1992; J. Mol. Biol. 227: 799-817; and Tomlinson et al. (1995) EMBO J. 14: 4628-4638. Yet another standard is the definition of ADM used by anti-aging modeling software Oxford Molecular's ADM body. See, in general, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed .: Duebel, S. and Kontermann, R., Springer -Verlag, Heidelberg) Modalities described with respect to Kabat CDRs can be implemented alternatively using similar described relationships with respect to Chothia hypervariable loops or loops defined by ADM.
[0075] [0075] Residues of the "Framework Region" or "FR" are residues of variable domain other than the hypervariable region. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. Thus, a "human framework region" is a framework region that is substantially identical (about 85% or more, usually 90 to 95% or more) to the framework region of a naturally occurring human immunoglobulin. The framework region of an antibody, that is, the combined framework regions of the constituent light and heavy chains, serves to position and align the CDR's. CDR's are primarily responsible for binding to an antigen antigen.
[0076] [0076] The terms "Fab", "Fab region", "Fab portion" or "Fab fragment" are understood to define a polypeptide that includes an immunoglobulin Vy domain, a Cy1, a Vi, and a C ,. Fab can refer to this region in isolation, or to this region in the context of an antibody molecule according to the invention, as well as a full-length immunoglobulin or an immunoglobulin fragment. entire light chain of an antibody An Fab region can be taken to define "an arm" of an immunoglobulin molecule. It contains the epitope-binding portion of that Ig. The Fab region of a naturally occurring immunoglobulin it can be obtained as a proteolytic fragment by papain di-management. A "F (ab ') portion;" is the proteolytic fragment of a pepsin-digested immunoglobulin. A "Fab'" is the product resulting from reduced binding disulfide of a portion F (ab ') .. As used here, in this application In the patent, the terms "Fab", "Fab region", "Fab portion" or "Fab fragment" may additionally include a hinge region that defines the C-terminal end of the antibody arm (cf. above). This joint region corresponds to the joint region found C- terminally of the CH1 domain within a full-length immunoglobulin in which the arms of the antibody molecule can be taken to define a Y. The term joint region is used in the art because an immunoglobulin has some flexibility in this region.
[0077] [0077] An "Fv" or Fv fragment "consists of only the V, and V4y domains of an" single arm "of an immunoglobulin. In this way an "Fv"
[0078] [0078] The term "Fc region" or "Fc fragment" is used here, in this patent application, to define a region of a C-terminal immunoglobulin heavy chain, including native sequence Fc regions
[0079] [0079] An antibody molecule according to the invention has two chains, a shorter chain, which in some modalities can be a light chain, and a main chain, which in some modalities can also be treated like the heavy chain. The antibody molecule is usually a dimer of these two chains. Based on the domains included in an antibody molecule of the invention, the antibody molecule can be taken to have a fragment.
[0080] [0080] The shortest chain of the antibody can be linked to the main chain of the antibody by means of one or more, including two or three, disulfide bonds. A respective disulfide bond can define a bridge between a shorter-chain C-terminal cysteine residue and a cysteine residue within the main chain hinge region of the antibody.
[0081] [0081] In an antibody molecule according to the invention the C-terminal region of the main chain can be defined by a fragment
[0082] [0082] The Fab fragment of an antibody molecule according to the invention is in some embodiments linked to the CH2 domain via a heavy chain domain of the Fab fragment. Therefore, the antibody main chain can have a heavy chain domain such as a CH1 domain (above), which is coupled to the CH2 domain. As explained above, a respective CH1 domain can be coupled to the CH2 domain through an articulation region. The heavy chain domain of the respective Fab fragment can in some embodiments be arranged at the N-terminus of the polypeptide chain of the antibody main chain. In some embodiments, the Fab fragment of an antibody molecule according to the invention is linked to the CH2 domain via a light chain domain of the Fab fragment. Therefore, the main chain of the antibody molecule may have a chain domain lightweight such as a CL domain, which is coupled to the CH2 domain. Again, a respective CL domain can be coupled to the CH2 domain through an articulation region. The light chain domain of the respective Fab fragment may in some embodiments be arranged at the N-terminus of the polypeptide chain of the main chain of the antibody molecule. In order to avoid dimerization of the molecules in bivalent modalities (Fig. 1h to 1E and 1N) the cysteine residues in the joint region providing inter-chain disulfide bonds can be exchanged. In tetravalent modes (Figs. 1F to 1M) these cysteine residues are preserved. In these embodiments, the antibody molecule can therefore be taken to define a dimer of a bivalent, dimeric antibody molecule as described above and each main chain and each shorter chain can be selected individually. As an example, the first of the shortest chains may have a VH domain at the N-terminus and a CL domain at the C-terminus. The first backbone can have a VL domain at the N-terminus and a CH1 C-terminal domain at this. In addition, the first backbone can have a CH2 domain and a CH3 domain, as well as a scFv C-terminal fragment. The scFv fragment can be coupled to the CH3 domain through the VL domain. The second of the shorter chains may have a VH domain at the N-terminus and a CH1 domain at the C-terminus. The second main chain can have a VL domain at the N-terminus and a CL domain C-terminally at this. The second main strand can also have a CH2 domain and a CH3 domain, as well as a C-terminal scFv fragment. The scFv fragment can be coupled to the CH3 domain through the VL domain.
[0083] [0083] A respective tetrameric antibody molecule can be composed of two dimeric antibody molecules that are linked to one another through one or more, such as two, disulfide bonds. A similar disulfide bond can define a bridge between a main chain cysteine residue of a first dimeric antibody molecule and a main chain cysteine residue of a second dimeric antibody molecule. Typically, the respective cysteine residues are positioned within the hinge region of the corresponding main chain of each dimeric antibody molecule. In some embodiments, one or both of the two main chains, that is, the main chain of the first dimeric molecule and the main chain of the second dimeric molecule of a tetrameric antibody molecule, have a cysteine residue in the 226 sequence position and / or in the 229 sequence position of a respective articulation domain, in line with the Kabat numbering [European Union index]. In one embodiment, a disulfide bond between the hinge domain of the first main chain and a hinge domain of the second main chain is defined by at least one of a cysteine residue in the sequence position 226 and a cysteine residue in the sequence position 229 of one of the articulation domains, according to the Kabat numbering [European Union Index]. In some embodiments, a tetrameric antibody molecule may have one or more disulfide bonds linking the hinge regions of the two main chains of the dimeric antibody molecules and a disulfide bond linking the hinge regions of the two main chains of the molecules. dimeric antibodies. In some modalities, two dimeric antibody molecules of a tetrameric antibody molecule according to the invention can be linked by means of a disulfide bond that is defined by a cysteine residue that is included in the CH2 domain of the main chain of a first dimeric antibody molecule and a cysteine residue that is included in the CH2 domain of the main chain of a second dimeric antibody molecule.
[0084] [0084] As an additional example, the first of the shortest chains
[0085] [0085] An "bispecific" or "bifunctional" antibody molecule is an antibody molecule that has two different epitope / antigen binding sites, and therefore has binding specificities for two different target epitopes. These two epitopes can be epitopes of the same antigen or different antigens. In contrast to this, a "divalent antibody" can have binding sites of identical antigen specificity.
[0086] [0086] A "bispecific antibody" can be an antibody molecule that binds an antigen or an epitope over one of two or more binding arms, defined by a first pair of heavy and light chain or main chain and shorter / smaller (above), and binds a different antigen or epitope on a second arm, defined by a second pair of heavy and light chains or main and smaller chains. A similar modality of a bispecific antibody has two distinct antigen binding arms, both in specificity and in CDR sequences. Typically, a bispecific antibody is monovalent for each antigen to which it binds. A bispecific antibody is a hybrid antibody molecule, which may have a first binding region that is defined by a first light chain variable region and a first heavy chain variable region, and a second binding region that is defined by a second variable region of light chain and a second variable region of heavy chain. In some embodiments, one of these link regions can be defined by a heavy / light chain pair. As explained above, in the context of the present invention the bispecific antibody molecule has a first binding site, defined by variable regions of a main and a minor chain, and a different second binding site defined by a variable region of a scFv fragment that is included in the main chain of the antibody molecule.
[0087] [0087] Methods of producing a bispecific antibody molecule are known in the art, for example, chemical conjugation of two different monoclonal antibodies or, for example, also chemical conjugation of two antibody fragments, for example, two fragments Fab. Alternatively, bispecific antibody molecules are produced recombinantly. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two pairs of immunoglobulin H chain - L chain, where the two H chains have different binding specificities. Due to the random selection of H and L chains, a potential mixture of ten different antibody structures is produced of which only one has the desired binding specificity. An alternative approach involves merging the variable domains with the desired binding specificities for the heavy chain constant region including at least part of the hinge region, CH2 and CH3 regions. In one mode, the CH1 region containing the site necessary for light chain binding is present in at least one of the fusions. DNA encoding these fusions, and if desired to the L chain are inserted into separate expression vectors and are then cotransfected into an organism.
[0088] [0088] The bispecific antibody molecule of the invention can act as a monoclonal antibody (MAb) with respect to each target. In some embodiments, the antibody is chimeric, humanized, or fully human.
[0089] [0089] A "double-specific antibody", which can be, for example, a full-length immunoglobulin or a construct with binding properties similar to immunoglobulin, is generally understood to have two binding arms, in particular arms defined by a pair of HC / LC, which can bind two different antigens or epitopes in each one of them (see the publication of the international patent application No. WO 02/02773). Therefore, a double-specific binding protein has two identical antigen binding arms, with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.
[0090] [0090] The T cell receptor (TCR) is a particular receptor that is present on the cell surface of T cells, that is, T lymphocytes / n the T cell receptor exists as a complex of several proteins. The T cell receptor generally has two separate peptide chains, typically alpha and beta T cell receptor chains (TOCRa and TCRB), over some T cell gamma and delta T cell receptors (TORE and TORI). The other proteins in the complex are the CD3 proteins: CD3ey and CD3eô heterodimers and, most importantly, a CD37 homodimer, which has a total of six motives | - TAM. ITAM motifs on CD36 can be phosphorylated by Lck and in turn recruit ZAP-70. Lck and / or ZAP-70 can also phosphorylate tyrosines on many other molecules, especially CD28, LAT and SLP-76, which allows the aggregation of signaling complexes around these proteins.
[0091] [0091] An antibody molecule according to the invention includes a light chain with a VL domain and a CL domain. The antibody molecule additionally includes a backbone that includes a VH domain, a CH1 domain and a hinge region. The VH domain is arranged at the N-terminus of the main chain, and the VH domain is linked to the CH1 domain, or directly linked to it or coupled via a binding peptide and typically 20 or less, including or less residues amino acid. The hinge region is linked to the C-terminal end of the CH1 domain. Therefore, the portion of the antibody molecule that is defined by the adjacent arrangement of the VL domain, the CL domain, the VH domain and the CH1 domain as well as the hinge region, can be taken to define a Fab fragment and therefore it is also referred to as such here, in this patent application. As with a naturally occurring immunoglobulin, the pairing of the VH domain and the VL domain together defines a single antigen binding site. Thus, the Fab fragment of an antibody of the invention includes the binding site for a first antigen. In a respective antibody molecule the light chain is linked to the main chain by a disulfide bond. In some embodiments, an antibody molecule according to the invention is a dimer that includes two main chains and two light chains as described above (cf. also below).
[0092] [0092] In some embodiments the sequence of a recombinant bispecific antibody molecule according to the invention can be compared against the IgG1 sequence, since the sequence of the antibody molecule according to the invention has a certain degree of similarity to the IgG1 sequence, as further illustrated below. In comparison to the IgG1 amino acid sequence according to Kabat et al. (1991, Sequences of Proteins of
[0093] [0093] According to the amino acid sequence of the main chain of an antibody of the invention, the Fab fragment, which consists of the VH domain, the CH1 domain and the articulation region, in these modalities typically covers amino acids 1 to 230. Within this Fab fragment, the VH domain is typically defined by amino acids 1 to 118, the CH1 domain is defined by amino acids 119 to 216, and the articulation region is defined by amino acids 217 to 231, according to Kabat numbering. The chain of antibodies with the sequence of SEQ ID NO: 6 can serve as an example of a respective modality. In some embodiments, the antibody molecule according to the invention has, at positions 342 and following on the main chain, a chimeric sequence composed of a VH domain and a VL domain. In some embodiments, the VL domain is arranged to define the C-terminal domain of this chimeric sequence. In some modalities, the antibody according to the invention has, in comparison with the amino acid sequence of IgG1 according to Kabat and others., A CH3 domain at positions 342 to 447, followed by a compound chimeric sequence of a VH domain and a C-terminal VL domain. In similar embodiments where a CH3 domain is included in the antibody according to the invention, this CH3 domain is defined by amino acids 342 to 448 according to the amino acid sequence of the main chain of the antibody molecule. The chimeric sequence composed of a VH domain and a VL domain, which can be in some C-terminal modalities (above), in these modalities is located at positions 449 and following of the amino acid sequence of the main chain of the antibody molecule.
[0094] [0094] A bispecific antibody molecule according to the invention can have two binding sites of any desired specificity. In some embodiments, one of the binding sites is capable of binding a tumor-associated antigen. In some embodiments, the binding site included in the Fab fragment is a specific binding site for a tumor-associated surface antigen. In some embodiments, the binding site included in the single chain Fv fragment is a specific binding site for a tumor-associated antigen such as a tumor-associated surface antigen.
[0095] [0095] The term "tumor-associated surface antigen" as used here, in this patent application, refers to an antigen that is or can be presented on a surface that is located on or within tumor cells. These antigens can be presented on the cell surface with an extracellular part, which is often combined with a transmembrane and cytoplasmic part of the molecule. These antigens in some modalities can be presented only by tumor cells and not by normal, that is, non-tumor cells. Tumor antigens can be expressed exclusively on tumor cells or they can represent a specific tumor mutation compared to non-tumor cells. In a similar embodiment, a respective antigen can be referred to as a tumor-specific antigen. Some antigens are presented by both tumor cells and non-tumor cells, which can be referred to as tumor-associated antigens. These tumor associated antigens can be overexpressed on tumor cells when compared to non-tumor cells or are accessible for antibody binding to tumor cells due to the less compact structure of the tumor tissue compared
[0096] [0096] Illustrative examples of a tumor-associated surface antigen are CD10, CD19, CD20, CD22, CD33, tyrosine kinase similar to Fms 3 (FLT-3, CD135), chondroitin sulfate proteoglycan 4 (CSPGA , chondroitin sulfate proteoglycan 4 associated with melanoma), Epidermal growth factor receptor (EGFR), Her2neu, Her3, IGFR, CD133, IL3R, fibroblast activating protein (FAP), CDCP1, Derlin1, Tenascin, frizzled 1-10, the vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-a (CD140a), PDGFR-B (CD140b) Endoglin, CLEC14, Tem1-8, and Tie2. Additional examples may include A383, CAMPATH-1 (CDw52), Carcinoembryonic antigen (CEA), Carboanidrase IX (MN / CA IX), CD21, CD25, CD30, CD34, CD37, CD44v6, CD45, CD133, de2-7 EGFR , EGFRvII, EBDCAM, Ep-CAM, Folate-binding protein, G250, Fms 3-like tyrosine kinase (FLT-3, CD135), c-Kit (CD117), CSF1IR (CD115), HLA-DR, IGFR , IL-2 receptor, IL3R, MCSP (cell surface chondroitin sulfate protoglycan associated with melanoma), Muc-1, prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA) , Prostate specific antigen (PSA), and TAG-72. Examples of antigens expressed on the extracellular matrix of tumors are Tenascin and the fibroblast activating protein (FAP).
[0097] [0097] In some embodiments one of the binding sites of an antibody molecule according to the invention is capable of binding a specific receptor molecule for T cells and / or a specific receptor molecule for natural killer cells (NK cells ). A specific receptor for T cells is called a "T cell receptor" (TCRs), which allows a T cell to bind to and, if additional signals are present,
[0098] [0098] An example of a specific receptor molecule for NK cells is CD16, a low affinity Fc receptor and NKG2D. An example of a receptor molecule that is present on the surface of both T cells and natural killer cells (NK) is CD2 and additional members of the CD2 superfamily. CD2 has the ability to act as a costimulatory molecule on T and NK cells.
[0099] [0099] In some embodiments the first binding site of the antibody molecule binds a tumor-associated surface antigen and the second binding site binds a T cell-specific receptor molecule and / or a cell-specific receptor molecule natural killer (NK). In some embodiments, the first binding site of the antibody molecule links one between A33, CAMPATH-1 (CDw52), Carcinoembryonic Antigen (CEA), Carboanidrase IX (MN / CA IX), CD10, CD19, CD20, CD21, CD22 , CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, CDCP1, Her3, chondroitin sulfate proteoglycan 4 (CSPGA, chondroitin sulfate proteoglycan 4 associated with melanoma), CLEC14, Derlin1, Factor receptor epidermal growth (EGFR), 2-7 EGFR, EGFRvIIl, EBCAM, Endodglin, Ep-CAM, Fibroblast-activating protein (FAP), Folate-binding protein, G250, Fms 3-like tyrosine kinase ( FLT-3, CD135), c-Kit (CD117), CSFIR (CD115), frizzled 1-10, Her2 / neu, HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (chondroitin sulfate proteoglycan from cell surface associated with melanoma), Muc-1, prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate specific antigen (PSA),
[00100] [00100] In some embodiments, the first binding site of the antibody molecule binds a specific receptor molecule for T cells and / or a specific receptor molecule for natural killer cells (NK) and the second binding site a tumor-associated surface antigen. In some embodiments, the first antibody binding site binds a specific receptor molecule for T cells and / or a specific receptor molecule for natural killer cells (NK) and the second binding site binds one between A33, CAMPATH-1 (CDw52), Carcinoembryonic Antigen (CEA), Carboanidrase IX (MN / CA IX), CD10, CD19, CD20, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, CDCP1, Her3, proteoglycan Chondroitin sulfate 4 (CSPGA, chondroitin sulfate proteoglycan 4 associated with melanoma), CLEC14, Derlin1, epidermal growth factor receptor (EGFR), 2-7 EGFR, EGFRvIIl, Ep-CAM, Endoglin, Ep-CAM, Fibroblast Activating Protein (FAP), Folate Binding Protein, G250, Fms 3-like Tyrosine Kinase (FLT-3, CD135), frizzled 1-10, Her2 / neu, HLA-DR, IGFR, I1L-2 receptor, IL83R, MCSP (cell surface chondroitin sulfate proteoglycan associated with melanoma), Muc-1, prosthesis-specific membrane antigen ta (PSMA), Prostate specific antigen (PSA), TAG-72, Tenascin, Tem1-8, Tie2 and VEGFR. In some modalities the first antibody binding site binds one between CD3, the T cell receptor (TOR), CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5 and CD95, and the second binding site binds a tumor-associated surface antigen.
[00101] [00101] The term "glycosylation" means the attachment of oligosaccharides (carbohydrates containing two or more simple sugars linked together, for example, from two to about twelve simple sugars linked together) to a glycoprotein. The oligosaccharide side chains are typically linked to the backbone of the glycoprotein via either N or O binding. The antibody oligosaccharides disclosed here, in this patent application, generally occur are attached to a CH2 domain of an Fc region as N-linked oligosaccharides "N-linked glycosylation" refers to the attachment of the carbohydrate moiety to an asparagine residue in a glycoprotein chain. The skilled technician will recognize that, for example, each of the CH2 domains of murine IgG1, IgG2a, IgG2b and I9gG3 as well as human IgG1, I9G2, I9G3, IgG4, IgA and IgD have a single site for N- glycosylation. bound at residue 297.
[00102] [00102] Sequences of domains or regions included in an antibody molecule according to the invention can be sequences of any desired species. Depending on the subsequent use of the antibody molecule, however, it may be desirable in some modalities to introduce changes that prevent undesirable side effects caused by the antibody. The use of intact non-human antibodies in the treatment of human diseases or disorders jointly carries the potential of currently well-established immunogenicity problems, which means that the patient's immune system can recognize the intact non-human antibody as non-human. own and mount a neutralizing response. This is particularly evident after multiple administration of the non-human antibody to a human patient. Several techniques have been developed over the years to overcome these problems and generally involve reducing the composition of non-human amino acid sequences in the intact antibody while maintaining the relative ease of obtaining non-human antibodies from of an immunized animal eg mouse, rat or rabbit. In general, two approaches have been used to achieve this. The first are chimeric antibodies, which generally have a non-human variable domain (for example, from rodents such as mice) fused to a constant human region. As the antigenic binding site of an antibody is defined by residues within the variable domains, the chimeric antibody maintains its binding affinity for the antigen but acquires the effector functions of the human constant region and is therefore able to perform effector functions as described above. Chimeric antibodies are typically produced using recombinant DNA methods. DNA encoding the antibodies (for example, cDNA) is isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that are capable of specifically binding genes encoding the H and L chains of the antibody of the invention. Hybridoma cells serve as a typical source of similar DNA.Once isolated, the DNA is placed in expression vectors which are then transfected into host cells such as E. coli cells, COS cells, CHO cells or myeloma cells that do not produce immunoglobulin protein differently to obtain antibody synthesis DNA can be modified by replacing the coding sequence for human L and H chains with the corresponding non-human H and L constant regions, for example, murine (see for example, Morrison; PNAS [1984] 81, 6851).
[00103] [00103] The second approach involves the generation of humanized antibodies in which the non-human content of the antibody is reduced by humanizing the variable domains. Two techniques for human-
[00104] [00104] Alternatively, humanization can be achieved by a process of "veneering" (coating). Statistical analysis of human and murine single immunoglobulin heavy and light chain variable domains revealed that the precise casings of exposed residues are different in human and murine antibodies, and most individual surface positions have a strong preference for a small number of different residues (see Padlan EA and others; (1991) Mol. Immunol. 28, 489-498 and Pedersen JT et al. (1994) J. Mol. Biol. 235; 959-973). Therefore, it is possible to reduce the immunogenicity of a non-human Fv by replacing residues exposed in their framework regions that differ from those generally found in human antibodies. Since protein antigenicity can be correlated with surface accessibility, the substitution of surface residues may be sufficient to make the mouse variable domain "invisible" to the human immune system (see also Mark GE et al. (1994 ) in Handbook of Experimental Pharmacology vol 113: The pharmacology of monoclonal Antibodies, Springer-Verlag, pp 105-134). This humanization procedure is referred to as "veneering" because only the surface of the antibody is altered, the support residues remain untouched.
[00105] [00105] An antibody molecule of the invention can be produced using any known and well-established expression system and recombinant cell culture technology, for example, by expression in bacterial hosts (prokaryotic systems), or eukaryotic systems such as yeasts, fungi , insect cells or mammalian cells. An antibody molecule of the present invention can be produced in transgenic organisms such as a goat, a plant or a transgenic mouse XENOMOUSE, a manipulated mouse strain that has large fragments of human immunoglobulin loci and is deficient in production of mouse antibodies. An antibody can also be produced by chemical synthesis.
[00106] [00106] For recombinant production of an antibody molecule of the invention typically a polynucleotide encoding the antibody is isolated and inserted into a replicable vector such as a plasmid for further cloning (amplification) or expression. An illustrative example of a suitable expression system is a glutamate synthase system (as sold by Lonza Biologics), with the host cell being, for example, CHO or NSO. A polynucleotide encoding the antibody is readily isolated and sequenced using conventional procedures. Vectors that can be used include plasmid, virus, phage, transposons, minicromosomes of which plasmids are a typical modality. Generally, the vectors referred to additionally include a signal sequence, origin of replication, one or more marker genes, a reinforcer element, a promoter and transcription termination sequences operably linked to the light and / or heavy chain polynucleotide so facilitating expression. Polynucleotides encoding the light and heavy chains can be inserted into separate vectors and transfected into the same host cell or, if desired, both the heavy chain and the light chain can be inserted into the same vector for transfection into the host cell. Both strands can be arranged, for example, under the control of a dicistronic operon and expressed to result in the functional and correctly folded antibody molecule as described in Skerra, A. (1994) Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli, Gene 151, 131-135, or Skerra, A. (1994) A general vector, pASK8A4, for cloning, bacterial production, and single-step purification of antibody Fab fragments, Gene 141, 79-8. Thus, according to one aspect of the present invention, it is
[00107] [00107] When using recombinant techniques, the antibody molecule can be produced intracellularly, in the periplasmic space, or directly secreted in the meiuo (cf. also Skerra 1994, supra). If the antibody is produced intracellularly, as a first step, particulate debris, or host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted into the E coli periplasmic space. The antibody can also be produced in any oxidizing environment. A similar oxidizing environment can be provided by the periplasm of Gram-negative bacteria such as E. coli, in the extracellular medium of Gram-positive bacteria or in the lumen of the endoplasmic reticulum of eukaryotic cells (including animal cells such as insects or mammalian cells) and generally favors the formation of structural disulfide bonds. However, it is also possible to produce an antibody molecule of the invention in the cytosol of a host cell such as E. coli. In this case, the polypeptide can either be obtained directly in a soluble state and folded or recovered in the form of inclusion bodies, followed by in vitro renaturation. An additional option is the use of specific host strains having an intracellular oxidizing medium, which can therefore allow the formation of disulfide bonds in the cytosol (Venturi M, Seifert C, Hunte C. (2002) “High level production of functional antibody Fab fragments in an oxidizing bacterial cytoplasm. ”J. Mol. Biol. 315, 1-8).
[00108] [00108] The antibody molecule produced by the cells can be purified using any conventional purification technology, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a preferred purification technique . Antibody molecules can be purified by affinity purification with proteins / ligands that specifically and reversibly bind constant domains such as CH1 domains or CL domains. Examples of similar proteins are bacterial immunoglobulin-binding proteins such as Protein A, Protein G, Protein A / G or Protein L, where Protein L binding is restricted to antibody molecules that contain light chains cover. An alternative method for purifying antibodies with light chains |) is the use of anti-capa antibodies coupled to beads (KappaSelect). The suitability of protein A as an affinity linker depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies (Lindmark et al., J. Imnmunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human gamma 3 (Guss et al., EMBO J. 5: 15671575 (1986)). The choice of the purification method that is used for a particular antibody molecule of the invention is within the knowledge of the person moderately skilled in the art.
[00109] [00109] It is also possible to equip one of the chains of the antibody molecule of the invention with an affinity tag. Affinity tags such as Strep-tag & or Strep-tagO Il (Schmidt, TGM and others. (1996) J. Mol. Biol. 255, 753-766), myc-tag, FLAG "" - tag, the His6-tag or the HA-tag allows easy detection and in addition simple purification of the recombinant antibody molecule.
[00110] [00110] The terms "mutated", "mutant" and "mutation" in reference to a nucleic acid or polypeptide refer to the exchange, deletion, or insertion of one or more nucleotides or amino acids, respectively, compared with naturally occurring nucleic acid or polypeptide, that is, with a reference sequence that can be taken to define the wild type.
[00111] [00111] It is understood in this regard that the term "position", when used in accordance with the present invention, means the position of an amino acid within a sequence of amino acids represented here, in this patent application. This position can be indicated in relation to a similar native sequence, for example, a sequence from a naturally occurring IgG domain or chain. The term "corresponding" as used here, in this patent application, also includes that a position is not necessarily, or not only, determined by the number of preceding nucleotides / amino acids. Thus, the position of a given amino acid according to the present invention which can be substituted can vary due to the elimination or addition of amino acids elsewhere in the antibody chain.
[00112] [00112] Thus, under a "corresponding position" according to the present invention it should be understood that the amino acids may differ in the number indicated, but may still have similar neighboring amino acids. The referred amino acids which can be exchanged, deleted or added are also included by the term "corresponding position". In order to determine whether an amino acid residue in a given amino acid sequence corresponds to a certain position in the amino acid sequence of a naturally occurring domain or chain of immunoglobulin, the skilled person can use commonly known means and methods in art, for example, alignments, either manually or using computer programs such as BLAST2.0, which represents Basic
[00113] [00113] In some modalities a substitution (or replacement) is a conservative substitution. Conservative substitutions are generally the following substitutions, listed according to the amino acid to be mutated, each followed by one or more replacements that can be made to be conservative: Ala - Gly, Ser, Val; Arg> Lys; Asn - Gln, His; Asp> Glu; Cys - Ser; Glh - Asn; Glu - Asp; Gly - Ala; His - Arg, Asn, Gln; Ile - Leu, Val; Leu - Ile, Val; Lys> Arg, Gln, Glu; Met - Leu, Tyr, lle; Phe - Met, Leu, Tyr; Ser - Thr; Thr - Ser; Trp - Tyr; Tyr - Trp, Phe; Val - Ile, Leu. Other substitutions are also permissible and can be determined empirically or according to other known conservative or non-conservative substitutions. As an additional guideline, the eight groups that follow each contain amino acids that can typically be taken to define conservative substitutions for each other: 1) Alanine (Ala), Glycine (Gly); 2) Aspartic acid (Asp), Glutamic acid (Glu); 3) Asparagine (Asn), Glutamine (Gln); 4) Arginine (Arg), Lysine (Lys); 5) Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val); 6) Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp); 7) Serine (Ser), Threonine (Thr); and 8) Cysteine (Cys), Methionine (Met)
[00114] [00114] If the substitutions referred to result in a change in biological activity, then more substantial changes, such as the following, or as further described below in reference to classes of amino acids, can be introduced, and the products screened for a characteristic desired. Examples of similar, more substantial changes are: Ala - Leu, Ile; Arg> Glh; Asn - Asp, Lys, Arg, His; Asp> Asn; Cys - Ward; Glh - Glu; Glu - Gln; His - Lys; lle> Met, Ala, Phe; Leu - Ala, Met, Norleucine; Lys - Asn; Met - Phe; Phe - Val, Ile, Ala; Trp> Phe; Tyr - Thr, Ser; Val Met, Phe, Ala.
[00115] [00115] In some embodiments an antibody molecule according to the invention includes one or more amino acid residues, including two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen , fifteen, sixteen, seventeen or eighteen amino acid residues, which are mutated to prevent dimerization through cysteine residues or to modulate the function of the Fc. In some of these modalities, one or more amino acid residues from the CH2 domain and / or from the joint region that is capable of mediating binding to Fc receptors are mutated. If present, the one or more amino acid residues capable of mediating binding to Fc receptors may be an amino acid residue that is capable of activating antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC). In some embodiments, a respective amino acid residue capable of mediating binding to Fc receptors is replaced by another amino acid, usually when comparing the sequence with the sequence of a corresponding domain that occurs naturally in an immunoglobulin, such as an IgG . In some embodiments, a similar amino acid residue capable of mediating binding to Fc receptors is eliminated, usually in relation to the sequence of a corresponding domain that occurs naturally in an immunoglobulin, such as an IgG. However, in other embodiments of the invention that refer to a bispecific antibody molecule consisting of a Fab fragment, a single chain Fv fragment and an immunoglobulin CH2 domain, it is within the scope of the invention to introduce mutations in the CH2 domain of Human E1, for example, that optimize antibody-dependent cytotoxicity (ADCC). The mutations referred to are described in International Patent Application Nos. WO 2011/076922 and WO2011 / 089211, for example.
[00116] [00116] In some embodiments the one or more mutated amino acid residues, for example, substituted or eliminated, is / are an amino acid located in one of the positions 226, 228, 229, 230, 231, 232, 233, 234, 235 , 236, 237, 238, 265, 297, 327, and 330. Again, the number of amino acids used corresponds to the sequence positions according to the Kabat numbering [European Union Index]. A corresponding elimination of an amino acid can be, for example, an elimination of amino acid 228, generally a proline in IgG, an elimination of amino acid 229, generally a cysteine in IgG, a deletion of amino acid 230, generally a proline in IgG, an elimination of amino acid 231, generally an alanine in IgG, an elimination of amino acid 232, generally an proline in IgG, an elimination of amino acid 233, generally an glutamic acid in IgG, an elimination of amino acid 234, generally an leucine in IgG , an elimination of amino acid 235, generally a leucine in IgG, an elimination of amino acid 236, generally an glycine in IgG, an elimination of amino acid 237, generally an glycine in IgG, an elimination of amino acid 238, generally an proline in IgG and an elimination of amino acid 265, usually an aspartic acid in IgG. A corresponding substitution of an amino acid can be, for example, a substitution of amino acid 226, generally a cysteine in IgG, a substitution of amino acid 228, generally a proline in IgG, a substitution of amino acid 229, generally a cysteine in IgG, a substitution for amino acid 230, generally a proline in IgG, a substitution for amino acid 231, generally an alanine in IgG, a substitution for amino acid 232,
[00117] [00117] Another type of amino acid variant of an antibody changes the original glycosylation pattern (if any) of the antibody molecule. By changing, it is indicated to eliminate one or more portions of carbohydrate found in the antibody, and / or to add one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the
[00118] [00118] In the context of the present invention in some embodiments the portion of the main chain of the antibody molecule of the invention, which represents the Fc region of an immunoglobulin, is typically inert, or at least essentially low in influence, with respect to binding to Fc receptors. As stated, this is achieved by eliminating and / or replacing (mutating) at least one of the selected amino acid residues in the CH2 domain that are capable of mediating binding to an Fc receptor. The molecules referred to are also referred to here, in this patent application, as “Fc-attenuated” antibody molecules or “Fc“ * “antibody molecules. The portion of an antibody chain according to the invention that can be taken to represent a portion of an Fc fragment, i.e., the CH2 domain, and, where present, the CH3 domain, can thus define a scaffold ”(Scaffolding) without providing a particular biological function such as an effector function, for example. However, it has been seen in the present invention, that this scaffolding can provide important advantages in terms of purification, production efficiency and / or stability of the antibody molecules of the invention compared to known antibody molecules (cf. the Examples).
[00119] [00119] In some modalities the recognition, and therefore the connection, of this Fc-corresponding portion to a given Fc receptor is about 2 times, about 5 times, about 8 times, about 10 times, about 12 times, about 15 times, about 20 times or less than the Fc region of a naturally occurring immunoglobulin. In some embodiments, this Fc-corresponding portion is entirely devoid of its ability to bind to Fc receptors. The binding of an antibody to Fc receptors, including determination of a dissociation constant, can be easily determined by the skilled technician using routine techniques such as superficial plasmon resonance, for example, using a Biameter measurement. core "M, Any other method of measuring biomolecular binding can also be used, which can, for example, be based on spectroscopic, photochemical, photometric or radiological means. Examples for the corresponding detection methods are correlation spectroscopy. fluorescence, photochemical cross-linking and the use of photoactive or radioactive markers respectively Some of these methods may include additional separation techniques such as electrophoresis or HPLC.
[00120] [00120] Where required, a replacement or elimination of amino acid residues, as explained above, can be performed for this purpose. Adequate mutations can be taken from Armor and others. (Eur. J. Immunol. [1999] 29, 2613-2624), for example. Additional suitable positions for mutations to an antibody chain sequence can be taken from published crystal structure data about the complex between FcERIII and the human I9G1 Fc fragment (Sondermann et al., Nature [2000] 406, 267- 273). In addition to measuring binding affinity as described above in order to assess the level of "Fc attenuation" or loss of binding affinity, it is also possible to functionally assess the (lack of) ability to mediate binding to an Fc receptor.
[00121] [00121] As noted above, substitutions or eliminations of cysteine residues can be performed in order to introduce or remove one or more disulfide bonds, including introducing or removing a potential disulfide bond or a previously existing disulfide bond . In this way, the link between a main chain and a shorter weight / shorter chain of an antibody molecule according to the invention can be controlled including including established, reinforced or abolished. By introducing or removing one or more cysteine residues, a disulfide bridge can be introduced or removed. As an illustrative example, a tetrameric antibody molecule according to the invention generally has one or more disulfide bonds that link two dimeric antibody molecules. A similar disulfide bond is typically defined by a cysteine in the main chain of a first dimeric antibody molecule and a cysteine in the hinge region of a second dimeric antibody molecule. In this regard, in some embodiments, an antibody according to the invention may include an amino acid substitution of a native cysteine residue at positions 226 and / or 229, in relation to the sequence of a human IgG immunoglobulin according to the Kabat numbering European Union], by another amino acid residue.
[00122] [00122] Substitutions or eliminations of amino acid residues such as arginine, asparagine, serine, threonine or tyrosine residues can also be performed in order to modify the glycosylation pattern of an antibody. As an illustrative example, an IgG molecule has a single N-linked biantennary carbohydrate in Asn297 of the CH2 domain. For IgG or serum or produced ex vivo in hybridomas or engineered cells, IgG are heterogeneous with respect to the carbohydrate bound by Asn297. For human IgG, the core oligosaccharide typically consists of GIcCNAc2Man3GIcNAc, with different numbers of external residues.
[00123] [00123] As indicated, in addition to binding antigens / epitopes, an immunoglobulin is known to have "effector functions", biological activities attributable to the Fc region (an Fc region of the native sequence or a Fc region variant of the amino acid sequence) of an immune - globulin, and vary with the immunoglobulin isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); binding of Fc receptor; antibody dependent cell cytotoxicity (ADCC); phagocytosis; hypo-regulation of cell surface receptors (for example, B cell receptors); and B cell activation. Effecting an antibody's effector functions usually involves recruiting effector cells. Several immunoglobulin effector functions are mediated by Fc receptors (FcRs), which bind to the Fc region of an antibody. FcRs are defined by their specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as FcyR, for IgE as FceyR, for IgA as FcaR and so on. Any of these effector functions (or the loss of similar effector functions) such as a CDC or ADCC can be used in order to assess whether an antibody molecule of the invention lacks Fc binding capacity.
[00124] [00124] In this context, it is noted that the term "Fc receptor" or "FcR" defines a receptor, usually a protein that is capable of binding to the Fc region of an antibody. Fc receptors are found on the surface of some cells of an organism's immune system, for example, natural killer cells, macrophages, neutrophils, and mast cells. / n vivo Fc receptors bind to immunoglobulins that are immobilized on infected cells or are present on invading pathogens. Its activity stimulates phagocytic or cytotoxic cells to destroy microbes, or cells infected by antibody-mediated phagocytosis or antibody-dependent cellular cytotoxicity. Some viruses such as flaviviruses use Fc receptors to help them infect cells, by a mechanism known as antibody-dependent infection booster. FcRs were reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Inmunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995).
[00125] [00125] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of the complement. The activation of the classical complement path is initiated by the binding of the first component of the complement system (Cla) to antibodies (of the appropriate subclass) which are linked to its cogenate antigen. In order to assess complement activation, a CDC test can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1997).
[00126] [00126] The term "complement system" is used in the art to refer to a series of small proteins - called complement factors - found in the blood, usually circulating as inactive precursors (pro-proteins). The term refers to the ability of this unalterable and non-adaptable system to “complement” the ability of antibodies and phagocytic cells to clear pathogens such as bacteria, as well as antigen-antibody complexes, from an organism. An example of complement factors is the C1 complex, which includes C1ig and two serine protases, C1r and C1s. The C1 complex is a component of the CDC path. C1iq is a hexavalent molecule with a molecular weight of approximately 460,000 and a structure similar to a bouquet of tulips in which six collagen "stems" are connected to their main globular regions. In order to activate the complement cascade, C1q must bind at least two molecules of IgG1, IgG2 or 19G3.
[00127] [00127] "Antibody dependent cell cytotoxicity" or ADCC refers to a form of cytotoxicity in which lg secreted bound to Fc receptors (FcRs) present on certain cytotoxic cells - such as natural killer (NK) cells, neutrophils and macrophages - allow these cytotoxic effector cells to specifically bind to a target cell bringing antigen and subsequently kill the target cell with cytotoxins. Antibodies "arm" cytotoxic cells and are required for destruction of the target cell by this mechanism. Primary cells to mediate ADCC, NK cells, express FCcERIII only, whereas monocytes express FcERI, FcERII and FCERIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991). In order to assess the ADCC activity of a molecule of interest, an in vitro ADCC test can be performed, as described in United States Patent Nos. 5,500,362 or
[00128] [00128] Various antibody effector functions are mediated by Fc receptors (FcRs), which bind the Fc region of an antibody. FcRs are defined by their specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as FcER, for IgE as FcyR, for IgA as FcaR and so on. Three subclasses of FcER have been identified: FCERI (CD64), FcERII (CD32) and FcERII! (CD16).
[00129] [00129] Turning now to nucleic acids of the invention, a nucleic acid molecule encoding one or more strands of an anti-
[00130] [00130] In some embodiments, a nucleic acid sequence encoding a strand, such as a major strand and / or a minor strand of an antibody according to the invention is included in a vector such as a plasmid. Where a substitution or deletion should be included in an antibody chain, when compared to a domain or a naturally occurring region of an antibody, the coding sequence for the respective native domain / the respective native region, for example, included in the sequence of an immunoglobulin, can be used as a starting point for mutagenesis. For the mutagenesis of selected amino acid positions, the person skilled in the art has at his disposal the various routine methods established for site-directed mutagenesis. A commonly used technique is the introduction of mutations by means of PCR (polymerase chain reaction) using mixtures of synthetic oligonucleotides, which have a degenerate base composition at the desired sequence positions. For example, the use of the codon
[00131] [00131] The use of nucleotide building blocks with reduced specificity of base pairs, such as, for example, inosine, 8-0x0-2'deoxyguanosine or G6 (2-deoxy-B -D-ribofuranosyl) -3, 4-dihydro-8H-pirimin-do-1,2-0xazine-7-one (Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603), is another option for introducing mutations in a chosen sequence segment. An additional possibility is the so-called triplet mutagenesis. This method uses mixtures of different nucleotide triplets, each of which encodes an amino acid, for incorporation into the coding sequence (Virnekãs B, et al., 1994 Nucleic Acids Res 22, 5600-5607).
[00132] [00132] A nucleic acid molecule encoding a strand, such as a main strand and / or a minor strand of an antibody according to the invention can be expressed using any suitable expression system, for example in a suitable host cell or in a cell-free system. The antibody molecule obtained is enriched by means of selection and / or isolation.
[00133] [00133] As explained above, an antibody molecule according to the invention can be targeted against any desired target epitopes / antigens. Depending on the epitopes / antigens selected, the antibody may be suitable for the treatment or prevention of disease. Therefore, in some embodiments, an antibody according to the invention can be used in a method of treating and / or preventing a medical condition such as a disorder or disease. In modalities where one of the antibodies incorporated into a bispecific molecule is able to activate immune cells in an FcR-dependent manner, it may be particularly useful to select an antibody molecule that has a corresponding Fc-portion that shows reduced binding to Fc receptors . Hereby an undesirable immune activation mediated by FCR binding is prevented. In some modalities, a disease to be treated or prevented can be a proliferative disease. Examples of a proliferative disease include, but are not limited to, hematopoietic malignancies, such as acute and chronic lymphatic and myeloid leukemias, as well as lymphomas, or solid tumors. Examples of solid tumors include, but are not limited to, tumors of the gastrointestinal tract, bone, lung, kidney, prostate, breast, brain, ovary, uterine, testis, mesenchymal and skin tumors, such as melanoma.
[00134] The invention also provides a pharmaceutical composition that includes an antibody molecule of the invention and, optionally, a pharmaceutically acceptable excipient.
[00135] [00135] The antibody molecule according to the invention can be administered via any parenteral or non-parenteral (enteral) route that is therapeutically effective for proteinaceous drugs. Parenteral application methods include, for example, intracutaneous, subcutaneous, intramuscular, intratracheal, intranasal, intravitreal or intravenous injection and infusion techniques, for example, in the form of solutions for injection, solutions for infusion or tinctures, as well as installation and inhalation of aerosols, for example in the form of mixtures of aerosols, sprays or powders. An overview of pulmonary drug delivery, that is, either through inhalation of aerosols (which can also be used in intranasal administration) or through intracheal instillation is given by J.S. Patton and others. The lungs as a portal of entry for systemic drug delivery. Proc. Amer. Thoracic Soc. 2004 Vol. 1 pgs. 338-344, for example). Non-parenteral delivery modes are, for example, orally, for example, in the form of pills, tablets, capsules, solutions or suspensions, or rectally, for example, in the form of suppositories. Antibody molecules of the invention can be administered systemically or topically in formulations containing conventional non-toxic excipients or carriers, additives and pharmaceutically acceptable vehicles as desired.
[00136] [00136] In one embodiment of the present invention the pharmaceutical product is administered parenterally to a mammal, and in particular to humans. Corresponding administration methods include, but are not limited to, for example, intracutaneous, subcutaneous, intramuscular, intratracheal or intravenous injection and infusion techniques, for example, in the form of solutions for injection, solutions for infusion or dyes as well as installation and inhalation of aerosols, for example, in the form of mixtures for aerosols, sprays or powders. A combination of intravenous and subcutaneous infusion and / or injection may be more convenient for compounds with a relatively short serum half-life. The pharmaceutical composition can be an aqueous solution, an oil-in-water emulsion or a water-in-oil emulsion.
[00137] [00137] In this regard, it is observed that transdermal delivery technologies, for example, iontophoresis, sonophoresis or enhanced release by micro bubbles, as described in Meidan VM and Michniak BB 2004 Am. J. Ther. 11 (4): 312-316, can also be used for transdermal delivery of an antibody molecule described here, in this patent application. Non-parenteral delivery modes are, for example, oral, for example, in the form of pills, tablets, capsules, solutions or suspensions, or rectal administration, for example, in the form of suppositories. The antibody molecules of the invention can be administered systemically or topically in formulations containing a variety of conventional non-toxic excipients or carriers, additives, and pharmaceutically acceptable vehicles.
[00138] [00138] The dosage of the applied antibody molecule can vary within wide limits in order to obtain the desired preventive effect or the desired therapeutic response. For example, it will depend on the affinity of the antibody molecule for a chosen target as well as on the half-life of the complex between the antibody molecule and the in vivo ligand. In addition, the optimal dosage will depend on the biodistribution of the antibody molecule or a conjugate thereof, the mode of administration, the severity of the disease / disorder being treated as well as the patient's medical condition. For example, when used in an ointment for topical applications, a high concentration of the antibody molecule can be used. However, if desired, the antibody molecule can also be administered in a gradual release formulation, for example, liposomal dispersions or hydrogel-based polymeric microspheres, such as PolyActiveTM or OctoDEXTM (cf. Bos et al. ., Business Briefing: Pharmate- ch 2003: 1-6). Other gradual release formulations available are, for example, PLGA-based polymers (PR pharmaceuticals), PLA-PEG-based hydrogels (Medincell) and PEA-based polymers (Medivas).
[00139] [00139] Therefore, the antibody molecules of the present invention can be formulated into compositions using pharmaceutically acceptable ingredients as well as established preparation methods (Gennaro, AL and Gennaro, AR (2000) Remington: The Science and Practice of Pharmacy, 20th Ed,., Lippincott Williams & Wilkins, Philadelphia, PA). In order to prepare the pharmaceutical compositions, inorganic or pharmaceutically inert organic excipients can be used. To prepare, for example, pills, powders, gelatin capsules or suppositories, for example, lactose, talc, stearic acid and their salts, fats, waxes, solid or liquid polyols, natural and hydrogenated oils can be used. Excipients suitable for the production of solutions, suspensions, emulsions, mixtures of aerosols or powders for reconstitution in aerosol solutions or mixtures before use include water, alcohols, glycerol, polyols, and suitable mixtures thereof as well as vegetable oils .
[00140] [00140] The pharmaceutical composition can also contain additives, such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and, in addition, solvents or solubilizers or agents to obtain an effect deposit. The last is that fusion proteins can be incorporated into slow or gradual or targeted release systems, such as liposomes and microcapsules.
[00141] [00141] The formulations can be sterilized by numerous means,
[00142] [00142] There are numerous possible applications for the antibody molecule of the invention in medicine. In addition to its use in in vitro diagnostics or drug delivery, an antibody molecule of the invention can be generated, which binds, for example, tissue-specific or tumor-specific cell surface molecules.
[00143] [00143] The invention is further illustrated by the following non-limiting Examples. EXAMPLE |
[00144] [00144] A bispecific bispecific Fc-attenuated molecule, also designated to be of the format bsFc Ҽ-1/2, with specificity for tumor X CD3, as schematically represented in Fig. 1E. Amino acid modifications of the joint region and the CH2 domain were introduced as shown in Fig. 10. Bispecific Fc-attenuated tetravalent molecules were also generated, also designated to be of the bsFc Ҽ * -1 format, with tumor specificity. X CD3, as schematically represented in Fig. 1G. Amino acid modifications of the hinge region and the CH2 domain were introduced as shown in Fig. 1P.
[00145] [00145] Plasmid cloning and amplification was performed using Escherichia coli DH5a (Invitrogen, Karlsruhe, Germany. The development of the respective vectors is represented in Fig. 2.
[00146] [00146] Co-transfection of expression vectors encoding major and minor chains, which can also be referred to as heavy and light chains, of specificities indicated were made in plasmacytoma cells Sp2 / 0, obtained from the American Type Culture Collection (ATCC, Manassas, VA). For the development of
[00147] [00147] Bispecific antibodies were purified from cell culture supernatants stably transfected through affinity chromatography using protein A for the Fe-1 format and KappaSelect for the bsFc Ҽ-1/2 format (both media chromatography samples were obtained from GE Healthcare, Munich, Germany). EXAMPLE | l
[00148] [00148] Immunoglobulin V regions have been combined with the desired constant C regions in an expression vector. The cloning procedure indicated here allows the introduction of complete Ig V regions and their expression in lymphoid cells without any changes in their amino acid sequence. For this purpose, the nucleotide sequence of a VDJ and VJ fragment of a monospecific antibody was used to designate primer pairs (C C '; D D'; Table 1). The re-amplified DNA fragments from segments V were digested (VJ directly and VDJ after re-amplification with the primer pair E E 'Table 1) with appropriate restriction nucleases (summarized in Table 1) and then ligated into the expression vectors. Alternatively, V domains were synthesized as fragments of DNA at GeneArt, Regensburg, Germany. This method was used for genes encoding EGFR-directed antibody V regions (clone C225). The vectors (Figure 2) contain genes from human heavy and human light constant regions. Thus, the insertion of amplified and digested V segments reconstitutes the original genomic organization of the lg genes in the vectors without alterations.
[00149] [00149] The original vector for the heavy chain contains the Ig heavy chain of the human € E1 isotype (Fig. 2A). Restriction sites were introduced at the required positions in introns in order to exchange the Aatll-Clal fragment with the VDJ fragment of the monoclonal antibody heavy chain 4G8 (anti-FIt3), BV10 (anti-FLT3), 4G7 (anti -CD19), C225 (anti- = EGFR) and 9.2.27 (anti-CSPG4) or any other monoclonal antibody. The region relevant for cloning the VDJ fragment is shown enlarged in Figure 2B. The fragment to be exchanged contains parts of the first intron with an Aatll restriction site, the second exon of the leader sequence, the VDJ region and part of the heavy chain intron with the Clal restriction site. To replace all exons in the human heavy chain constant region [1 € 1 human, restriction sites were introduced at the required position in the heavy chain intron (Mlul) and in the 5-UTR heavy chain polyA region (pA region; Spel), as shown in Figure 2A and 2C.
[00150] [00150] In addition, with the expression vectors constructed, it is possible to exchange the entire constant region of the human Ig & 1 isotype (Mlul-Spel fragment; see Figure 2A) or against constant regions of all other antibody isotypes or against Fc parts with optimized or reduced effective function. In the case of antibodies optimized to trigger ADCC, amino acid substitutions have been introduced in the CH2 domain of the human E1 constant region as shown in International Patent Requirements Nos. WO 2011/076922 and WO 2011/089211. In order to generate bispecific antibodies as shown in Figs. 1A to IN, fragments of DNA flanked by Mlul and Spel containing or exons encoding for wild or modified constant domains of the Ig heavy chain can be inserted. The Mlul-Spel fragment to be exchanged is shown enlarged in Figure 2C. Adding the second antigen specificity of an anti-
[00151] [00151] The original vector for the light chain contains the human VJ light chain region and the gene 11) C region (Figure 2D). Restriction sites were introduced at the required locations (Xhol and Spel) in order to replace the Xhol-Spel light chain fragment with the appropriate VJ fragment of the 4G8 monoclonal antibody light chain (anti-FLT3), BV10 (anti -FLT3), 4G7 (anti-CD19), C225 (anti-EGFR) or
[00152] [00152] In this way, bispecific antibody molecules were obtained with FLT3xCD3 (4G8xUCHT1, BVIOXUCHT1), FLT3XTCRO / RB (4G8xBMAO31, BVIOXBMAO31), FLT3XCD28 (4G8x9.3, BV10x9CD8G3 (FL103), 4G7xUCHTI1), CD19xTCRo / B (4G7xBMAO31), CD1I9xCD28 (4G67x9.3), CD19xCD16 (4G7x3G8), - “CSPG4xCD3 (9.2.27xUCHT1I) CSPG4xXTCTCW BB (9.2.27XCDPG4) (9.2.27 - CSPG4) () 9.2.27x3G8), - EGFRxCD3 (C225xUCHT1I) EGFRXTCRW BB (C225xBMAO031), EGFRXCD28 (C225x9.3), EGFRXCD16 (C225x3G8) as bsFc '* º-1 tetravalente e bsFc' * 1-b e * bivalent e bs Sequences of the corresponding strands are represented as SEQ ID NO: 1 to SEQ ID NO: 26 and in Fig. 6.
[00153] [00153] Co-transfection of expression vectors encoding the heavy and light chimeric chain (I9G1 / 7) or modified heavy chains in the non-lg Sp2 / 0 myeloma cell line produced stable transfectomas secreting bispecific monoclonal antibodies which are capable to bind specifically to the desired antigen. The functional characterization of these antibody molecules is illustrated in the experiments that follow using bispecific antibody molecules FLT3xCD3, CD19xTCRo / B and CSPGXCD3. EXAMPLE Ill
[00154] [00154] T cell activation was determined by the two antibody formats of Example |, the bsFc "º-1/2 format and the bsFc" -1 format, with and without FLT3 / CD19 positive REH cells. The data are shown in Fig. 3. The bispecific antibody molecules used had the FLT3 binding site (first binding site) of clone 4G8 and a CD3 binding site (second binding site) of clone U-
[00155] [00155] REH cells (Deutsche Sammlung fúr Mikroorganismen und Zellkulturen, DSMZ, Braunschweig, Germany) and PBMCs cells were grown in RPMI 1640 medium, supplemented with 10% calf fetus serum (PAN-Biotech, Aidenbach, Germany), 1 % of penicillin and streptomycin (Lonza, Basel, Switzerland). EXAMPLE IV
[00156] [00156] Lysis of REH cells expressing FLT3 / CD19 (Fig. 4A) and SKMel63 cells expressing CSPG (Fig. 4B) was determined by bispecific antibodies and activated CD8 + T killer cells.
[00157] [00157] Human mononuclear cells (PBMCs) were stimulated using the UCHT1 monospecific CD3 antibody (10 ng / ml) for three days. Then activated CD8 + T cells were isolated by positive selection using magnetic cell classification. The cells were added to FLT3 / CD19 positive REH cells marked with “Cr (Fig. 4A) or CSPG4 positive SKMel63 cells (Fig. 4B), and incubated with antibodies in concentrations as indicated. After 4 hours, cell supernatants were harvested on scintillation plates and radioactivity was determined in a scintillation counter.
[00158] [00158] Percent specific lysis was analyzed under experimental conditions defined as follows: cpmí (exp) -cpom (bg) / cpm (100) -cpm (bg), where cpm (bg) corresponds to the release of chromium without antibody and effector cells, and with (100) corresponds to the release of chromium after incubation of target cells with a detergent.
[00159] [00159] SKMel63 cells were obtained from Dr. B. Gúckel, Klinik fúr Gynâkologie, University of Túbingen, Germany.
[00160] [00160] EXAMPLE V
[00161] [00161] The rate of aggregation and production of FLT3 X CD3 antibodies (FLT binding site: clone 4G8, CD3 binding site: UCHT1 clone) having identical specificity was compared between three different formats: bispecific single chain (bs-scFv), bsFc “º-1/2 format, bsFc“ º-1 format. The antibody molecule of the “bsFcº-1/2 format” consisted of the SEQ ID NO: 1 and SEQ ID NO: 6 chains and the “bsFc format“ º-1 ”antibody molecule consisted of the SEQ chains ID NO: 1 and SEQ ID NO: 26.
[00162] [00162] Bispecific single chain molecules were purified by affinity chromatography using protein L.
[00163] [00163] Gel filtration was performed using a superdex 200 PC3.2 / 80 column and a SMARTSystem (GE-Healthcare, Munich, Germany). The standard proteins used were catalase (232 kDa, from bovine liver), aldolase (158 kDa; from rabbit muscle), albumin (67 kDa; from bovine serum) and ribonuclease A (13.7 kDa; from bovine pancreas). The results are shown in Fig. 5A. It is evident from Fig. 5A that the formation of aggregates is considerably more pronounced if the antibody is expressed as bs-scFv (43% aggregation rate) instead of bsFcko-1/2 (no aggregation detected) or bsFcko- 1 (2% aggregation rate), i.e., the bispecific antibody molecules of the present invention remain monomeric molecules with essentially no tendency to aggregate.
[00164] [00164] To compare the production rates of genes encoding bispecific molecules containing the specificity for 4G8 (anti-FLT3) and the specificity for UCHT1 (anti-CD3) - in the formats represented, they were introduced in Sp2 / 0 cells and antibodies were purified using affinity chromatography. The amount of antibody purified from the supernatants of clones selected for maximum production is shown in Fig. 5B. Antibody concentrations were determined by optical spectroscopy assuming an optical density at 280nm of 1.4 for an antibody concentration of 1 mg / ml. The production rates for the bsFcko-1/2 and bsFcko-1 antibody molecules of the invention were significantly higher than for the respective bs-scFv molecule.
[00165] [00165] A person skilled in the art will readily recognize that the present invention is well adapted to achieve the objectives and obtain the mentioned purposes and advantages, as well as those inherent in it. Furthermore, it will be readily apparent to a person skilled in the art that variable substitutions and modifications can be made to the invention disclosed here, in this patent application, without departing from the scope and spirit of the invention. The specific compositions, methods, procedures, treatments, molecules and compounds described here, in this patent application, are currently representative of certain modalities are examples and are not intended to be considered as limitations of the scope of the invention. Modifications of the same and other uses will occur for people skilled in the art which are included in the spirit of the invention are defined by the scope of the claims. The listing or discussion of a document previously published in this specification should not necessarily be taken as an acknowledgment that the document is part of the state of the art or is common knowledge.
[00166] [00166] The invention illustratively described here, in this patent application, can be practiced conveniently in the absence of any element or elements, limitations or limitations, not specifically disclosed here, in this patent application. Thus, for example, the terms "comprising", "including," containing ", etc. should be read expansively and without limitation. In addition, the terms and expressions used here, in this patent application, were used as terms of description and not of limitation, and there is no intention in using such terms and expressions to exclude any equivalents of the characteristics shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Thus, it should be understood that although the present invention has been specifically revealed by exemplary modalities and optional features, modification and variation of the inventions incorporated therein can be used by those skilled in the art, and that similar modifications and variations are considered to be within the scope of this invention.
[00167] [00167] The invention has been widely and generically described here, in this patent application. Each of the more limited species and subgeneric groups that are within the generic revelation is also part of the invention. This includes the generic description of the invention with a negative condition or limitation removing any subject of its kind, regardless of whether or not the excised material is specifically mentioned here, in this patent application.
[00168] [00168] Other modalities are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, persons skilled in the art will recognize that the invention is also described in this way in terms of any individual member or subgroup of members of the Markush group. Reference List 1 Davis, L., Vida, R. and Lipsky, P. E. Regulation of human T imymphocyte mitogenesis by antibodies to CD3, J. Immunol., 137: 3758- 3767, 1986.
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权利要求:
Claims (16)
[1]
1. Recombinant bispecific antibody molecule, characterized by the fact that it consists of a Fab fragment that comprises a first binding site for a first antigen, a single chain Fv fragment comprising a second binding site a second antigen and an immunoglobulin CH2 domain, in which the Fab fragment and the single-chain Fv fragment are linked through the CH2 domain, in which at least one amino acid residue from the CH2 domain is capable of mediating binding to receptors of Fc is absent or mutated, and in which additionally one or more amino acid residue (s) of sequence positions 226, 228 and 229 is absent or mutated.
[2]
2. Antibody molecule according to claim 1, characterized in that: a) in which either the first binding site or the second binding site binds to a tumor-associated antigen, in which (i) the antigen associated with a tumor, it is preferably located on the vasculature of a tumor; and / or (ii) the tumor-associated antigen is preferably a surface antigen or an extracellular matrix antigen / and / or (ili) the tumor-associated antigen is preferably selected from the group consisting of CD10 , CD19, CD20, CD 21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CDw5 2, tyrosine kinase similar to Fms 3 (FLT-3, CD135), c-Kit (CD117 ), CSF1IR, (CD115), CD133, PDGFR-a (CD140a), PDGFR-B (CD140b), chondroitin sulfate proteoglycan 4 (CSPGA, chondroitin sulfate proteoglycan 4 associated with melanoma), Muc-1, EGFR, de2- 7-EGFR, EGFRvII, Folate-binding protein, Her2neu, Her3, PSMA, PSCA, PSA, TAG-72, HLA-DR, IGFR, CD133, IL3R, fibroblast activation protein (FAP), Carboanidrase IX ( MN / CA IX), Carcinoembryonic antigen (CEA), ECAM, CDCP1, Derlin1, Tenascina, frizzled 1-10, vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), Endoglin, CLEC14, Tem1-8 , and Tie2; and / or b) in which either the first binding site or the second binding site preferably binds to a receptor molecule specific for T cells or NK (natural killer) cells,
wherein the specific T cell or NK cell receptor molecule is preferably one of CD3, the T cell receptor (TCR), CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5 and CD95 ,
wherein the TCR is preferably TCR (alpha / beta) or TCR (gamma / delta); and / or c) the Fab fragment is linked to the CH2 domain via the CH1 and VH domains of the Fab fragment heavy chain or via the light chain CL and VL domains of the Fab fragment,
wherein the Fab fragment heavy chain domains or the Fab fragment light chain domains are preferably arranged at the N-terminus of the polypeptide chain;
wherein the CH2 domain is preferably linked to the scFv fragment via the variable domain of the light chain (VL domain) of the scFvy fragment comprising the second binding site or via the variable domain of the heavy chain (VH domain) of the frag - scFv element comprising the second binding site; or
(iv) the Fab fragment that comprises the first binding site for the first antigen, preferably consists of the VL domain fused to the CH1 domain and the VH domain fused to the CL domain,
wherein the CH1 domain of the Fab fragment is preferably fused to the CH2 domain, and / or where the VL-CH1 chain of the Fab fragment is preferably arranged at the N-terminus of the polypeptide chain; and / or d) in which the Fab fragment comprises an articulation region; and / or e) where the first binding site binds to a tumor-associated surface antigen and the second binding site binds to one between CD3, the T cell receptor (TOR), CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5 and CD95.
[3]
Antibody molecule according to claim 1 or 2, characterized by the fact that at least one amino acid residue in the CH2 domain that is capable of mediating binding to Fc receptors is absent or mutated, is selected from the group consisting of sequence position 230, 231, 232, 233, 234, 235, 236, 237, 238, 265, 297, 327, and 330 (numbering of sequence positions according to the European Union Index (EU-index) ).
[4]
4. Antibody molecule according to claim 1, characterized in that a cysteine in one or both of positions 226 and 229 is replaced by a different amino acid.
[5]
5. Antibody molecule according to claim 3 or 4, characterized in that it comprises at least one mutation selected from the group consisting of an elimination of amino acid 228, an elimination of amino acid 229, an elimination of amino acid 230, an elimination of amino acid 231, an elimination of amino acid 232, an elimination of amino acid 233, a substitution Glu233 — Pro, a substitution Leu234—- Val, an elimination of amino acid 234, a substitution Leu235 — Ala, an elimination of amino acid 235, an elimination of amino acid 236, an elimination of amino acid 237, an elimination of amino acid 238, an Asp265-Gly substitution, an Asn297> Gln substitution, an Ala327> Gln substitution, and an Ala330-Ser substitution.
[6]
6. Recombinant bispecific antibody molecule, characterized by the fact that it consists of a Fab fragment that comprises
has a first binding site for a first antigen, a single chain Fv fragment comprising a second binding site for a second antigen, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain, in which the fragment Fab and the single chain Fv fragment are linked to each other via the CH2 domain and CH3 domain, and at least one amino acid residue from the CH2 domain that is capable of mediating binding to an Fc receptor is absent or mutated, wherein the at least one amino acid residue of the CH2 domain that is capable of mediating binding to an absent or mutated Fc receptor is selected from the group consisting of sequence positions 228, 230, 231, 232, 233, 234 , 235, 236, 237, 238, 265, 297, 327 and 330 (numbering of sequence positions according to the European Union Index), in which a) the first binding site binds to a receptor molecule associated with T cell or NK cell and where the second binding site binds to a sup antigen tumor-associated skin; or b) the second binding site binds to a T cell or NK cell-associated receptor molecule and where the first binding site binds to a tumor-associated surface antigen.
[7]
7. Antibody molecule according to claim 6, characterized by the fact that: a) it comprises at least one mutation selected from the group consisting of an elimination of amino acid 228, an elimination of amino acid 230, an elimination of amino acid 231, an elimination of amino acid 232, an elimination of amino acid 233, a substitution Glu233—> Pro, an elimination of amino acid 234, a substitution of amino acid Leu234 -— Val, an elimination of amino acid 235, a substitution Leu235 — Ala, an elimination of amino acid 236, a deletion of amino acid 237, a deletion of amino acid 238, a substitution Asp265 — Gly, a substitution
Asn297 5Gln, an Ala327-GInh replacement, and an Ala330 replacement — Ser; and / or b) in which the tumor-associated surface antigen is selected from the group consisting of CD10, CD19, CD20, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CDw52, ti - Fms 3-like rosina kinase (FLT-3, CD135), c-Kit (CD117), CSF1IR (CD115), CD133, PDGFR-a (CD140a), PDGFR-B (CD140b), chondroitin sulfate proteoglycan 4 ( CSPGA4, chondroitin sulfate proteoglycan 4 associated with melanoma), Muc-1, EGFR, de2-7-EGFR, EGFRvIIl, Folate-binding protein, Her2neu, Her3, PSMA, PSCA, PSA, TAG-72, HLA-DR , IGFR, CD133, IL3R, fibroblast activation protein (FAP), Carboanidrase IX (MN / CA IX), Carcinoembryonic antigen (CEA), EBPCAM, CDCP1, Derlin1, Tenascin, frizzled 1-10, VEGFR2 vascular antigens (KDR / FLK1), VEGFR3 (FLT4, CD309), Endoglin, CLEC14, Tem1-8, and Tie2; and / or c) where the T cell or NK cell associated molecule is one among CD3, the T cell receptor (TCR), CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5 and CD95, wherein the TCR is preferably TOR (alpha / beta) or TCR (gamma / delta).
[8]
Antibody molecule according to any one of claims 1 to 7, characterized in that the Fab fragment is linked to the CH2 domain via a heavy chain domain of the Fab fragment or via a light chain domain of the Fab fragment , wherein the Fab fragment heavy chain domains are preferably arranged at the N-terminus of the polypeptide chain.
[9]
Antibody molecule according to claim 6, characterized in that the CH2 / CH3 domains are linked to the scFv fragment via the variable domain of the light chain (VL domain) of the scFv fragment comprising the second binding site or via the heavy chain variable domain (VH domain) of the scFv fragment comprising the second binding site.
[10]
Antibody molecule according to any one of claims 6 to 9, characterized in that the first binding site binds to a tumor-associated surface antigen and the second binding site binds one between CD3, the T cell receptor (TCR), CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5 and CD95, wherein a) the antibody molecule preferably comprises a cysteine residue at the 226 and / or 229 sequence position (numbering of sequence positions according to the European Union index), and / or in which the antibody molecule preferably comprises at least a modification in the CH3 domain that prevents the dimerization of this domain; or b) at least one cysteine amino acid residue that is capable of forming a disulfide bridge for dimerization is preferably absent or mutated.
[11]
11. Tetrameric antibody molecule, characterized in that it consists of a dimer of the bispecific antibody molecule, as defined in any one of claims 6 to 9, wherein the tetrameric antibody molecule preferably comprises a disulfide bridge between the articulation regions of the bispecific antibody molecule, where the disulfide bridge is preferably formed by at least one between the cysteine residue in the sequence position 226 or 229 (sequence position numbering according to the index of the European Union).
[12]
12. Pharmaceutical composition, characterized by the fact that it comprises an antibody molecule, as defined in any one of claims 1 to 11.
[13]
13. Antibody molecule, as defined in any of claims 1 to 11, characterized by the fact that it is for use in the treatment of a disease, in which the disease is preferably a proliferative disease selected from the group that consists of hematopoietic malignancies, such as acute and chronic lymphatic and myeloid leukemias, as well as lymphomas, solid tumors such as tumors of the gastrointestinal tract, lung, kidney, prostate, breast, brain, ovarian, uterine, mesenchymal tumors and melanoma.
[14]
14. Nucleic acid molecule, characterized by the fact that it encodes an antibody molecule, as defined in any one of claims 1 to 11, preferably comprised in a vector or a host cell comprising said nucleic acid molecule or the said vector.
[15]
15. Method of producing an antibody molecule, as defined in any of claims 1 to 11, characterized by the fact that it comprises the expression of a nucleic acid encoding the antibody molecule under conditions that allow the expression of the nucleic acid , in which the antibody molecule is preferably expressed in a host cell or cell-free system.
[16]
16. Invention, characterized by any of its concretizations or categories of claim encompassed by the material initially revealed in the patent application or in its examples presented here.

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同族专利:

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公开号 | 公开日

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ES2628075T3|2017-08-01|

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DK2794658T3|2017-06-19|

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US20150119555A1|2015-04-30|

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WO2013092001A1|2013-06-27|

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SI2794658T1|2017-05-31|

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US9718893B2|2017-08-01|

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JP2015502373A|2015-01-22|

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HRP20170658T1|2017-06-30|

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LT2794658T|2017-05-10|

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JP6441079B2|2018-12-19|

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CA2859767A1|2013-06-27|

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EA201400709A1|2016-08-31|

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EP2794658A1|2014-10-29|

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CA2859767C|2018-09-11|

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CN104203981A|2014-12-10|

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US20180057608A1|2018-03-01|

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HUE033245T2|2017-11-28|

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EP2794658B1|2017-03-15|

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法律状态:
2020-11-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-01-19| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-03-16| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-04-27| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201161577327P| true| 2011-12-19|2011-12-19|

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US61/577,327|2011-12-19|

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PCT/EP2012/072364|WO2013092001A1|2011-12-19|2012-11-12|Bispecific antibody molecule|

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