Anti-alpha beta TCR antibodies

Abstract

The present invention relates to anti-[alpha] [beta] TCR antibodies, in particular to humanized monoclonal antibodies of CDR comprising the murine antibody BMA031, which bind to the [alpha] [beta] TCR.CD3 complex and have improved biological properties.

Description

[0001] This invention application is a divisional application based on an invention patent application with an application date of September 12, 2012, application number 201910265294.3, and name “Anti-αβTCR Antibody”. Technical Field

[0002] The present invention relates to antibodies specific for αβT cell receptor (αβTCR). In particular, the present invention relates to humanized anti-αβTCR antibodies derived from murine monoclonal antibody BMA031 and the use of the humanized antibodies in immunosuppressive therapy. Background Art

[0003] introduce

[0004] The use of immunosuppressants in autoimmune disease and organ transplant therapy is well documented; however, this approach is far from optimal. Toxicity, opportunistic infections, cytokine storms, and increased cancer risk are prevalent in patients treated with these agents. The use of biologics in this setting has improved patient outcomes to some extent, but these side effects are still evident.

[0005] For immunosuppression, the use of polyclonal antisera directed against lymphocytes is well known. However, the production of antisera is extremely laborious, they show properties that vary between different batches, and the specificity that can be obtained using polyclonal antisera is limited.

[0006] Monoclonal antibodies were first produced by hybridoma technology. and Milstein (Nature 256: 495-497 (1975)). Compared with polyclonal antisera, monoclonal antibodies (mAbs) are more specific and have more consistent properties. mAbs have been most frequently and successfully used for immunosuppressive therapy in clinical organ transplantation. However, most mAbs used as immunosuppressants for the treatment of autoimmune diseases and for transplant patients have broad immunosuppressive capacity, thus undesirably affecting the function of many immune cells, which are not presumed to be all involved in graft rejection.

[0007] Mouse monoclonal antibodies against T cell surface receptor antigens were first produced in 1979 using hybridoma technology (Kung et al. (1979) Science 206:347-349). Among the three monoclonal antibodies discovered by Kung et al., one antibody was named muromonab-CD3 (OKT3), which has a defined specificity for the CD3 receptor of T cells, reacting with more than 95% of peripheral mature T cells without affecting immature thymocytes. Binding of OKT3 to the CD3 complex causes internalization of the CD3 receptor and loss of CD3-positive cells from the periphery. Successful OKT3 treatment is associated with a rapid decline in CD3-positive T cells from approximately 60% to less than 5%.

[0008] OKT3 has been widely used to treat patients who experience acute allograft rejection after renal transplantation (Russell, PS, Colvin, RB, Cosimi, AB (1984) Annu. Rev. Med. 35:63 and Cosimi, AB, Burton, RC, Colvin, RB, et al. (1981) Transplantation 32:535). In addition, OKT3 and rabbit complement are used to eliminate mature T cells from donor bone marrow to prevent acute graft-versus-host disease (GVHD) in allogeneic bone marrow transplantation (Prentice, HG, Blacklock, HA, Janossy, G., et al. (1982) Lancet 1:700 and Blacklock, HA, Prentice, HG, Gilmore, MJ, et al. (1983) Exp. Hematol. 11:37). Although OKT3 treatment appears to be effective in preventing GVHD in allogeneic bone marrow transplantation for acute leukemia, combined in vitro / in vivo treatment with OKT3 failed to prevent GVHD during treatment of severe combined immunodeficiency (Hayward, AR et al. (1982) Clin. Lab. Observ. 100: 665). Treatment of T cells with OKT3 induces several responses inconsistent with immunosuppression, including T cell activation, production of immune mediators, and T3 regulation. It is assumed that the T3 antigen complex recognized by CD3-mAb (e.g., OKT3) plays a key role during T cell activation. α / β T lymphocytes recognize peptide-MHC ligands by means of a multimeric protein collectively referred to as the αβ T cell antigen receptor (TCR)·CD3 complex. This structure consists of a variable αβTCR dimer that binds antigen and three invariant dimers (CD3γε, δε, and ζζ), which are involved in TCR·CD3 surface transport, stability, and signal transduction. The αβT cell receptor (αβTCR) is expressed on most (about 95%) T cells and plays a key role in T cell activation through engagement of antigens displayed on MHC. The remaining 5% of cells are γδT cell receptor (γδTCR) positive. The γδTCR-positive cell population plays an important role in the innate immune response that defends against opportunistic infections of bacterial, viral and fungal origin. γδT cells do not play a role in graft rejection in transplantation. Therefore, targeting the αβTCR-positive cell population and sparing the γδTCR-positive population should allow for significant therapeutic effects while maintaining baseline immune protection against opportunistic infections.

[0009] The mouse IgG2b monoclonal antibody BMA031 (Borst et al. (November 1990) Hum. Immunol. 29 (3): 175-88; EP0403156) is specific for a common determinant on the TCRα / β / CD3 complex and does not bind to the γ-δ TCR. BMA031 is highly immunosuppressive and can induce apoptosis of activated T cells via an activation-induced cell death (AICD) mechanism (Wesselborg et al. (May 1993) J. Immunol. 150 (10): 4338-4345). It inhibits mixed lymphocyte reactions in vitro and has shown preliminary clinical efficacy in the prevention of graft rejection in a number of solid organ transplant settings and in the treatment of acute graft-versus-host disease (aGVHD) (Kurrle et al. (February 1989) Transplant Proc. 21 (1): 1017-1019). BMA031 does not bind human Fcγ receptors (FcγRs) in the majority of the human population (approximately 10% of humans have FcγRs that bind the mouse IgG2b isotype). Thus, the antibody does not cause T cell activation via T cell receptor cross-linking and, therefore, does not induce T cell activation or associated cytokine release. In this regard, its profile is highly preferred relative to that of OKT3. However, BMA031 is a murine antibody and, therefore, is not suitable for repeated dosing in human subjects due to the human anti-mouse antibody (HAMA) response it elicits in human subjects.

[0010] Several humanized forms of BMA031 have been described (see, EP 0403156; see also Shearman et al., (1991) J. Immunol. 147:4366-4373). As described in EP0403156, CDR grafting alone did not successfully maintain antigen binding. A clone, EUCIV3, with significant framework modifications, successfully bound to T cells; however, as described in EP0403156, binding to the αβ TCR was not as effective as the parental BMA031 antibody, as determined by flow cytometry competition assays. We also showed that the ability of EuCIV3 to inhibit in vitro immune responses was significantly reduced compared to BMA031 (see Figure 2 ). In addition, EuCIV3 was originally produced on a wild-type human IgG1 or IgG4 backbone that still retains FcγR binding. Therefore, these humanized antibodies allow T cell activation, proliferation and concomitant cytokine release and thus differ significantly from the original properties of BMA031.

[0011] Modification of antibody glycosylation is known in the art. For example, it is known that aglycosylated antibodies can have extensively modified functions; see Boyd et al. (1996) Mol. Immunol. 32: 1311-1318. However, aglycosylated forms of humanized BMA031 or derivatives with modified glycosylation patterns have not been described previously.

[0012] Therefore, there is a need in the art for anti-αβ TCR humanized antibodies that improve the binding properties of EUCIV3 and advantageously retain the immunosuppressive and non-T cell activating properties of BMA031. Summary of the invention

[0013] In a first aspect, a humanized monoclonal antibody is provided that comprises the CDRs of BMA031 and retains the binding affinity of BMA031 to its cognate antigen. In a first embodiment, the humanized antibody comprises a heavy chain variable region comprising the CDRs described in SEQ ID NO: 7, 12 or 13 and the human IGH3-23 framework described in SEQ ID NO: 17, wherein framework position 6 is a donor residue; in an alternative embodiment, framework position 18 is a donor residue. Optionally, framework positions 49 and / or 69 are donor residues.

[0014] In a second embodiment, the humanized monoclonal antibody comprises a heavy chain variable region comprising the CDRs set forth in SEQ ID NO: 15 or 16 and the human IGHV1-3*01 framework set forth in SEQ ID NO: 18, wherein one or more of framework positions 38, 44 and / or 48 are donor residues; in an alternative embodiment, framework positions 44 and 48 are donor residues.

[0015] In a third embodiment, the humanized monoclonal antibody comprises a light chain variable region comprising the CDRs set forth in SEQ ID NO: 14 and the human IGKV3-11*01 framework set forth in SEQ ID NO: 19, wherein framework positions 70 and / or 71 are donor residues. Optionally, position 46 is a donor residue.

[0016] Examples of the antibody according to the first embodiment include antibodies comprising a heavy chain variable region selected from the heavy chains comprising the sequences described in SEQ ID NO:7, SEQ ID NO:12 and SEQ ID NO:13, and a light chain variable region sequence comprising the sequence described in SEQ ID NO:14.

[0017] Examples of antibodies of the second embodiment include antibodies comprising a heavy chain variable region and a light chain variable region sequence, wherein the heavy chain variable region is selected from a heavy chain comprising the sequence described in SEQ ID NO: 15 and SEQ ID NO: 16, and the light chain variable region sequence comprises the sequence described in SEQ ID NO: 14. The humanized antibody according to the embodiment is a humanized form of BMA031. Its primary structure is different from that of the humanized antibody EuCIV3, and EuCIV3 has reduced binding to αβTCR compared to BMA031.

[0018] In the sequence listing, CDRs are indicated by annotation or underlining. Frameworks are all sequences outside of CDRs, which are defined according to the "Kabat" numbering system and extended by the use of the "IMGT" CDR definition when applicable. If there is no indication to change a framework residue to match a donor sequence, the residue should generally be understood as an acceptor residue.

[0019] A humanized antibody may comprise a constant region. In one embodiment, the constant region is of human origin.

[0020] The humanized antibodies of the present invention can be further modified by Fc engineering. Immunoglobulins are prone to cross-linking Fcγ receptors, which can lead to constitutive T cell activation of anti-T cell antibodies. To avoid Fcγ cross-linking, antibodies can be modified to remove the Fc region (e.g., by producing Fab or Fv fragments); however, truncated immunoglobulins lack beneficial effector functions and exhibit shorter serum half-lives. Therefore, the Fc region of the humanized antibody can be modified to prevent Fcγ cross-linking. Exemplary techniques include producing non-glycosylated immunoglobulins (e.g., by modifying the Fc region by N297Q mutations). Immunoglobulins that cannot bind to Fcγ are also described by Armour et al. (1999) Eur. J. Immunol. 29: 2613-2624. The modification to achieve IgG1 is called Δab modification and consists of a combination of Δa mutations (wherein IgG residues are substituted at positions 327, 330 and 331, and IgG2 residues are substituted at positions 233-236) and Δb mutations (wherein residue 236 is deleted). In another embodiment, the glycosylation pattern of the antibodies according to the invention may be modified.

[0021] In one embodiment, the antibody comprises one or more of the mutations S298N, T299A and Y300S.

[0022] In an embodiment, the antibody comprises two or more of the mutations N297Q, S298N, T299A and Y300S. For example, a humanized antibody comprising multiple mutations N297Q / S298N / Y300S, S298N / T299A / Y300S or S298N / Y300S is provided.

[0023] In a second aspect, a humanized monoclonal antibody is provided that comprises the CDRs of BMA031 and retains the T cell inhibitory properties of BMA031. The humanized antibody preferably comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:12, 13, 15 or 16 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14.

[0024] In a third aspect, a nucleic acid encoding at least the heavy chain variable region of a humanized monoclonal antibody according to the aforementioned aspects of the embodiment is provided. The nucleic acid may encode the variable region and constant region of the humanized antibody. The heavy chain and light chain may be encoded on different nucleic acids or on the same nucleic acid molecule.

[0025] According to a fourth aspect, a cell expressing a nucleic acid according to the preceding aspects is provided. The cell is, for example, a cell in culture suitable for expressing an antibody molecule. The nucleic acid may include a signal sequence and / or other sequences or modifications that are necessary for, or regulate, the expression of the antibody molecule in the cell and / or the secretion of the antibody molecule from the cell.

[0026] In yet another embodiment, a humanized antibody is provided as described in the above aspects for use in inhibiting a T cell-mediated response in a subject.

[0027] For example, T cell-mediated responses may be involved in a condition selected from the group consisting of tissue transplantation (including solid organ transplantation and composite tissue transplantation), tissue grafting, multiple sclerosis, and type 1 diabetes.

[0028] Furthermore, another embodiment provides a method for treating a subject suffering from a condition involving an aberrant T cell-mediated response, comprising administering to a subject in need thereof a therapeutically effective dose of an antibody according to said embodiments.

[0029] Therefore, humanized non-activated anti-αβTCR antibodies that do not induce cytokine release have been produced, which can selectively regulate αβTCR and induce apoptosis of activated αβTCR positive T cells. These antibodies have been produced for use as immunosuppressants in T cell-mediated diseases. These antibodies have been produced by humanization of mouse anti-αβTCR antibody BMA031 and by engineering the Fc of humanized antibodies to prevent engagement of Fcγ receptors. Unlike the humanized form of BMA031 available in the art, the antibody according to the embodiment retains the binding affinity of BMA031. In addition, as shown in an in vitro education assay, the immunosuppressive properties of the antibody according to the embodiment are superior to BMA031. Moreover, unlike the humanized BMA031 antibody of the prior art, the antibody according to the embodiment does not induce cytokine release in normal PBMCs.

[0030] According to a fifth aspect, there is provided an antibody comprising a modified Fc, wherein the modified Fc comprises a modified glycosylation pattern that reduces FcγR receptor binding, the antibody comprising one or more of the mutations S298N, T299A and Y300S.

[0031] In one embodiment, the antibody comprises two or more of the mutations N297Q, S298N, T299A and Y300S.

[0032] In an embodiment, the antibody comprises the multiple mutations N297Q / S298N / Y300S, S298N / T299A / Y300S or S298N / Y300S.

[0033] For example, the antibody may be an antibody as described in the preceding aspects of the invention.

[0034] According to a sixth aspect, a multispecific antibody is provided, which comprises at least the heavy chain of the first binding domain according to the aforementioned aspect of the present invention and a second binding domain having specificity for a tumor-specific antigen.

[0035] In one embodiment, the first binding domain comprises a heavy chain according to the second aspect of the invention.

[0036] Multispecific antibodies can comprise many different configurations; in one embodiment, they comprise an anti-TCR / CD3 scFv and an anti-tumor scFv.

[0037] In one embodiment, the multispecific antibody has bispecificity.

[0038] Specifically, the present invention relates to the following:

[0039] 1. A humanized monoclonal antibody specific for a human αβTCR / CD3 complex, comprising a heavy chain variable region comprising the CDRs described in SEQ ID NO: 7, 12 or 13 and the human IGH3-23 framework described in SEQ ID NO: 17, wherein framework position 6 is a donor residue.

[0040] 2. A humanized monoclonal antibody specific for a human αβTCR / CD3 complex, comprising a heavy chain variable region comprising the CDRs described in SEQ ID NO:7, 12 or 13 and the human IGH3-23 framework described in SEQ ID NO:17, wherein framework position 18 is a donor residue.

[0041] 3. A humanized monoclonal antibody specific for the human αβTCR / CD3 complex according to item 1 or 2, wherein framework positions 49 and / or 69 are donor residues.

[0042] 4. A humanized monoclonal antibody specific for a human αβTCR / CD3 complex, comprising a heavy chain variable region comprising the CDRs set forth in SEQ ID NO: 15 or 16 and the human IGHV1-3*01 framework set forth in SEQ ID NO: 18, wherein one or more of framework positions 38, 44 and / or 48 are donor residues.

[0043] 5. A humanized monoclonal antibody specific for a human αβTCR / CD3 complex, comprising a heavy chain variable region comprising the CDRs described in SEQ ID NO: 15 or 16 and the human IGHV1-3*01 framework described in SEQ ID NO: 18, wherein framework positions 44 and 48 are donor residues.

[0044] 6. A humanized monoclonal antibody specific for a human αβTCR / CD3 complex, comprising a light chain variable region comprising the CDRs described in SEQ ID NO: 14 and the human IGKV3-11*01 framework described in SEQ ID NO: 19, wherein framework positions 70 and / or 71 are donor residues.

[0045] 7. A humanized monoclonal antibody specific for the human αβTCR / CD3 complex according to item 6, wherein framework position 46 is a donor residue.

[0046] 8. A humanized monoclonal antibody specific to human αβTCR / CD3 complex, comprising a heavy chain variable region and a light chain variable region sequence, wherein the heavy chain variable region is selected from the heavy chain comprising the amino acid sequence described in SEQ ID NO:7, SEQ ID NO:12 and SEQ ID NO:13, and the light chain variable region sequence comprises the amino acid sequence described in SEQ ID NO:14.

[0047] 9. A humanized monoclonal antibody specific to the human αβTCR / CD3 complex, comprising a heavy chain variable region and a light chain variable region sequence, wherein the heavy chain variable region is selected from the heavy chain comprising the amino acid sequence described in SEQ ID NO: 15 and SEQ ID NO: 16, and the light chain variable region sequence comprises the amino acid sequence described in SEQ ID NO 14.

[0048] 10. The humanized antibody according to any preceding item, further comprising a constant region of human origin.

[0049] 11. The humanized antibody according to item 10, further comprising an Fc modification that reduces Fcγ receptor binding.

[0050] 12. The humanized antibody according to item 11, comprising a non-glycosylated Fc region or a δab modification.

[0051] 13. A humanized antibody according to item 11, comprising a modified glycosylation pattern.

[0052] 14. A humanized antibody according to item 13, comprising one or more of the mutations S298N, T299A and Y300S.

[0053] 15. A humanized antibody according to item 14, comprising two or more of the mutations N297Q, S298N, T299A and Y300S.

[0054] 16. A humanized antibody according to item 14, comprising multiple mutations N297Q / S298N / Y300S, S298N / T299A / Y300S or S298N / Y300S.

[0055] 17. A nucleic acid encoding at least the heavy chain variable region of the humanized monoclonal antibody according to any one of items 1 to 16.

[0056] 18. A cell expressing the nucleic acid according to item 17.

[0057] 19. A humanized antibody according to any one of items 1 to 16 for use in inhibiting a T cell mediated response in a subject.

[0058] 20. A humanized antibody according to item 19, wherein the T cell-mediated response is involved in a condition selected from the group consisting of: tissue transplantation, including solid organ transplantation and composite tissue transplantation, tissue grafting, multiple sclerosis and type 1 diabetes.

[0059] 21. An antibody comprising a modified Fc, wherein the modified Fc comprises a modified glycosylation pattern that reduces Fcγ receptor binding, the antibody comprising one or more of the mutations S298N, T299A and Y300S.

[0060] 22. An antibody according to item 21, comprising two or more of the mutations N297Q, S298N, T299A and Y300S.

[0061] 23. An antibody according to item 14, comprising the multiple mutations N297Q / S298N / Y300S, S298N / T299A / Y300S or S298N / Y300S.

[0062] 24. The antibody according to any one of items 21-23, wherein the antibody is a humanized antibody according to any one of items 1 to 10.

[0063] 25. A multispecific antibody comprising at least the heavy chain of a first binding domain according to any one of items 1 to 9 and a second binding domain specific for a tumor-specific antigen.

[0064] 26. A multispecific antibody according to item 25, which comprises at least the heavy chain of the binding domain according to any one of items 4, 5 or 9.

[0065] 27. The multispecific antibody according to item 25 or item 26, which comprises an anti-αβTCR / CD3 scFv and an anti-tumor scFv.

[0066] 28. The multispecific antibody according to any one of items 25-27, which has bispecificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 .BMA031 binds to αβTCR more strongly than EuCIV3.

[0069] BMA031 MoIgG2b, BMA031 HuIgG1 and EuCIV3 HuIgG1 antibodies competed with PE-labeled BMA031 antibody for binding. EuCIV3 had a lower potency than BMA031.

[0070] Figure 2. In the in vitro acclimation (IVE) assay, EuCIV3 was less potent than BMA031.

[0071] The curve shows the performance loss of the EuCIV3 humanized antibody in the biological assay when compared to the parental BMA031 antibody.CD8+ T cells were treated with anti-αβTCR antibodies at different concentrations (x-axis) and co-cultured with autologous dendritic cells pulsed with CMV peptide 495-503 (pp65) for 7 days.

[0072] Figure 3 In competition assays, HEBE1 binds αβ TCR comparably to BMA031.

[0073] BMA031 HuIgG1, HEBE1 HuIgG1 and EuCIV3 HuIgG1 antibodies competed with PE-labeled BMA031 antibody for binding. EuCIV3 had a lower potency than BMA031 and HEBE1.

[0074] Figure 4 . In the in vitro acclimation (IVE) assay, HEBE1 and EuCIV3 had similar potency.

[0075] like Figure 2 The IVE assay was performed as described in.

[0076] Figure 5 . Schematic representation of multiple rounds of mutagenesis of the anti-αβ TCR variable domain.

[0077] The framework in the shaded box depicts the FR regions with certain mouse residues. The shaded residues in the first row of mutations are mouse amino acids that can be used to maintain the off-rate. The shaded residues in the second row of mutations are mouse residues that were retained during the final germlining process around the CDR regions.

[0078] Figure 6 . The optimized humanized antibody has an improved dissociation rate compared to BMA031.

[0079] The kinetics of antibody dissociation from the αβTCR on T cells were measured by flow cytometry. BMA031 has a superior dissociation rate compared to EuCIV3 and HEBE1. The dissociation rate of HEBE1.H10 was improved compared to BMA031 by optimizing the binding domain of HEBE1.

[0080] Figure 7 . The optimized humanized antibody has an improved dissociation rate compared to BMA031.

[0081] The kinetics of antibody dissociation from the αβTCR on T cells were measured by flow cytometry.The off-rate of HEBE1.H66 was improved compared to BMA031 by optimizing the binding domain of HEBE1.

[0082] Figure 8 The optimized humanized antibody had an improved off-rate compared to BMA031 in terms of both Δab and aglycosylation pattern.

[0083] The kinetics of antibody dissociation from the αβ TCR on T cells were measured by flow cytometry.

[0084] Fig. 9 HEBE1 is optimized to be functionally equivalent to BMA031.

[0085] IVE assay Figure 4 BMA031 inhibits the domestication of CD8+ T cells because it is unable to lyse the specific target in a dose-dependent manner. The parental humanized antibody HEBE1 was less potent than BMA031 and was only able to inhibit domestication at the highest dose (similar results were observed with the second unmodified humanized Ab HEBE1 H13). Additional modifications were made to the humanized antibody HEBE1 H10, which was equivalent in potency to BMA031 in this assay.

[0086] Fig.10 .IVE data of anti-αβTCR antibodies.

[0087] Both HEBE1 and GL1 BM series antibodies showed improved IVE results compared to BMA031.

[0088] Fig.11 . Antigen-positive cells from IVE assays as determined by antigen-specific tetramer binding.

[0089] Antigen positive cells (ie, have been trained in the IVE assay) are able to bind MHC-tetramer molecules. When the IVE assay is performed in the presence of antibodies that have prevented T cells from training against the antigen, fewer cells are able to bind MHC-tetramers at the end of the assay.

[0090] Fig.12 . Proliferation of PBMCs in the presence of anti-αβTCR antibody OKT3 and stimulatory beads.

[0091] No stimulatory activity of OKT3 was seen with the anti-αβ TCR antibodies in this comparison.

[0092] Fig.13 . Cytokine release from PBMCs in the presence of anti-αβTCR antibodies.

[0093] The cytokine release profile of the anti-αβTCR antibody was similar to that shown by BMA031.

[0094] Fig.14 . IFN-γ release from T cells in IVE assay.

[0095] In vitro acclimation (IVE) assays were performed using different concentrations (see Figure 2 CD8+ T cells were treated with anti-αβTCR antibodies (Figure , x-axis) and co-cultured with autologous dendritic cells pulsed with CMV peptide 495-503 (pp65) for 7 days. IFN-γ release was measured in this assay.

[0096] Fig.15 .Apoptosis induced by activation of anti-αβTCR antibodies.

[0097] The combination of anti-αβTCR antibody BMA031 and HEBE1 H66 induced apoptosis of antigen-stimulated CD8+T cells. HEBE1 H66 had a greater ability to induce apoptosis than BMA031.

[0098] Fig.16 .Separation of glycosylation mutants and aglycosylated antibodies

[0099] Coomassie-blue stained gel showing expression and purification of glycosylation mutants

[0100] Fig.17 Binding of αβTCR antibody mutants to human FcγRIIIa using Biacore.

[0101] Biacore was used to assess binding to recombinant human FcγRIIIa (V158 and F158).

[0102] Fig.18 Binding of αβTCR antibody mutants to human FcγRI using Biacore.

[0103] Biacore was used to assess binding to recombinant human and FcγRI.

[0104] Fig.19 . Cytokine release from PBMCs in the presence of glycosylation mutant anti-αβTCR antibodies (day 2).

[0105] The cytokine release profiles of TNFa, GM-CSF, IFNy and IL10 by the anti-αβTCR antibodies were similar to those shown by BMA031 and H66δAB.

[0106] Fig. 20. Cytokine release from PBMCs in the presence of glycosylation mutant anti-αβTCR antibodies (day 2).

[0107] The cytokine release profiles of IL6, IL4 and IL2 by the anti-αβTCR antibodies were similar to those shown by BMA031 and H66δAB.

[0108] Fig.21 . Cytokine release from PBMCs in the presence of glycosylation mutant anti-αβTCR antibodies (day 4).

[0109] The cytokine release profiles of TNFa, GM-CSF, IFNy and IL10 by the anti-αβTCR antibodies were similar to those shown by BMA031 and H66δAB.

[0110] Fig. 22 . Cytokine release from PBMCs in the presence of glycosylation mutant anti-αβTCR antibodies (day 4).

[0111] The cytokine release profiles of IL6, IL4 and IL2 by the anti-αβTCR antibodies were similar to those shown by BMA031 and H66δAB.

[0112] Fig.23 : Overview of TRACER integration.

[0113] The binding profile of the bispecific antibodies to both tumor target cells and human T cells was assessed by flow cytometry.

[0114] Fig.24 : Cytotoxic activity of different T cell recruitment arms.

[0115] A panel of humanized BMA031 antibodies has been generated, and a number of antibodies that exhibit cytotoxic activity against tumor antigen expressing cell lines have been selected from this panel.

[0116] Fig.25 : Overview of cytokine release in different T cell recruitment arms.

[0117] A panel of TRACER with different T cell recruitment arms showed similar cytokine release profiles. Significant amounts of cytokines were detected upon T cell activation in the presence of target cells, whereas in the presence of unstimulated human PBMCs alone, significantly lower cytokine levels were observed.

[0118] Fig.26 : Binding of CD52 antibody mutants to FcγRIIIa.

[0119] Anti-CD52 antibodies containing S298N / Y300S mutations on the Fc domain were used to assess the effector function of the modified molecules. A: Binding to CD52 peptide. B: Binding to FcγRIII-Val158. C: Control binding to mouse FcRn. DETAILED DESCRIPTION OF THE INVENTION

[0121] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any methods and materials similar or equivalent to those described herein may be used in the methods and techniques of the present invention. All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the methods, reagents, and tools reported in the publications that may be used in conjunction with the present invention.

[0122] The methods and techniques of the present application are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. For example, see Gennaro, AR, ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing; Hardman, JG, Limbird, LE, and Gilman, AG, ed. (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill; Colowick, S. et al., ed., Methods In Enzymology, Academic Press; Weir, DM, and Blackwell, CC, ed. (1986) Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications; Maniatis, T. et al., ed. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, FM et al., ed. (1999) Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons; Ream et al., ed. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, CR and Graham, A. (1997) PCR (Introduction to Biotechniques Series), 2nd edition, Springer-Verlag.

[0123] The humanized monoclonal antibodies referred to herein are antibodies composed of a human antibody framework onto which the complementarity determining regions (CDRs) from non-human antibodies have been grafted. The human acceptor framework may also be altered. The procedures for designing and producing humanized antibodies are well known in the art and have been described in, for example, the following documents: Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 0 125 023; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent Application 0 120 694; Neuberger, MS et al., WO 86 / 01533; Neuberger, MS et al., European Patent Application 0 194276B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent Application 0 239 400; Padlan, EA et al., European Patent Application 0 519 596. Additional details regarding antibodies, humanized antibodies, human engineered antibodies, and methods for their preparation can be found in Kontermann, R. and Dübel, S., eds. (2001, 2010) Antibody Engineering, 2nd ed., Springer-Verlag, New York, NY.

[0124] Unless otherwise indicated, the term "antibody" is used to refer to whole antibodies as well as antigen-binding fragments of such antibodies. For example, the term encompasses four-chain IgG molecules as well as antibody fragments.

[0125] The term "antibody fragment" as used herein refers to a portion of an intact full-length antibody, for example as further described below.

[0126] Antibodies can be of any class, such as IgG, IgA, or IgM; and any subclass, such as IgG1 or IgG4. Different classes and subclasses of immunoglobulins have different properties that can be advantageous in different applications. For example, IgG4 antibodies have reduced binding to Fc receptors.

[0127] In the context of the antibodies described herein, specificity means that the claimed antibody is able to selectively bind to its defined cognate antigen, which is the αβTCR.CD3 complex. The antibodies of the invention bind to the αβTCR.CD3 complex expressed on cells.

[0128] The human αβTCR / CD3 complex is a T cell receptor complex presented on the surface of T cells. See Kuhns et al. (2006) Immunity 24: 133-139. The murine monoclonal antibody BMA031 targets this complex (see European Patent Application EP 0 403 156; SEQ ID NOs: 1 and 2).

[0129] Natural immunoglobulins have a common core structure in which two identical light chains (about 24 kD) and two identical heavy chains (about 55 or 70 kD) form a tetramer. The amino-terminal portion of each chain is called the variable (V) region and is distinguished from the more conserved constant (C) region of the rest of each chain. The C-terminal portion, called the J region, is located within the variable region of the light chain (also called the V L In the heavy chain variable region (also called V H In addition to the J region, there is a D region in the V region (domain). Most of the amino acid sequence variation of immunoglobulins is limited to three separate positions directly involved in antigen binding within the V region, called the hypervariable region or complementarity determining region (CDR). Proceeding from the amino terminus, these regions are named CDR1, CDR2, and CDR3. The CDRs are held in place by more conserved framework regions (FRs). Proceeding from the amino terminus, these regions are named FR1, FR2, FR3, and FR4. The position and numbering system of the CDR and FR regions have been defined by Kabat et al. (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, US Government Printing Office (1991) and updated content that can be found online). In addition, the boundaries of the CDR regions have been further defined by the IMGT nomenclature.

[0130] The variable regions of the antibodies according to the embodiments are shown in SEQ ID NOs: 5-7 and 12-16 and can be obtained by humanizing BMA031, i.e. by transferring the CDRs of BMA031 to a human framework. Two series of humanized antibodies are described, the HEBE1 series, comprising SEQ ID NOs: 5-7, 12 and 13, and the GL1BM series, comprising the heavy chain variable regions as shown in SEQ ID NOs: 8, 15 and 16. In both cases, the light chain variable region used is as shown in SEQ ID NO: 14 (GL1BM VK43).

[0131] The human frameworks used were IGH3-23 in the case of HEBE1 and IGHV1-3*01 and IGKV3-11*01 in the case of GL1BM.

[0132] The constant region can be derived from any human antibody constant region. The variable region gene and the constant region gene can be cloned in frame into an expression vector to express the immunoglobulin heavy chain and light chain. These expression vectors can be transfected into antibody-producing host cells for antibody synthesis.

[0133] Human antibody variable and constant regions can be derived from sequence databases, for example, immunoglobulin sequences can be obtained in the IMGT / LIGM database (Giudicelli et al., (2006) Nucleic Acids Res. 34:(Suppl 1):D781-D784) or VBase (vbase.mrc-cpe.cam.ac.uk).

[0134] Aglycosylated antibodies may have extensively modified functions; see Boyd et al. (1996) Mol. Immunol. 32: 1311-1318. "δab" or Δab modifications referred to herein are Fc modifications as described in Armour et al. (1999) Eur. J. Immunol. 29: 2613-2624. Techniques for modifying the glycosylation of the Fc region of an antibody are known in the art and include chemical, enzymatic, and mutational means, e.g., mutation of the N297 position in the CH2 domain. Techniques for mutating antibody genes to produce aglycosylated IgG molecules are described in Tao and Morrison (1989) J. Immunol. 143: 2595-2601.

[0135] The "nucleic acid" mentioned herein includes a DNA molecule encoding an antibody of the present invention. Preferably, it is an expression vector suitable for expressing the antibody gene in a host cell. Expression vectors and host cells for antibody gene expression are known in the art; see, for example, Morrow, KJ Genetic Engineering & Biotechnology News (June 15, 2008) 28 (12), and Backliwal, G. et al. (2008) Nucleic Acids Res. 36 (15): e96-e96.

[0136] 1. Antibodies

[0137] The present invention encompasses antigen-binding fragments of humanized anti-αβTCR antibodies. Antibody fragments are capable of binding to the αβTCR.CD3 complex. They encompass Fab, Fab', F(ab')2 and F(v) fragments, or individual light or heavy chain variable regions or portions thereof. Fragments include, for example, Fab, Fab', F(ab')2, Fv and scFv. These fragments lack the Fc portion of the intact antibody, are cleared from circulation more rapidly than intact antibodies, and may have lower nonspecific tissue binding. These fragments can be generated from intact antibodies using well-known methods, for example by proteolytic cleavage with enzymes such as papain (producing Fab fragments) or pepsin (producing F(ab')2 fragments).

[0138] Antibodies and fragments also encompass single-chain antibody fragments (scFv) that bind to the αβTCR.CD3 complex. scFv comprises a light chain variable region (V L ) of the antibody heavy chain variable region (V H ), wherein the heavy chain variable region and the light chain variable region together or separately form a binding site for binding to αβTCR. scFv may comprise a V at the amino terminus H V L Alternatively, the scFv may comprise a V L V H In addition, although the two domains V L and V H are encoded by different genes, but they can be joined using recombinant methods by synthetic linkers that enable them to function as V L and V H The heavy chain variable region and the light chain variable region are paired to form a single protein chain of a monovalent molecule (called a single chain Fv (scFv)). The scFv may optionally further comprise a polypeptide linker between the heavy chain variable region and the light chain variable region.

[0139] Antibodies and fragments also encompass domain antibody (dAb) fragments as described in Ward, ES et al. (1989) Nature 341:544-546, which are composed of V H Domain composition.

[0140] Antibodies and fragments also encompass heavy chain antibodies (HCAbs). These antibodies are reported to form antigen binding regions using only the heavy chain variable region, since these functional antibodies are only heavy chain dimers (referred to as "heavy chain antibodies" or "HCAbs"). Therefore, antibodies and fragments may be heavy chain antibodies (HCAbs) that specifically bind to the αβTCR.CD3 complex.

[0141] Antibodies and fragments also encompass antibodies that are SMIPs or binding domain immunoglobulin fusion proteins specific for the αβTCR.CD3 complex. These constructs are single-chain polypeptides comprising the antigen binding domain fused to an immunoglobulin domain necessary for carrying out the antibody effector functions (see WO 2005 / 017148).

[0142] Antibodies and fragments thereof also include double-chain antibodies (diabodies). These double-chain antibodies are bivalent antibodies, wherein V H and V L The domains are expressed on a single polypeptide chain, but a linker is used that is too short to allow pairing between the two domains on the same chain. This forces the domains to pair with the complementary domains of another chain and thereby create two antigen binding sites (see, e.g., WO 93 / 11161). Diabodies can be bispecific or monospecific.

[0143] The antibody or antibody fragment thereof does not cross-react with any target other than the αβTCR.CD3 complex.

[0144] The antibody or fragment thereof may be modified to increase its serum half-life, for example, by the addition of molecules such as PEG or other water soluble polymers, including polysaccharide polymers, to increase the half-life.

[0145] Antibodies and fragments thereof may have bispecificity. For example, bispecific antibodies may be similar to single antibodies (or antibody fragments), but have two different antigen binding sites (variable regions). Bispecific antibodies may be produced by various methods (e.g., chemical techniques, "polydoma" techniques, or recombinant DNA techniques). Bispecific antibodies may have binding specificity for at least two different epitopes, at least one of which is an αβTCR.CD3 complex. The other specificity may be selected from any useful or desired specificity, including, for example, specificity for human serum albumin that prolongs half-life in vivo.

[0146] The clinical use of bispecific antibodies in oncology applications has now become a reality, with the trifunctional Catumaxomab Approved for use in cases of malignant ascites, and the bispecific antibody Blinatumomab is now in Phase II trials for hematological malignancies. These molecules share a binding arm that binds to T cells and a second arm that binds to tumor target cells, resulting in T cell-mediated tumor target lysis. These molecules also share the recruitment of T cells via the CD3 protein located on the cell surface. An alternative to CD3 recruitment utilizes the αβT cell receptor (αβTCR) also expressed on the cell surface. Therefore, the antibodies according to the present invention can be used to produce anti-tumor antibodies by combining the specificity for tumor-associated antigens with the specificity for the αβT cell receptor (αβTCR).

[0147] 2. Antibody Production

[0148] The amino acid sequences of the variable domains of the antibodies described herein are set forth in SEQ ID NOs: 5-7 and 12-16. Antibody production can be performed by any technique known in the art, including in transgenic organisms, such as sheep (see Pollock et al. (1999) J. Immunol. Methods 231: 147-157), chickens (see Morrow, KJJ (2000) Genet. Eng. News 20: 1-55), mice (see Pollock et al., supra), or plants (see Doran, PM (2000) Curr. Opinion Biotechnol. 11: 199-204, Ma. JK-C. (1998) Nat. Med. 4: 601-606, Baez, J. et al. (2000) BioPharm. 13: 50-54, Stoger, E. et al. (2000) Plant Mol. Biol. 42: 583-590). Antibodies can also be produced by chemical synthesis or by expressing genes encoding the antibodies in host cells.

[0149] The polynucleotide encoding the antibody is separated and inserted into a reproducible construct or vector (e.g., a plasmid) for further propagation or expression in a host cell. The art can be used with a construct or vector (e.g., an expression vector) suitable for expressing the humanized immunoglobulin according to the embodiment. A variety of vectors can be used, including a vector that is maintained in a host cell with a single copy or multiple copies or becomes integrated into the chromosome of the host cell. The construct or vector can be introduced into a suitable host cell, and cells expressing the humanized immunoglobulin can be produced and maintained in culture. A single vector or multiple vectors can be used to express the humanized immunoglobulin.

[0150] The polynucleotides encoding the antibody are easily isolated and sequenced using conventional procedures (e.g., oligonucleotide probes). Useful vectors include plasmids, viruses, bacteriophages, transposons, minichromosomes, of which plasmids are typical embodiments. Typically, these vectors further include a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, which are operably linked to the light chain and / or heavy chain polynucleotides to promote expression. The polynucleotides encoding the light chain and the heavy chain can be inserted into different vectors and introduced into the same host cell simultaneously or sequentially (e.g., by transformation, transfection, electroporation or transduction), or if desired, both the heavy chain and the light chain can be inserted into the same vector before this introduction.

[0151] A promoter for expression in a suitable host cell can be provided. The promoter can be constitutive or inducible. For example, the promoter can be operably linked to a nucleic acid encoding a humanized immunoglobulin or immunoglobulin chain so that it directs the expression of the encoded polypeptide. A variety of suitable promoters for prokaryotic and eukaryotic hosts can be used. Prokaryotic promoters include lac, tac, T3, T7 promoters for E. coli; promoters for 3-phosphoglycerate kinase or other glycolytic enzymes (e.g., enolase, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose 6-phosphate isomerase, 3-phosphoglycerate mutase and glucokinase). Eukaryotic promoters include inducible yeast promoters, such as alcohol dehydrogenase 2 promoter, isocytochrome C promoter, acid phosphatase promoter, metallothionein promoter, and promoters of enzymes responsible for nitrogen metabolism or maltose / galactose utilization; RNA polymerase II promoters, including viral promoters, such as polyoma virus promoter, fowl pox virus and adenovirus (e.g., adenovirus 2) promoter, bovine papilloma virus promoter, avian sarcoma virus promoter, cytomegalovirus promoter (particularly, immediate early gene promoter), retroviral promoter, hepatitis B virus promoter, actin promoter, Rous sarcoma virus (RSV) promoter, and early or late simian virus 40; and non-viral promoters, such as EF-1α promoter (Mizushima and Nagata (1990) Nucleic Acids Res. 18 (17): 5322). Those skilled in the art will be able to select appropriate promoters for expressing humanized antibodies or portions thereof.

[0152] If appropriate, for example, for expression in higher eukaryotic cells, other enhancer elements may be included to replace those found in the above-mentioned promoters or include other enhancer elements and those found in the above-mentioned promoters. Suitable mammalian enhancer sequences include enhancer elements from globulin, elastase, albumin, alpha-fetoprotein, metallothionein and insulin. Alternatively, enhancer elements from eukaryotic cell viruses, such as SV40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers, baculovirus enhancers or murine lgG2a loci (see WO 04 / 009823) may be used. Although these enhancers are usually located at sites upstream of the promoter on the vector, they may also be located elsewhere, for example, in an untranslated region or downstream of a polyadenylation signal. Enhancers may be selected and positioned based on compatibility with the host cell used for expression.

[0153] In addition, the vector (e.g., expression vector) may include a selectable marker and (in the case of a replicable vector) an origin of replication for selecting a host cell carrying the vector. Genes encoding products that confer antibiotic resistance or drug resistance are common selectable markers and can be used for prokaryotic cells (e.g., f3-lactamase gene (ampicillin resistance), Tet gene (tetracycline resistance) and eukaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin or hygromycin resistance (hygromycin resistance) genes). The dihydrofolate reductase marker gene allows selection in a variety of hosts with methotrexate. Genes encoding the gene products of the host's auxotrophic markers (e.g., LEU2, URA3, HIS3) are commonly used as selectable markers in yeast. It is also contemplated that a virus (e.g., baculovirus) or phage vector and a vector (e.g., retroviral vector) can be integrated into the host cell genome.

[0154] In eukaryotic systems, polyadenylation and termination signals are operably linked to the polynucleotide encoding the antibody of the present invention. Such signals are usually located at the 3' of the open reading frame. In mammalian systems, non-limiting examples of polyadenylation / termination signals include those derived from growth hormone, elongation factor 1α and viral (e.g., SV40) genes or retroviral long terminal repeats. In yeast systems, non-limiting embodiments of polyadenylation / termination signals include those derived from phosphoglycerate kinase (PGK) and alcohol dehydrogenase 1 (ADH) genes. In prokaryotic systems, polyadenylation signals are usually not required, but shorter and more fully defined terminator sequences are usually used. Polyadenylation / termination sequences can be selected based on compatibility with the host cell used for expression. In addition to the above features, other features that can be used to improve yields also include chromatin remodeling elements, introns, and host cell-specific codon modifications. The codon usage of the antibodies of the invention can be modified to adapt to the codon preference of the host cell, thereby increasing transcript and / or product yield (e.g., Hoekema, A. et al. (1987) Mol. Cell Biol. 7(8):2914-24). Codons can be selected based on compatibility with the host cell used for expression.

[0155] Thus, the present invention relates to isolated nucleic acid molecules encoding humanized immunoglobulins or heavy or light chains thereof. The present invention also relates to isolated nucleic acid molecules encoding antigen binding portions of immunoglobulins and chains thereof.

[0156] Antibodies can be produced, for example, by expressing one or more recombinant nucleic acids encoding the antibodies in a suitable host cell. Host cells can be produced using any suitable method. For example, an expression construct described herein (e.g., one or more vectors, e.g., mammalian cell expression vectors) can be introduced into a suitable host cell, and the resulting cell can be maintained (e.g., in culture, in an animal, in a plant) under conditions suitable for the expression of the construct or vector. The host cell can be a prokaryotic cell, including a bacterial cell, such as Escherichia coli (e.g., strain DH5a TM ) (Invitrogen, Carlsbad, CA), PerC6 (Crucell, Leiden, NL), Bacillus subtilis (B. subtilis), and / or other suitable bacteria; eukaryotic cells, such as fungi or yeast cells (e.g., Pichia pastoris, Aspergillus sp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa) or other lower eukaryotic cells and higher eukaryotic cells, such as those from insects (e.g., Drosophila Schnieder S2 cells, Sf9 insect cells) (WO 94 / 126087 (O'Connor)), BTI-TN-5B1-4 (High Five TM) insect cells (Invitrogen), mammalian cells (e.g., COS cells, such as COS-1 (ATCC Accession No. CRL-1650) and COS-7 (ATCC Accession No. CRL-1651), CHO (e.g., ATCC Accession No. CRL-9096), CHO DG44 (Urlaub, G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA, 77 (7): 4216-4220), 293 (ATCC Accession No. CRL-1573), HeLa (ATCC Accession No. CCL-2), CVI (ATCC Accession No. CCL-70), WOP (Dailey, L. et al. (1985) J. Virol., 54: 739-749), 3T3, 293T (Pear, WS, et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 8392-8396), NSO cells, SP2 / 0 cells, HuT 78 cells, etc.; or plant cells (e.g., tobacco cells, lemna (duckweed) cells and algae cells). For example, see Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons (1993). In some embodiments, the host cell is not part of a multicellular organism (eg, a plant or an animal), for example, it is an isolated host cell or is part of a cell culture.

[0157] Host cells can be cultured in spinner flasks, shaking flasks, roller bottles, wave reactors (e.g., System 1000, from wavebiotech.com), or hollow fiber systems, but for large-scale production, stirred tank reactors or bag reactors (e.g., Wave Biotech, Somerset, New Jersey USA) are preferred, especially for suspension cultures. Stirred tank reactors can be adapted to be aerated using, for example, sparger, baffle, or low shear impeller. For bubble columns and airlift reactors, direct aeration using air or oxygen bubbles can be used. If host cells are cultured in serum-free medium, the medium can be supplemented with a cell protectant, such as pluronic F-68, to help prevent the cells from being damaged by the aeration process. Depending on the characteristics of the host cells, microcarriers can be used as a growth matrix for anchorage-dependent cell lines, or these cells can be adapted to suspension culture. Host cells, particularly vertebrate host cells, can be cultured using a variety of operating modes, such as batch, fed-batch, repeated batch processing (see Drapeau et al. (1994) Cytotechnology 15: 103-109), extended batch processes, or perfusion culture. Although recombinantly transformed mammalian host cells can be cultured in serum-containing media (such media include fetal calf serum (FCS)), it is preferred to culture in serum-free media (such as disclosed in Keen et al. (1995) Cytotechnology 17: 153-163) or commercially available media such as ProCHO-CDM or UltraCHO TM (Cambrex NJ, USA), supplementing these serum-free media or commercially available media with energy sources (e.g., glucose) and synthetic growth factors (e.g., recombinant insulin) if necessary. Serum-free culture of host cells may require that the cells are adapted to grow under serum-free conditions. One adaptation method is to culture these host cells in serum-containing media and repeatedly exchange 80% of the media for serum-free media so that the host cells learn to adapt to serum-free conditions (e.g., see Scharfenberg, K. et al. (1995) Animal Cell Technology: Developments Towards the 21st Century (Beuvery, EC et al., eds.), pp. 619-623, Kluwer Academic publishers).

[0158] The antibody of the embodiment can be secreted into the culture medium and recovered and purified from it using a variety of techniques to provide a degree of purification suitable for the desired use. For example, when compared with a culture medium comprising a therapeutic antibody, the use of therapeutic antibodies for treating human patients generally requires at least 95% purity (as determined by reducing SDS-PAGE), more typically 98% or 99% purity. In the first case, centrifugation can be used, and then the clarification step of the supernatant can be used (for example, microfiltration, ultrafiltration and / or deep filtration) to remove cell debris from the culture medium. Alternatively, antibodies can be harvested by microfiltration, ultrafiltration or deep filtration without pre-centrifugation. A variety of other techniques can be used, such as dialysis and gel electrophoresis and chromatography techniques, such as hydroxyapatite (HA), affinity chromatography (optionally involving affinity tag systems, such as polyhistidine) and / or hydrophobic interaction chromatography (HIC) (see US 5,429,746). In one embodiment, after various clarification steps, the antibody is captured using protein A or G affinity chromatography followed by other chromatography steps (e.g., ion exchange and / or HA chromatography, anion or cation exchange, size exclusion chromatography, and ammonium sulfate precipitation). Various virus removal steps (e.g., nanofiltration using, for example, a DV-20 filter) may also be employed. After these various steps, a purified preparation comprising at least 10 mg / ml or greater (e.g., 100 mg / ml or greater) of an antibody of the invention is provided and thus forms another embodiment of the invention. Concentrations of 100 mg / ml or greater may be produced by ultracentrifugation. Such preparations are substantially free of aggregated forms of the antibodies of the invention.

[0159] Bacterial systems are particularly suitable for expressing antibody fragments. These fragments are located intracellularly or periplasmically. Insoluble periplasmic proteins can be extracted and refolded to form active proteins according to methods known to those skilled in the art, see Sanchez et al. (1999) J. Biotechnol. 72: 13-20; Cupit, PM et al. (1999) Lett. Appl. Microbiol. 29: 273-277.

[0160] The present invention also relates to cells comprising nucleic acids of the present invention (e.g., vectors, e.g., expression vectors). For example, nucleic acids encoding the heavy and light chains of the humanized immunoglobulins of the embodiments (i.e., one or more nucleic acids) or constructs comprising such nucleic acids (i.e., one or more constructs, e.g., one or more vectors) can be introduced into suitable host cells by methods suitable for the selected host cells (e.g., transformation, transfection, electroporation, infection), wherein the nucleic acids are or become operably linked to one or more expression control elements (e.g., in a vector, in a construct produced by a process in a cell, integrated into the host cell genome). The host cells can be maintained under conditions suitable for expression (e.g., in the presence of an inducer, a suitable culture medium supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), thereby producing the encoded polypeptide. If desired, the encoded humanized antibodies can be isolated from, for example, host cells, culture medium, or milk. This approach encompasses expression in host cells (eg, mammary cells) of transgenic animals or plants (eg, tobacco) (eg, see WO 92 / 03918).

[0161] 3. Therapeutic applications

[0162] In many cases where immunosuppression and / or autoimmune conditions are warranted, inhibition of T cell activity is desirable. Thus, targeting the αβTCR.CD3 complex is indicated in the treatment of diseases involving inappropriate or undesirable immune responses, such as inflammation, autoimmunity, and other conditions involving such mechanisms. In one embodiment, this disease or disorder is an autoimmune and / or inflammatory disease. Examples of such autoimmune and / or inflammatory diseases are systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), multiple sclerosis (MS), scleroderma and type 1 diabetes (T1D); and other diseases and disorders, such as PV (pemphigus vulgaris), psoriasis, atopic dermatitis, celiac disease, chronic obstructive pulmonary disease, Hashimoto's thyroiditis, Graves' disease (thyroid), Sjogren's syndrome, Guillain-Barré syndrome, Goodpasture's syndrome, Addison's disease, Wegener's granulomatosis, and leukemia. granulomatosis), primary biliary cirrhosis, sclerosing cholangitis, autoimmune hepatitis, polymyalgia rheumatica, Raynaud's phenomenon, temporal arteritis, giant cell arteritis, autoimmune hemolytic anemia, pernicious anemia, polyarteritis nodosa, Behcet's disease, primary biliary cirrhosis, uveitis, myocarditis, rheumatic fever, ankylosing spondylitis, glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, alopecia areata, and vitiligo.

[0163] In one embodiment, the disease or disorder is SLE, RA or IBD. In one embodiment, the disease or disorder is MS.

[0164] In another embodiment, the antibodies of the embodiments are used to assist transplantation by immunosuppression of the individual. This application alleviates graft-versus-host disease. For a description of existing treatments for graft-versus-host disease, see, for example, Svennilson, Bone Marrow Transplantation (2005) 35: S65-S67 and references cited therein. Advantageously, the antibodies of the invention can be used in combination with other available therapies.

[0165] For the treatment of autoimmune diseases, the combination therapy may include the administration of an antibody of the invention and an agent, which together with the antibody comprises an effective amount for preventing or treating such autoimmune diseases. If the autoimmune disease is type 1 diabetes, the combination therapy may include one or more agents that promote pancreatic β cell growth or enhance β cell transplantation, such as β cell growth or survival factors or immunomodulatory antibodies. If the autoimmune disease is rheumatoid arthritis, the combination therapy may include one or more of the following: methotrexate, anti-TNF-β antibodies, TNF-β receptor-Ig fusion proteins, anti-IL-15 or anti-IL-21 antibodies, nonsteroidal anti-inflammatory drugs (NSAIDs) or disease-modifying antirheumatic drugs (DMARDs). For example, another agent may be a biological agent, such as an anti-TNF agent (e.g., Infliximab (recombinant human immunodeficiency virus ) and adalimumab or rituximab If the autoimmune disease is hematopoietic transplant rejection, hematopoietic growth factors (e.g., erythropoietin, G-CSF, GM-CSF, IL-3, IL-11, thrombopoietin, etc.) or antimicrobial agents (e.g., antibiotics, antivirals, antifungal drugs) may be administered. If the autoimmune disease is psoriasis, the other agent may be one or more of the following: tar and its derivatives, phototherapy agents, corticosteroids, cyclosporin A, vitamin D analogs, methotrexate, p38 mitogen-activated protein kinase (MAPK) inhibitors, and biological agents (e.g., anti-TNF agents and If the autoimmune disease is inflammatory bowel disease (IBD) (e.g., Crohn's disease or ulcerative colitis), the other agent may be one or more of the following: an aminosalicylate, a corticosteroid, an immunomodulator, an antibiotic, or a biologic agent (e.g., a remdesivir, a cefotaxime ... and ).

[0166] Combination therapy can be implemented in any manner deemed necessary or convenient by a person skilled in the art, and for the purposes of this specification, it is not intended to limit the order, amount, repetition or relative amount of the compounds to be used in combination. Thus, the antibodies of the embodiments can be formulated into pharmaceutical compositions for use in therapy.

[0167] 4. Pharmaceutical Composition

[0168] In a preferred embodiment, a pharmaceutical composition comprising an antibody of the invention or one or more ligands identifiable by an analytical method as defined in the previous aspects of the invention is provided. The ligand may be an immunoglobulin, a peptide, a nucleic acid or a small molecule, as described herein. It is referred to as a "compound" in the following discussion.

[0169] The pharmaceutical composition of the present invention is a target composition comprising one or more compounds capable of modulating T cell activity as active ingredients. The compound is in any pharmaceutically acceptable salt form, or, for example, if appropriate, in analog form, free base form, tautomer form, enantiomer form, racemic isomer form, or a combination thereof. When administered in an amount depending on the specific situation, the active ingredients of the pharmaceutical composition comprising the active ingredients of the present invention are expected to exhibit therapeutic activity, for example, for treating graft-versus-host disease.

[0170] In another embodiment, one or more compounds of the present invention may be used in combination with any art-recognized compound known to be suitable for treating a particular indication for the treatment of any of the above conditions. Thus, one or more compounds of the present invention may be combined with one or more art-recognized compounds known to be suitable for treating the above indications so that a convenient single composition can be administered to a subject. The dosage regimen may be adjusted to provide the optimal therapeutic response.

[0171] For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0172] The active ingredient may be administered in a convenient manner, for example, by the oral, intravenous (if water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository route or by implantation (for example, using a sustained release molecule).

[0173] Depending on the route of administration, the active ingredients may need to be coated with a material to protect these ingredients from the action of enzymes, acids, and other natural conditions that could inactivate the ingredients.

[0174] To administer the active ingredient by means other than parenteral administration, it will be coated with or administered with a material that prevents its inactivation. For example, the active ingredient may be administered in an adjuvant, co-administered with an enzyme inhibitor, or administered in liposomes. Adjuvant is used in its broadest sense and includes any immunostimulatory compound, such as interferon. Adjuvants contemplated herein include resorcinol; nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin.

[0175] Liposomes include water-in-oil-in-water type CGF emulsions and conventional liposomes.

[0176] The active ingredient may also be administered parenterally or intraperitoneally.

[0177] Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0178] The pharmaceutical form suitable for injection includes sterile aqueous solution (if soluble in water) or dispersion and sterile powder for preparing sterile injectable solution or dispersion in real time. In all cases, the form must be sterile and its fluidity must make it easy to inject. It must be stable under manufacturing and storage conditions and must be preserved against the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures thereof and vegetable oils. Appropriate fluidity can be maintained by, for example, using coatings such as lecithin, by maintaining the desired particle size (in the case of dispersions) and by using surfactants.

[0179] The prevention of the action of microorganisms may be achieved by various antibacterial or antifungal agents, for example, parahydroxybenzoic acid, chlorobutanol, phenol, sorbic acid, thirmerosal, or the like. In some cases, it may be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may be brought about by the use of agents in the composition that delay absorption, for example, aluminum monostearate and gelatin.

[0180] Sterile injectable solutions are prepared by incorporating the active ingredient and, if necessary, a number of other ingredients listed above in the desired amount in an appropriate solvent, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and the desired other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying techniques, which can produce a powder of the active ingredient and any additional desired ingredients from a previously sterile filtered solution.

[0181] Various other materials may be present as coatings or their presence may otherwise modify the physical form of the dosage unit. Of course, any material used in preparing any dosage unit form should be of pharmaceutical purity and the amount employed should be substantially nontoxic. In addition, the active ingredient may be incorporated into sustained release preparations and formulations.

[0182] As used herein, "pharmaceutically acceptable carriers and / or diluents" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except for any conventional media or agents that are incompatible with the active ingredient, the present invention contemplates their use in the therapeutic compositions. Supplementary active ingredients may also be incorporated into the compositions.

[0183] Formulating parenteral compositions in dosage unit form is particularly advantageous for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suitable as unitary dosages for mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the novel dosage unit forms of the present invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such active materials to treat diseases in living subjects with diseased states of compromised biological health.

[0184] The main active ingredient is formulated in an effective amount with a suitable pharmaceutically acceptable carrier in a dosage unit form for convenient and effective administration. In the case of compositions containing supplementary active ingredients, the administration is determined by reference to the conventional dosage and mode of administration of these ingredients.

[0185] To facilitate delivery of peptide compounds (including antibodies) to cells, peptides can be modified to improve their ability to cross cell membranes. For example, US 5,149,782 discloses the use of fusion peptides, ion channel forming peptides, membrane peptides, long chain fatty acids and other membrane adjuvants to increase protein transport across cell membranes. These and other methods are also described in WO 97 / 37016 and US 5,108,921, which are incorporated herein by reference.

[0186] In another aspect, the active ingredient of the invention as defined above is provided, alone or in combination with a known compound suitable for treating a specific indication recognized in the art for use in treating a disease. Thus, the use of the active ingredient of the invention is provided for the manufacture of a medicament for treating a disease associated with an abnormal immune response.

[0187] Additionally, methods of treating conditions associated with an aberrant immune response are provided, comprising administering to a subject a therapeutically effective amount of a ligand identifiable using the assays described above.

[0188] For purposes of illustration only, the present invention is further described in the following examples.

[0189] Comparative Example 1

[0190] The binding and biological activity of EuCIV3 are lower than those of BMA031

[0191] Flow cytometry has been used to show that EuCIV3 is inferior to BMA031 in T cell binding ( Figure 1). In this competition assay, T cells were incubated on ice in the presence of a fixed concentration of directly phycoerythrin-labeled MoIgG2b-BMA031 (murine competitor) and increasing concentrations of anti-αβTCR antibodies. After a 20-minute incubation, cells were washed and surface-bound directly phycoerythrin-labeled MoIgG2b-BMA031 was detected by flow cytometry. The competition of the BMA031 HuIgG1 chimeric antibody was much more effective than EuCIV3.

[0192] To assess its ability to suppress T cell activity in vivo, different concentrations of Figure 2 , x-axis) and co-cultured with autologous dendritic cells (DCs) pulsed with CMV peptide 495-503 (pp65) for 7 days.

[0193] Obtained from HemaCare, Van Nuys, CA +Blood separation (aphaeresis) product of normal donors of individuals. PBMCs were separated by centrifugation on Ficoll (GE Healthcare, Piscataway, NJ). Magnetic beads (Invitrogen, Carlsbad, California) were used to separate CD8+T cells according to the manufacturer's instructions. To produce autologous immature dendritic cells, PBMCs were resuspended in RPMI 1640 / 5% human AB serum (Sigma), plated in a triple flask (Triple flask) (Corning) and incubated at 37°C / 5% CO2 for more than 2 hours. Adherent monocytes were then rinsed with PBS and cultured for 6 days in RPMI 1640 / 5% human AB serum supplemented with GM-CSF (Immunex, Seattle, WA) and IL-4 (PeproTech, Rocky Hill, NJ). Before establishing T cell / DC co-cultures, DCs were pulsed for 4 hours with peptides (10 μg / ml) and then matured. Mature dendritic cells were generated by adding 50ng / ml TNF-α, 25ng / ml IL-1β, 10ng / ml IL-6, 500ng / ml PGE-2 (PeproTech, Rocky Hill, NJ) and the dendritic cells were cultured for an additional 24 hours. Peptide-pulsed DCs were then added to previously isolated CD8+ T cells at a T:DC ratio of 10:1. The cultures were immediately fed with IL-2 (100 IU / ml) added to the cultures. The cultures were supplemented with IL-2 (100 IU / ml) on day 4. The bulk cultures were analyzed for peptide reactivity in a chromium release assay on day 7.

[0194] Figure 2 The graph in shows lysis data from a chromium release assay, where untreated T cells were successfully educated with the pp65 peptide and were able to lyse >50% of the specific target. BMA031 inhibited the educated T cells due to its inability to lyse the specific target in a dose-dependent manner. The humanized antibody EuCIV3 was less potent than BMA031 and was able to inhibit educated only at the highest dose.

[0195] Example 2

[0196] Fc engineering of BMA031 chimeric antibody

[0197] In vitro profile

[0198] The in vitro profile of BMA031 has been evaluated in a panel of assays. Table 1 shows the in vitro profile of BMA031. BMA031 was compared to OKT3 in these assays.

[0199] In the PBMC proliferation assay, human PBMCs were cultured with increasing concentrations of therapeutic antibodies for 72 hours. 3 H-thymidine and cells were harvested 18 h later.

[0200] For the T cell depletion / cytokine release assay, human PBMCs were cultured with increasing concentrations of therapeutic antibodies and analyzed daily for cell count and viability (Vi-Cell, Beckman Coulter) until day 7. Cell supernatants were also harvested, stored at -20°C and analyzed on an 8-plex cytokine panel (Bio-Rad).

[0201] BMA031 does not induce: (i) PBMC proliferation; (ii) T cell exhaustion; (iii) CD25 expression; or (iv) cytokine release. In contrast, OKT3 induces all of the above effects. BMA031 and OKT3 can block CD8+ cell training to peptides in an in vitro training (IVE) assay and can also block mixed lymphocyte reactions (MLR). BMA031 also induces apoptosis of activated T cells (activation-induced cell death; AICD).

[0202] Unlike BMA031, the chimeric form of BMA031 with the wild-type human IgG1 constant region (HuIgG1) has an in vitro profile comparable to OKT3 (Table 1). We hypothesized that the involvement of FcγRs is crucial for this change in the in vitro profile of HuIgG1 BMA031 compared to BMA031 MoIgG2b. Therefore, we prepared F(ab')2 fragments of BMA031 HuIgG1 and found that these fragments restored the profile of BMA031 MoIgG2b. By Fc engineering we incorporated modifications that removed FcγR binding in the mutation (called "δab") (Armour et al. (1999) Eur. J. Immunol., 29: 2613-2624) and produced a non-glycosylated form of HuIgG4 (N297Q). HuIgG1 δab and HuIgG4 agly anti-αβTCR antibodies have the same in vitro profile as BMA031 MoIgG2b (Table 1).

[0203]

[0204] Table 1

[0205] Example 3

[0206] Construction of humanized antibodies with improved binding

[0207] Two series of humanized forms of BMA031 have been generated, referred to as the HEBE1 series (IGH3-23) and the GL1BM series (IGHV1-3*01 & IGKV3-11*01; see VBase, vbase.mrc-cpe.cam.ac.uk). Initial grafting of the BMA031 heavy chain CDR regions onto the IGH3-23 framework regions (see SEQ ID NOs: 5 and 6) improved binding of the antibody to the αβ TCR, as shown by competition assays ( Figure 3 ); see Example 2. However, this improvement did not translate into improved antibody function, as shown by the IVE assay ( Figure 4 ).

[0208] Example 4

[0209] Optimization of humanized antibodies

[0210] The optimization strategy of humanized antibodies is based on mutagenesis and functional screening. Optimization begins with a block change of amino acid residues in each of the 4 framework regions of the variable domain from mouse to human. The key framework regions in each of the GL1BMHC, GL1BM LC and HEBE1 HC series are identified. Based on this identification, individual residues in those framework regions are mutated from the original mouse sequence to human germline residues. It is found that the framework residues that are essential for maintaining the binding properties of the antibody with the consistency of the mouse sequence are kept as mouse residues. Otherwise, the framework residues are changed to match the human germline amino acid sequence. This continues within the sequence until the minimum number of mouse residues that maintain the original binding properties of the antibody are identified. See Figure 5 Several antibodies from these series have been shown to have improved binding compared to BMA031 as measured by antibody dissociation rates on T cells ( Figure 6 , 7 and 8).

[0211] For the off-rate assay, 10 5 Individual human T cells were incubated for 30-60 minutes in 100 μL of complete growth medium containing 2 μg / ml of antibody denoted HuIgG1-Δab. The cells were then washed, resuspended in 50 μL of complete growth medium and 20 μg / ml HEBE1 F(ab')2 was added to prevent rebinding of the dissociated candidate antibody. At the end of this time course assay, the cells were fixed and the level of residual HuIgG1-Δab antibody bound to the cell surface was measured by flow cytometry via a PE-labeled goat anti-HuIgG secondary antibody.

[0212] It has also been shown that in IVE( Fig. 9 , 10and 11) and in the MLR assay, the antibody is active in preventing immune responses. In the IVE assay, tetramer binding is used as a quantitative measure of IVE. The percentage of cells with antigen specificity is determined by staining T cells with directly labeled tetramers specific for the training peptide. Briefly, on day 7, CD8+ T cells from IVE were stained with tetramers by a standard flow cytometry staining protocol and analyzed on a BD FACSCalibur. In addition, the humanized antibody showed comparable levels of proliferation potential for PBMC and cytokine release compared to BMA031 ( Fig.12 and 13 ).

[0213] These antibodies also showed the ability to inhibit IFNγ release from T cells in the IVE assay ( Fig.14 In addition, we have shown that many of these antibodies have improved ability to induce activation-induced cell death (AICD) of activated αβTCR-positive T cells compared to BMA031 ( Fig.15 ). In the AICD assay, antigen-specific CD8+ T cells are cultured with therapeutic antibodies. At 24 hours, 48 ​​hours, and 72 hours, cells are stained for apoptosis markers Annexin V and 7-AAD. Cells are also stained with tetramers to track apoptosis that is an effector of antigen-specific T cells.

[0214] In summary, we have made significant improvements over previous attempts to humanize BMA031. An unexpected discovery was made by this approach of antibodies with improved off-rates compared to BMA031. This improvement in binding correlated with improved potency in suppressing immune responses, as shown in the IVE assay ( Fig.10 and 11 ). The specificity of the antibodies for the αβ TCR, reduced immunogenicity by humanization, specific apoptosis of activated T cells, and lack of T cell activation upon antibody binding make these antibodies excellent candidates for therapeutic purposes.

[0215] Example 5

[0216] Generation of Fc mutants with reduced effector function

[0217] Engineered Fc variants were designed and generated in which a glycosylation site was introduced at amino acid position Ser298, adjacent to the naturally occurring Asn297 site. Glycosylation at Asn297 was maintained or knocked out by mutation. The mutation and glycosylation results are described in Table 2.

[0218]

[0219] Table 2

[0220] The αβT cell receptor antibody clone 66 was mutated by Quikchange using the pENTR_LIC_IgG1 template. The VH domain of HEBE1Δab IgG1 66 was amplified using LIC primers and cloned into mutated or wild-type pENTR_LIC_IgG1 by LIC to generate full-length Ab mutants or wild-type. Subclones were verified using DraIII / XhoI double digestion, resulting in approximately 1250 bp insertions in successful clones. The full-length mutants were then cloned into the expression vector pCEP4(-E+I)Dest via Gateway cloning. Mutations were then confirmed by DNA sequencing.

[0221] Two constructs, HEBE1 Agly IgG4 and HEBE1 Δab IgG1 in pCEP4 were used as controls in HEK293 transfections.

[0222] Mutants, wild type and controls (Agly and Δab) were transfected into HEK293-EBNA cells for expression in three-necked flasks. Proteins were purified from 160 ml of conditioned medium (CM) using a 1 ml HiTrap Protein A column (GE) on a multichannel peristaltic pump. 5 μg of each supernatant was analyzed on 4-20% Tris-glycine reducing and non-reducing SDS-PAGE (see Fig.16 ). The heavy chains of the non-glycosylated mutants (N297Q, T299A and Agly controls) are lower (black arrows), consistent with the loss of glycans in these antibodies. However, the heavy chains of the engineered glycosylated antibodies (NSY, STY, SY, Δab and wild-type controls, red arrows) migrated in the same manner as the wild-type control. This result is consistent with the expected result for the engineered glycosylation site at position 298. SEC-HPLC analysis indicated that all mutants were expressed as monomers.

[0223] Glycosylation analysis by LC-MS

[0224] The engineered H66 IgG1 Fc variants were partially reduced with 20 mM DTT at 37°C for 30 minutes. Samples were analyzed by capillary LC / MS on an Agilent 1100 capillary HPLC system coupled to a QSTAR qq TOF hybrid system (Applied Biosystem). Data analysis was performed using Bayesian protein reconstruction with baseline correction and computer modeling in Analyst QS1.1 (Applied Bisoystem). For the mutant S298N / T299A / Y300S H66 antibody lead, a glycosylation site was observed at amino acid 298, with biantennary and triantennary complex glycans detected as the major species, as well as G0F, G1F, and G2F.

[0225] Binding of αβTCR antibody mutants to human FcγRIIIa and FcγRI using Biacore

[0226] Biacore was used to evaluate binding to recombinant human FcγRIIIa (V158 and F158) and FcγRI. All 4 flow cells of the CM5 chip were immobilized with anti-HPC4 antibodies via the standard amine coupling procedure provided by Biacore. Anti-HPC4 antibodies were diluted to 50 μg / mL in 10 mM sodium acetate (pH 5.0) for coupling reactions and injected at 5 μL / min for 25 minutes. Approximately 12,000 RU of antibodies were immobilized to the chip surface. Recombinant human FcγRIIIa-V158 and FcγRIIIa-F158 were diluted to 0.6 μg / mL in binding buffer HBS-P with 1 mM CaCl2 and injected into flow cells 2 and 4, respectively, at 5 μL / min for 3 minutes to capture 300-400 RU of receptors to the anti-HPC4 chip. To distinguish low binders, three times as much rhFcγRIIIa as that normally used in this assay was captured onto the anti-HPC4 surface. Flow cells 1 and 3 were used as reference controls. Each antibody was diluted to 200 nM in binding buffer and injected over all 4 flow cells for 4 minutes, followed by 5 minutes of dissociation in buffer. The surface was regenerated for 3 minutes using 10 mM EDTA in HBS-EP buffer at 20 μL / min.

[0227] Results are shown in Fig.17 middle.

[0228] Biacore was also used to compare FcγRI binding. The anti-tetra-His antibody was buffer exchanged to 10 mM sodium acetate (pH 4.0) using a Zeba desalting column and diluted to 25 μg / mL in acetate buffer for amine coupling. After 20 minutes of injection at 5 μL / min, two flow cells of the CM5 chip were fixed with approximately 9000RU of anti-tetra-His antibody. Similar to the previous experiment, 10 times FcγRI was captured to the anti-tetra-His surface to compare weak binders. Recombinant human FcγRI was diluted to 10 μg / mL in HBS-EP binding buffer and injected into flow cell 2 at 5 μL / min for 1 minute to capture approximately 1000RU receptors to the anti-tetra-His chip. A single concentration of antibody (100 nM) was injected onto the capture receptor and control surface at 30 μL / min for 3 minutes. Dissociation was monitored for 3 minutes. The surface was regenerated with two 30 second injections of 10 mM glycine, pH 2.5, at 20 [mu]L / min.

[0229] Results are shown in Fig.18 middle.

[0230] The results showed that the glycoengineered mutants had very little binding to FcγRIIIa or FcγRI. Specifically, H66S298N / T299A / Y300S had almost no binding to either receptor. This mutant was selected as a lead for detailed characterization.

[0231] Characterizing Stability Using Circular Dichroism (CD)

[0232] The stability of the S298N / T299A / Y300S antibody mutant was monitored by Far-UV CD thermal melting experiments, wherein the CD signals at 216nm and 222nm were monitored as the temperature increased (which ultimately led to the unfolding of the antibody). The CD spectrum was collected in a quartz cuvette (Hellma) with a path length of 10mm on a Jasco 815 spectrophotometer with a protein concentration of about 0.5mg / mL (in PBS buffer). The temperature was controlled by a thermoelectric Peltier (Jasco model AWC100) and it was linearly changed from 25°C to 89°C at a rate of 1°C / min. Both CD signals and HT voltages were collected. Data were obtained from 210-260nm, with a data interval of 0.5nm and a temperature interval of 1°C. The scanning speed was 50nm / min and the data pitch was 0.5nm. A bandwidth of 2.5nm and a medium sensitivity setting were used. Four repetitions were scanned for each sample. The results showed that both the δAB H66 and S298N / T299A / Y300S H66 mutants showed similar thermal properties and had the same degradation onset temperature of about 63° C. In other words, the mutants were as stable as the δAB form.

[0233] See also Fig.18 .

[0234] Example 6

[0235] Functional analysis of Fc-engineered mutants

[0236] PBMC proliferation and cytokine release assays were performed as described in Example 2. Normal donor PBMCs were thawed and treated under the following conditions (all in medium containing complement):

[0237] Untreated

[0238] BMA031, moIgG2b 10μg / ml

[0239] OKT3, moIgG2a 10μg / ml

[0240] ·H66, huIgG1δAB 10μg / ml, 1μg / ml and 0.1μg / ml

[0241] ·H66, huIgG1 S298N / T299A / Y300 10μg / ml, 1μg / ml and 0.1μg / ml

[0242] Cytokines were harvested on days 2 (D2) and 4 (D4) for Bioplex analysis (IL2, IL4, IL6, IL8, IL10, GM-CSF, IFNg, TNFa). Cells were stained on D4 for CD4, CD8, CD25 and abTCR expression.

[0243] Shown in Figure 19-22 The results in show that in all cell-based assays, H66S298N / T299A / Y300S behaved similarly to H66δAB, showing minimal T cell activation by CD25 expression; binding to abTCR, but with slightly different kinetics than δAB; minimal cytokine release at D2 and D4 time points; and for several cytokines, the mutant was actually superior to δAB at D4.

[0244] Thus, the S298N / T299A / Y300S mutant abolished effector function as effectively as the δAB mutation.

[0245] Example 7

[0246] Bispecific Antibodies

[0247] A bispecific molecule was constructed consisting of two single chain antibodies (scFv) linked together via a short amino acid linker, wherein one arm can bind to the tumor target and the other can bind to T cells via the αβ TCR. The bispecific molecule is referred to herein as TRACER (T cell receptor activating cytotoxicity EnableR).

[0248] The following humanized anti-αβ TCR scFv constructs were prepared:

[0249] GL1BMΔSxVK1

[0250] GL1BMΔSxVK27

[0251] GL1BMΔSVH11xVK1

[0252] GL1BMΔSVH15xVK1

[0253] GL1BMΔSVH28xVK43

[0254] GL1BMΔSVH31xVK43

[0255] The sequences of the heavy and light chains are set forth in SEQ ID nos 14-16 and 20-24.

[0256] Characterization of these molecules includes assessment of binding to tumor targets and T cells, in vitro cytotoxic activity, and cytokine release profiles in the presence and absence of tumor target cells.

[0257] Binding profiles assessed by flow cytometry showed that the anti-αβTCR bispecific antibody was able to bind both tumor target cell lines and T cells. Fig.23 .

[0258] In vitro cytotoxic activity measured by flow cytometry showed that T cells recruited by the anti-αβTCR bispecific antibody were able to induce T cell-mediated lysis. Fig.24 .

[0259] Analysis of cytokine release profiles has shown that upon binding of the two arms of the bispecific antibody, high levels of TH1 / TH2 cytokines are released from T cells that are not observed in the absence of target cells. Overall, this mechanism of action shows a similar profile to the CD3-based bispecifics described in the literature.

[0260] Example 8

[0261] Preparation and characterization of engineered Fc variants in anti-CD52 antibodies

[0262] To test the generality of the applicability of the Fc mutations described in this application, the glycosylation mutant S298N / Y300S was also prepared in the anti-CD52 antibody (clone 2C3) to see if the modulation of effector function with loss of FcγRIII binding is applicable to different antibody backbones. The S298N / Y300S2C3 variant DNA was prepared by rapid change mutagenesis. After transient transfection of HEK293, the protein was purified from the conditioned medium. Anti-CD52 2C3 wild-type antibody was produced in parallel as a control. Biacore was used to characterize the binding properties, antigen-binding and FcγRIII of the purified antibody (see Fig.26 ).

[0263] The S298N / Y300S2C3 variant bound tightly to the CD52 peptide, and the binding sensorgram was indistinguishable from that of the wild-type control, indicating that this mutation in the Fc domain did not affect its antigen binding ( Fig.26 A).

[0264] To determine Fc effector function, the FcγRIII receptor (Val158) was used for binding studies. Mutant and wild-type control antibodies were diluted to 200 nM and injected into HPC4-tag captured FcγRIIIa. FcγRIII binding was barely detectable for the S298N / Y300S mutant, indicating a loss of effector function for this variant ( Fig.26 B). This mutant also binds to the FcRn receptor with the same affinity as the wild-type antibody control, so we expect no changes in its circulating half-life or other pharmacokinetic properties (see Fig.26 C). We conclude that the S298N / Y300S mutation is generally applicable to antibodies to reduce or eliminate undesired Fc effector functions, for example, through engagement of human Fcγ receptors.

Claims

1. A humanized monoclonal antibody specific for a human αβTCR / CD3 complex, comprising a heavy chain variable region comprising the CDRs set forth in SEQ ID NO:7, 12 or 13 and the human IGH3-23 framework set forth in SEQ ID NO:17, wherein framework position 6 or 18 is a donor residue.

2. A humanized monoclonal antibody specific for a human αβTCR / CD3 complex, comprising a heavy chain variable region comprising the CDRs set forth in SEQ ID NO: 15 or 16 and the human IGHV1-3*01 framework set forth in SEQ ID NO: 18, wherein one or more of framework positions 38, 44 and / or 48 are donor residues.

3. A humanized monoclonal antibody specific for a human αβTCR / CD3 complex, comprising a heavy chain variable region comprising the CDRs described in SEQ ID NO: 15 or 16 and the human IGHV1-3*01 framework described in SEQ ID NO: 18, wherein framework positions 44 and 48 are donor residues.

4. A humanized monoclonal antibody specific for a human αβTCR / CD3 complex, comprising a light chain variable region comprising the CDRs described in SEQ ID NO: 14 and the human IGKV3-11*01 framework described in SEQ ID NO: 19, wherein framework positions 70 and / or 71 are donor residues.

5. A humanized monoclonal antibody specific to human αβTCR / CD3 complex, comprising a heavy chain variable region and a light chain variable region sequence, wherein the heavy chain variable region is selected from the heavy chain comprising the amino acid sequence described in SEQ ID NO:7, SEQ ID NO:12 and SEQ ID NO:13, and the light chain variable region sequence comprises the amino acid sequence described in SEQ ID NO:

14.

6. A humanized monoclonal antibody specific to the human αβTCR / CD3 complex, comprising a heavy chain variable region and a light chain variable region sequence, wherein the heavy chain variable region is selected from the heavy chain comprising the amino acid sequence described in SEQ ID NO: 15 and SEQ ID NO: 16, and the light chain variable region sequence comprises the amino acid sequence described in SEQ ID NO 14.

7. A nucleic acid encoding at least the heavy chain variable region of the humanized monoclonal antibody according to any one of claims 1 to 6.

8. A cell expressing a nucleic acid according to claim 7.

9. An antibody comprising a modified Fc, wherein the modified Fc comprises a modified glycosylation pattern that reduces Fcγ receptor binding, the antibody comprising one or more of the mutations S298N, T299A and Y300S.

10. A multispecific antibody comprising at least the heavy chain of a first binding domain according to any one of claims 1 to 6 and a second binding domain having specificity for a tumor-specific antigen.

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