Anti-αβTCR binding polypeptides with reduced fragmentation

Abstract

The present disclosure relates to improved compositions and methods for treating T cell-mediated diseases and disorders (e.g., autoimmune disorders, graft-versus-host disease, and graft rejection). The following anti-αβTCR binding polypeptides including antibodies are provided, which comprise at least one amino acid substitution or modification, wherein at least one amino acid substitution or modification increases the stability of the binding polypeptide by reducing the breakage of the light chain variable region. The methods provided herein generally relate to administering to a subject in need thereof an effective amount of a stabilized humanized binding polypeptide specific for an αβT cell receptor (αβTCR).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent No. 62 / 828,601, filed April 3, 2019, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on March 30, 2020 is named 704503_SA9-254PC_ST25.txt and is 32,602 bytes in size. Background Art

[0005] Monoclonal antibodies are an important class of conjugated peptides and biotherapeutics. In general, the peptide backbone is highly stable under physiological conditions. However, the fragmentation of heavy and light chain peptides is a major problem for therapeutic monoclonal antibodies.

[0006] In general, protein backbones are highly stable under physiological conditions. However, fractures can be caused by a variety of mechanisms, for example, due to the destruction of natural covalent bonds, resulting in spontaneous or enzymatic cleavage of the polypeptide backbone. In addition, specific regions and motifs (e.g., Asn-Pro motifs) are more susceptible to fracture due to the following reasons: the flexibility of the amino acid sequence, the two-dimensional or three-dimensional polypeptide structure, and incompatible solvents and environmental conditions (e.g., temperature and pH).

[0007] Fragmentation can occur at any step during the manufacture or storage of a biological composition. Because fragmentation can lead to reduced potency or the presence of unwanted and potentially immunogenic material, reducing fragmentation is a key consideration in the production of any biotherapeutic.

[0008] Therefore, there is a need in the art for antibody compositions with improved stability that exhibit reduced antibody polypeptide chain fragmentation during manufacturing and subsequent storage. Summary of the invention

[0009] The present disclosure provides improved compositions and methods for treating T cell-mediated diseases and disorders. Humanized binding polypeptides that specifically bind to αβT cell receptors (αβTCRs) are provided. The anti-αβTCR compositions provided herein are improvements to known compositions, wherein the improvements are that the improved compositions comprise at least one amino acid substitution or modification, and the at least one amino acid substitution or modification increases the stability of the binding polypeptide by reducing the breakage of the light chain variable region. Methods for treating T cell-mediated diseases and disorders (e.g., graft-versus-host disease, autoimmune diseases, and transplant rejection) with improved compositions are also provided. The methods provided herein generally relate to administering to a subject in need thereof an effective amount of a humanized binding polypeptide that specifically binds to αβTCR.

[0010] Surprisingly, the inventors have found that removal of the Asn cleavage site in the light chain of anti-human αβTCR antibody VH31 reduced fragmentation of the antibody. This finding is particularly unexpected because the putative Asn cleavage site occurs within the complementarity determining region (CDR), and previous attempts to remove the Asn cleavage site in an unrelated antibody (i.e., sFLT01) did not prevent antibody fragmentation.

[0011] In one aspect, a binding polypeptide that specifically binds to a human αβTCR / CD3 complex is provided, the binding polypeptide comprising a heavy chain variable region, a light chain variable region and a constant region, wherein:

[0012] The light chain variable region comprises three complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 26, 27 and 28, respectively;

[0013] SEQ ID NO:28 comprises the amino acid sequence QQWSSX 1 -X 2 -LT, where X 1 is an amino acid selected from Q, D, H, S, Y and A, and X 2 is an amino acid selected from P and A; and

[0014] The constant regions are of human origin.

[0015] In certain embodiments, X 1 It’s S.

[0016] In certain embodiments, X 2 It's P.

[0017] In certain embodiments, X 1 is S, and X 2 It's P.

[0018] In certain embodiments, the light chain variable region further comprises a human light chain framework region shown in SEQ ID NO:14.

[0019] In certain embodiments, the binding polypeptide has increased stability at a pH greater than 5.0 compared to VH31.

[0020] In certain embodiments, the binding polypeptide has increased stability at temperatures greater than 4°C compared to VH31.

[0021] In certain embodiments, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:7, 12, 13, 15 and 16.

[0022] In certain embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:12, and SEQ ID NO:13.

[0023] In certain embodiments, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:15 and SEQ ID NO:16.

[0024] In certain embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:15.

[0025] In certain embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:16.

[0026] In certain embodiments, the constant region comprises an Fc modification with a modified glycosylation pattern that reduces Fc gamma receptor binding.

[0027] In certain embodiments, the Fcγ receptor is one or more receptors selected from FcγRIIIa and FcγRI.

[0028] In certain embodiments, the Fc modification is selected from N297Q / S298N / Y300S, S298N / T299A / Y300S, and S298N / Y300S.

[0029] In certain embodiments, the Fc modification is N297Q / S298N / Y300S.

[0030] In certain embodiments, the Fc modification is S298N / T299A / Y300S. In one embodiment, the Fc modification is S298N / Y300S.

[0031] In certain embodiments, the binding polypeptide is humanized.

[0032] In certain embodiments, the binding polypeptide is a monoclonal antibody.

[0033] In certain embodiments, the binding polypeptide is multispecific.

[0034] In certain embodiments, the binding polypeptide is bispecific.

[0035] In another aspect, a pharmaceutical composition is provided, comprising a binding polypeptide as described herein and a pharmaceutically acceptable carrier or diluent.

[0036] In another aspect, a formulation comprising a pharmaceutical composition described herein is provided. In certain embodiments, the formulation is a liquid formulation. In certain embodiments, the formulation is a lyophilized formulation.

[0037] On the other hand, a method for treating a T cell-mediated disease or disorder in a subject is provided, the method comprising administering to the subject an effective amount of a binding polypeptide or pharmaceutical composition as described herein, thereby achieving treatment. In certain embodiments, the T cell-mediated disease or disorder is selected from systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease (CD), multiple sclerosis (MS), scleroderma, type 1 diabetes (T1D), pemphigus vulgaris (PV), psoriasis, atopic dermatitis, celiac disease, chronic obstructive pulmonary disease, Hashimoto's thyroiditis, Graves' disease (thyroid), Sjögren's syndrome, Guillain-Barré syndrome, Goodpasture's hemorrhage-nephritis syndrome, Addison's disease, Wegener's granulomatosis, primary biliary sclerosis, 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, vitiligo, graft-versus-host disease (GvHD), and allograft rejection.

[0038] In another aspect, nucleic acids encoding the binding polypeptides described herein are provided.

[0039] In another aspect, a vector comprising a nucleic acid described herein is provided.

[0040] In another aspect, a cell comprising a nucleic acid as described herein is provided. In one embodiment, the cell is a mammalian cell. In one embodiment, the mammalian cell is selected from Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK) cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0042] Figure 1 is a photograph of a protein gel showing the appearance of low molecular weight (LMW) fragments of antibodies formulated with reference humanized anti-αβTCR antibody VH31 under accelerated conditions including elevated temperature (45°C) and specified pH for five weeks. HC, heavy chain; LC, light chain.

[0043] Figure 2 is a line graph plotting the potency of reference VH31 formulated antibodies at different concentrations under accelerated conditions for five weeks. Open squares correspond to reference VH31 antibodies stored at 45°C, pH 8.0; open circles correspond to reference VH31 antibodies stored at 45°C, pH 5.0; and open diamonds correspond to reference VH31 antibodies stored under control conditions, i.e., ≤0°C, pH 5.0.

[0044] Figure 3 is a photograph of a protein gel showing the assigned identities based on molecular weight and N-terminal sequencing of reference VH31 bands present under the indicated accelerated conditions. HC, heavy chain; LC, light chain.

[0045] Figure 4 The reference VH31 light chain (LC) amino acid sequence (SEQ ID NO:25) and the N93 / P94 putative splice sites are depicted.

[0046] Figure 5A-Figure 5C is a line graph showing various light chain fragments generated by cleavage at N93 / P94 of a reference VH31 light chain tryptic digest (amino acids 61-102 or 61-106 according to Kabat numbering). These fragments correspond to amino acid residues 94-102 ( Figure 5A ), residues 61-93( Figure 5B ) and residues 94-106 ( Figure 5C ). The C-termini of 102 and 106 are due to incomplete trypsin digestion.

[0047] Figure 6 Depicted is a proposed method by which cleavage at Asn 93 (N93) can occur in solution.

[0048] Figure 7 Depicted are various VH31 LC amino acid mutations made to enhance LC stability.

[0049] Figure 8is a pair of protein gel photographs showing high expression and purity of VH31 antibodies containing mutant (lanes 1-8) or non-mutated LC. 1, N93Q; 2, N93D; 3, N93H; 4, N93S; 5, N93Y; 6, N93A; 8, N93A / P94A.

[0050] Fig. 9 are photographs of a pair of protein gels (left panels) and three SEC-HPLC graphs (right panels) showing that the purity of antibodies comprising the N93S LC mutation is comparable to that of wild-type (WT) and control (VH31) antibodies.

[0051] Fig.10 is a line graph plotting the potency of VH31 antibodies comprising the indicated LC mutations. Antibodies comprising the LC variant N93S (open diamond symbols) are only slightly less potent than the reference VH31 (open square symbols) or the wild-type (WT) antibody (open octagon symbols).

[0052] Fig.11 is a photograph of a protein gel showing (left panel) that the N93S mutant has significantly reduced degradation compared to the wild-type (WT) or VH31 control antibody after storage for 6 weeks at 45°C, pH 8. The right panel shows that the N93S mutant has significantly less potency loss (3-fold loss) under accelerated conditions compared to the wild-type or VH31 control antibodies (both greater than 20-fold loss).

[0053] Fig.12 is a photograph of a protein gel showing the fragmentation of the reference VH31 antibody and the N93S mutant after six weeks of storage at elevated temperature (45°C) and pH 5.5. DETAILED DESCRIPTION

[0054] The present disclosure provides improved compositions and methods for treating T cell-mediated disorders (e.g., graft-versus-host disease, autoimmune diseases, and allogeneic graft rejection). The methods provided herein generally relate to administering to a subject in need thereof an effective amount of a humanized binding polypeptide specific for an αβT cell receptor (αβTCR). The anti-αβTCR compositions provided herein are improvements to known compositions, wherein the improvements are that the improved compositions comprise one or more amino acid substitutions or modifications, and the one or more amino acid substitutions or modifications improve the stability of the binding polypeptide by reducing the breakage of the light chain variable region.

[0055] I. Definitions

[0056] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present disclosure belongs. Any methods and materials similar or equivalent to those described herein may be used in the methods of the technology of the present disclosure. For the purpose of describing and publishing methods, reagents and tools reported in publications that may be relevant to the present disclosure, all publications cited herein are incorporated herein by reference in their entirety.

[0057] 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. See, e.g., Gennaro, AR, ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, JG, Limbird, LE, and Gilman, AG, ed. (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., ed., Methods In Enzymology, Academic Press, Inc.; 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 and Sons, Inc. Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, CR and Graham, A., eds. (1997) PCR (Introduction to Biotechniques Series), 2nd Edition, Springer-Verlag.

[0058] The human αβTCR / CD3 complex is a T cell receptor complex present on the surface of T cells. See Kuhns et al. (2006) Immunity 24: 133-139. The complex is targeted by the mouse monoclonal antibody BMA031 (see European patent application EP0403156; SEQ ID NO: 1 and 2, incorporated herein by reference in its entirety) and the humanized stabilized antibodies disclosed herein.

[0059] Mouse IgG2b monoclonal antibody BMA031 (Borst et al. (1990) Hum. Immunol. 29 (3): 175-88; EP0403156) is specific for the common determinant cluster of the α-β TCR / CD3 complex and does not bind to the γ-δ TCR. BMA031 is highly immunosuppressive and can induce activated T cell apoptosis through an activation-induced cell death (AICD) mechanism (Wesselborg et al. (1993) J. Immunol. 150 (10): 4338-4345). In vitro, it inhibits mixed lymphocyte reactions and shows preliminary clinical efficacy in preventing graft rejection and treating acute graft-versus-host disease in many solid organ transplant scenarios (Kurrle et al. (1989) Transplant Proc. 21 (1): 1017-1019). BMA031 does not bind to human Fcγ receptors (FcγRs) in most populations. Therefore, BMA031 does not cause T cell activation by cross-linking of T cell receptors, and therefore, it does not induce T cell activation or related cytokine release. In this respect, its characteristics are more preferred than those of OKT3. However, BMA031 is a murine antibody and is therefore not suitable for repeated administration in human subjects in view of the human anti-mouse antibody (HAMA) response elicited therein.

[0060] Several humanized forms of BMA031 have been described (see, e.g., WO 2013 / 037484; EP 0403156; also Shearman et al. (1991) J. Immunol. 147: 4366-4373). As described in EP 0403156, CDR transplantation alone was not successful in maintaining antigen binding. A clone with significant "civilizing" framework modifications, EUCIV3, successfully bound to T cells; however, as described in EP 0403156, binding to αβTCR was not as effective as the parent BMA031 antibody, as determined by flow cytometry competition assays. In addition, EUCIV3 was originally generated on a wild-type human IgG1 or IgG4 backbone that still retained FcγR binding. Therefore, these humanized antibodies allow T cell activation, proliferation, and concomitant cytokine release, and are therefore significantly different from the original properties of BMA031.

[0061] Unless otherwise indicated, the term "binding protein" or "binding polypeptide" is used to refer to a polypeptide (e.g., an antibody or antigen-binding fragment thereof) that contains at least one binding site responsible for selectively binding to a target antigen of interest (e.g., a human antigen). Exemplary binding sites include antibody variable domains, ligand binding sites of receptors, or receptor binding sites of ligands. In certain aspects, the binding polypeptides described herein comprise multiple (e.g., two, three, four or more) binding sites.

[0062] Unless otherwise indicated, the term "antibody" is used to refer to intact antibodies as well as antigen-binding fragments of such antibodies. For example, the term includes four-chain IgG molecules as well as antibody fragments.

[0063] As used herein, the term "antibody fragment" refers to a portion of an intact full-length antibody, eg, as further described below.

[0064] The antibody can be of any class, such as IgG, IgA, IgE, IgD, or IgM; and of any subclass, such as IgG1 or IgG4. Different classes and subclasses of immunoglobulins have different properties, which may be advantageous in different applications. For example, IgG4 antibodies have reduced binding to Fc receptors.

[0065] Naturally occurring immunoglobulins have a common core structure in which two identical light chains (about 24 kDa) and two identical heavy chains (about 55 or 70 kDa) form a tetramer. The amino-terminal portion of each chain is called the variable (V) region and can be distinguished from the more conserved constant (C) region of the rest of each chain.

[0066] Most amino acid sequence variations in immunoglobulins are limited to three independent positions in each V region, called hypervariable regions or complementary determining regions (CDRs), which are directly involved in antigen binding. Starting from the amino terminus, these regions are respectively referred to as CDR1, CDR2 and CDR3. CDR is fixed in place by a more conservative framework region (FR). Starting from the amino terminus, these regions are respectively referred to as FR1, FR2, FR3 and FR4. Starting from the amino terminus, these combined regions contained in the V region are referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Kabat et al. have defined the position and numbering system of CDR and FR regions. (Kabat, EA et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (USGovernment Printing Office) (1991), and its updated version that can be found online). In addition, the IMGT nomenclature further defines the CDR region boundaries.

[0067] As used herein, a "humanized monoclonal antibody" is an antibody consisting of a human antibody framework into which complementary determining regions (CDRs) from non-human antibodies have been grafted. The human acceptor framework may also be altered. The design and production procedures of humanized antibodies are well known in the art and have been described in, for example, U.S. Patent No. 4,816,397; U.S. Patent No. 4,816,567; and U.S. Patent No. 5,225,539; European Patent Application No. 0 120 694; European Patent Application No. 0 125023; European Patent Application No. 0 194 276B1; European Patent Application No. 0 239 400; European Patent Application No. 0 519 596; and International Patent Application WO 86 / 01533. More details about antibodies, humanized antibodies, human engineered antibodies, and methods for making them can be found in Kontermann, R. and Dijbel, S., eds. (2001, 2010) Antibody Engineering, 2nd ed., Springer-Verlag, New York, N.Y. The entire contents of each of the patents and patent application publications listed above are incorporated herein by reference.

[0068] The variable region of the antibody according to the described embodiment can be obtained at least in part by humanizing BMA031, i.e., by transferring the CDRs of BMA031 to a human framework, and further modifying one or more VL CDRs to improve the stability of the binding polypeptide by reducing the breakage of the light chain variable region. Two series of humanized BMA031 antibodies are described in PCT Publication WO 2013 / 037484, which is incorporated herein by reference. The two series are: the HEBE1 series, which comprises SEQ ID NOs: 5-7, 12 and 13 of the present invention; and the GL1BM series, which comprises the heavy chain variable region as shown in SEQ ID NOs: 8, 15 and 16 of the present invention. In both cases, the light chain variable region used is as shown in SEQ ID NO: 14 (GL1BM VK43) of the present invention. The human frameworks used were IGH3-23 (SEQ ID NO: 17 of the present invention) in the case of HEBE1, and IGHV1-3*01 and IGKV3-11*01 (SEQ ID NO: 18 and 19 of the present invention) in the case of GL1BM.

[0069] The constant region can be derived from any human antibody constant region. The variable region gene can be cloned into an expression vector in the same frame as the constant region gene to express the immunoglobulin heavy chain and light chain. Such expression vectors can be transfected into antibody-producing host cells for antibody synthesis.

[0070] 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 VBase30 (vbase.mrc-cpe.cam.ac.uk). Deglycosylated antibodies can have extensively modified functions; see Boyd et al. (1996) Mol.Immunol.32: 1311-1318. As used herein, "Δab" or Δab modification is an Fc modification described in Armour et al., (1999) Eur.J.Immunol.29: 2613-2624. Techniques for modifying the glycosylation of antibody Fc regions are known in the art and include chemical, enzymatic and / or mutational means, such as mutations at position N297 in the CH2 domain. Techniques for mutating antibody genes to produce deglycosylated IgG molecules are described in Tao and Morrison (1989) J. Immunol. 143:2595-2601.

[0071] In the context of the antibodies described herein, specificity means that the claimed antibody is able to selectively bind to its defined cognate antigen, i.e., the αβTCR / CD3 complex. The antibodies described herein bind to the αβTCR / CD3 complex expressed on cells, including on T cells.

[0072] The terms "stable", "stability" and "stabilization", as used herein in the context of binding polypeptides, refer to the resistance of binding polypeptides to thermal and chemical degradation or cleavage under given manufacturing, preparation, transportation and storage conditions. Under given manufacturing, preparation, transportation and storage conditions, "stable" compositions retain greater than or equal to 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or 99.9% biological activity. The stability of binding polypeptides can be evaluated, for example, in terms of the degree of degradation or cleavage, or the level of specific fragments or the type or size of aggregates compared to a control or compared to the starting material, using methods and measurements known to those skilled in the art. Such methods and measurements include, but are not limited to, reduced area under the curve (AUC), size exclusion chromatography (SEC), high performance (or high pressure) size exclusion chromatography (HPSEC), liquid chromatography-mass spectrometry (LC-MS), capillary gel electrophoresis (CGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) compared to a reference.

[0073] As used herein, the term "nucleic acid" includes DNA molecules encoding the antibodies described herein. Preferred DNA molecules encoding the antibodies described herein are expression vectors suitable for expressing the antibody genes in host cells. 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.

[0074] As used herein, the terms "treat" and "treatment" refer to the care of a patient or subject suffering from a disease, disorder, or condition. Treatment may involve, but is not limited to, any one or any combination of: cure of the disease, disorder, or condition; amelioration of at least one symptom of the disease, disorder, or condition; and / or prophylactic or preventative behavior, the purpose of which is to prevent or reduce the occurrence of the disease, disorder, or condition. In certain embodiments, treatment may involve, but is not limited to, cure of the disease, disorder, or condition; or amelioration of at least one symptom of the disease, disorder, or condition.

[0075] As used herein, the term "subject" refers to any mammal, including mice, rats, gerbils, hamsters, guinea pigs, rabbits, cats, dogs, sheep, goats, pigs, cattle, horses, and primates. In certain embodiments, the subject is a mammal other than a human. In certain embodiments, the subject is a non-human primate. In certain embodiments, the subject is a human.

[0076] II. Antibodies

[0077] The present disclosure includes methods of administering one or more antigen-binding fragments of the humanized anti-αβTCR antibodies described herein to a subject in need thereof. The antibody fragments are capable of binding to the αβTCR / CD3 complex. They include Fab, Fab', F(ab')2 and F(v) fragments, or individual light chain or heavy chain variable regions or any portion thereof. Fragments include, for example, Fab, Fab', F(ab')2 and F(v) fragments. 2 , Fv, scFv, etc. In some aspects, the fragment lacks the Fc portion of the intact antibody, is cleared from the circulation faster, and / or may have less nonspecific tissue binding than the intact antibody. In some aspects, fragments can be generated from intact antibodies using well-known methods, such as by using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments) by proteolytic cleavage.

[0078] In some aspects, antibodies and / or antibody fragments include single-chain antibody fragments (scFv) bound to αβTCR / CD3 complexes. In some aspects, scFv includes an antibody heavy chain variable region (VH) operably connected to an antibody light chain variable region (VL), wherein one or both of the heavy chain variable region and the light chain variable region form a binding site for binding αβTCR together or alone. ScFv can include a VH region at the amino terminal and a VL region at the carboxyl terminal. Alternatively, scFv can include a VL region at the amino terminal and a VH region at the carboxyl terminal. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic joints using recombinant methods, so that they can be made into a single protein chain, wherein the VL and VH regions are paired to form a monovalent molecule (referred to as single-chain Fv (scFv)). ScFv can also optionally include a polypeptide joint between the heavy chain variable region and the light chain variable region.

[0079] Antibodies and antibody fragments also include domain antibody (dAb) fragments composed of VH domains (as described in Ward, ES et al. (1989) Nature 341: 544-546). Antibodies and antibody fragments also include heavy chain antibodies (HCAb). It is reported that HCAb only uses the heavy chain variable region to form an antigen binding region because these functional antibodies are dimers of only heavy chains (referred to as "heavy chain antibodies" or "HCAb"). Therefore, in some aspects, antibodies and antibody fragments can be HCAbs that specifically bind to the αβTCR / CD3 complex. Antibodies and antibody fragments also include antibodies that act as small modular immunopharmaceuticals (SMIPs) or binding domain immunoglobulin fusion proteins that are specific for the αβTCR / CD3 complex. These constructs are single-chain polypeptides that include an antigen binding domain fused to an immunoglobulin domain necessary for performing antibody effector functions (see WO 2005 / 017148). Antibodies and antibody fragments also include diabodies. Diabodies refer to bivalent antibodies in which the VH and VL 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, thereby creating two antigen binding sites (see, e.g., WO 93 / 11161). Diabodies can be bispecific or monospecific.

[0080] In certain aspects, the antibody or antibody fragment specifically binds to the human αβTCR / CD3 complex, ie, the antibody or antibody fragment does not cross-react with any target other than the human αβTCR / CD3 complex.

[0081] The antibody or antibody fragment may be modified to increase its serum half-life, for example by adding molecules such as PEG or other water-soluble polymers (including polysaccharide polymers, etc.) to increase the half-life of the antibody or antibody fragment.

[0082] Antibodies and antibody fragments can be multi-specific or bispecific. For example, a bispecific antibody or antibody fragment can be similar to a single antibody (or antibody fragment) comprising two different binding sites (variable regions). Bispecific antibodies can be produced by various methods, such as chemical techniques, "polydoma" techniques or recombinant DNA techniques. Bispecific antibodies can have binding specificity for at least two different epitopes, at least one of which is an αβTCR / CD3 complex. Other specificities can be selected from any useful or desired specificity, including, for example, specificity for human serum albumin to extend the half-life in vivo.

[0083] With the trifunctional catumaxomab Approved for use in malignant ascites settings and the bispecific antibody blinatumomab Approved as a second-line treatment for Philadelphia chromosome-negative relapsed or refractory acute lymphoblastic leukemia (ALL), bispecific antibodies are now becoming a reality for clinical use in oncology. These antibodies have in common one binding arm that binds to T cells and a second arm that binds to tumor target cells, resulting in T cell-mediated lysis of the tumor target. Also in common is that these molecules recruit T cells via the CD3 protein located on the cell surface. Another alternative to recruitment via CD3 is to utilize the αβ T cell receptor (αβTCR) also expressed on the cell surface.

[0084] In certain exemplary embodiments, antibodies according to the present disclosure can be used to develop anti-tumor antibodies by combining specificity for tumor-associated antigens with specificity for αβ T cell receptor (αβ TCR).

[0085] III. Anti-αβTCR Antibodies

[0086] As described above, two series of such humanized anti-αβTCR antibodies are described in PCT Publication WO 2013 / 037484, which is incorporated herein by reference. The two series are based on the murine anti-αβTCR antibody BMA031 and include: the HEBE1 series, which comprises SEQ ID NOs: 5-7, 12 and 13; and the GL1BM series, which comprises 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 shown in SEQ ID NO: 14 (GL1BMVK43). The human framework used is IGH3-23 (SEQ ID NO: 17) in the case of HEBE1, and IGHV1-3*01 (SEQ ID NO: 18) and IGKV3-11*01 (SEQ ID NO: 19) in the case of GL1BM.

[0087] In the sequences listed in Table 1, selected CDRs are shown in bold and are set forth as SEQ ID NOs: 26-28.

[0088] Table 1. VH, VL and CDR amino acid sequences of exemplary anti-αβ TCR antibodies.

[0089]

[0090]

[0091]

[0092] The LCDR3 of GL1BM VK43 in SEQ ID NO: 14 (QQWSSNPLT (SEQ ID NO: 29)) and the LCDR3 of stabilized GL1BM VK43 (QQWSSX 1 -X 2 -LT, where X 1 is Q, D, H, S, Y, or A, and X 2 is P or A (SEQ ID NO: 28)) revealed that amino acid X in SEQ ID NO: 28 1 and X 2 They correspond to N and P in SEQ ID NO: 29, respectively.

[0093] In certain embodiments, a binding polypeptide that specifically binds to a human αβTCR / CD3 complex is provided, the binding polypeptide comprising a heavy chain variable region, a light chain variable region, and a constant region, wherein:

[0094] The light chain variable region comprises three complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences shown in SEQ ID NOs: 26, 27 and 28, respectively;

[0095] SEQ ID NO:28 comprises the amino acid sequence QQWSSX 1 -X 2 -LT, where X 1 is an amino acid selected from Q, D, H, S, Y and A, and X 2 is an amino acid selected from P and A; and

[0096] The constant regions are of human origin.

[0097] In certain embodiments, X 1 It's Q.

[0098] In certain embodiments, X 1 It is D.

[0099] In certain embodiments, X 1 It's H.

[0100] In certain embodiments, X 1 It’s S.

[0101] In certain embodiments, X 1 It is Y.

[0102] In certain embodiments, X 1 It is A.

[0103] In certain embodiments, X 2 It's P.

[0104] In certain embodiments, X 2 It is A.

[0105] In certain embodiments, X 1 is Q, and X 2 It's P.

[0106] In certain embodiments, X 1 is Q, and X 2 It is A.

[0107] In certain embodiments, X 1 is D, and X 2 It's P.

[0108] In certain embodiments, X 1 is D, and X 2 It is A.

[0109] In certain embodiments, X 1 is H, and X 2 It's P.

[0110] In certain embodiments, X 1 is H, and X 2 It is A.

[0111] In certain embodiments, X 1 is S, and X 2 It's P.

[0112] In certain embodiments, X 1 is S, and X 2 It is A.

[0113] In certain embodiments, X 1 is Y, and X 2 It's P.

[0114] In certain embodiments, X 1 is Y, and X 2 It is A.

[0115] In certain embodiments, X 1 is A, and X 2 It's P.

[0116] In certain embodiments, X 1 is A, and X 2 It is A.

[0117] In certain embodiments, the light chain variable region further comprises a human light chain framework region according to SEQ ID NO:14.

[0118] In certain embodiments, the binding polypeptide has increased stability at a pH greater than 5.0 compared to reference antibody VH31.

[0119] In certain embodiments, the binding polypeptide has increased stability at temperatures greater than 4°C compared to reference antibody VH31.

[0120] In certain embodiments, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:7, 12, 13, 15 and 16.

[0121] In certain embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:12, and SEQ ID NO:13.

[0122] In certain embodiments, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:15 and SEQ ID NO:16.

[0123] In certain embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:15.

[0124] In certain embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:16.

[0125] IV. Modified anti-αβTCR antibodies

[0126] The anti-αβTCR antibody may comprise one or more modifications. The modified anti-αβTCR antibody according to the present invention may be prepared using any technique known in the art.

[0127] i) Reduce fracture

[0128] The term "fragmentation" as used herein in the context of a binding polypeptide composition refers to the cleavage of a binding polypeptide or a first portion thereof into two or more portions, each portion having a lower molecular weight than the original binding polypeptide or the first portion thereof. Such fragmentation forms include, but are not limited to, a full-length unpaired heavy chain, a full-length unpaired light chain, a first full-length heavy chain paired with a second full-length heavy chain, a full-length heavy chain paired with a partial heavy chain, a first partial heavy chain paired with a second partial heavy chain, a full-length heavy chain paired with a full-length light chain, a full-length heavy chain paired with a partial light chain, a partial heavy chain paired with a full-length light chain, a partial heavy chain paired with a partial light chain, a first full-length heavy chain and a full-length light chain paired with a second full-length heavy chain, a first full-length heavy chain and a partial light chain paired with a second full-length heavy chain, a first full-length heavy chain paired with a first partial light chain and a second full-length heavy chain paired with a second partial light chain, a first full-length heavy chain and a full-length light chain paired with a partial heavy chain, a first full-length heavy chain and two full-length light chains paired with a partial heavy chain, a first full-length heavy chain and a partial light chain paired with a partial heavy chain, a first partial heavy chain and a full-length light chain paired with a second partial heavy chain, and a first partial heavy chain and a partial light chain paired with a second partial heavy chain.

[0129] As used herein, "fragment" refers to at least one amino acid. Typically, a fragment will contain two or more amino acids linked together, more typically at least 10, 20, 30, 40 or 50 such amino acids. Particularly for fragments containing more than one polypeptide chain, a fragment may contain, for example, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600 or at least 700 amino acids.

[0130] Fragmentation can be caused by a variety of mechanisms, for example, due to the destruction of natural covalent bonds, resulting in the cleavage of the polypeptide backbone by spontaneous (e.g., non-enzymatic) or enzymatic reactions. In general, protein backbones are highly stable under physiological conditions. However, specific regions and motifs are more susceptible to fracture due to the following reasons: their amino acid sequence, the flexibility of the two-dimensional or three-dimensional polypeptide structure, and incompatible solvents and environmental conditions (e.g., temperature and pH).

[0131] For example, the amino acids Asp (D), Gly (G), Ser (S), Thr (T), Cys (C), and Asn (N) have been shown to be particularly sensitive to cleavage in the polypeptide backbone of antibodies. See, e.g., Liu H., et al. J. Am. Soc. Mass Spectrom. 2009; 20: 2258-2264. In addition, the Asn-Pro (NP) amide bond is known to completely cleave in the presence of ammonia. See, e.g., Tarelli E. and Corran PHPeptide Res. 2003; 62: 245-251.

[0132] Fragmentation can occur at any step during the manufacture or storage of a biological composition. Because fragmentation can lead to reduced potency or the presence of unwanted and potentially immunogenic material, reducing fragmentation is a key consideration in the production of any biotherapeutic.

[0133] In some embodiments, less than 5% (e.g., less than 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4.0%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, 3.0%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2.0%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%). , 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) antibodies are stored at 2°C to 8°C for at least one month (e.g., at least two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, or longer).

[0134] Methods for determining the amount of monomeric binding polypeptides and the amount of monomeric, oligomeric, aggregated or fragmented forms of binding polypeptides present in solution are described herein and exemplified in the working examples. For example, a skilled artisan can use, for example, size exclusion chromatography high performance liquid chromatography (SEC-HPLC), static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and differential scanning calorimetry (DSC) to determine the percentage of intact, fragmented, unfolded intermediates and / or aggregated species present in a given solution. In the working examples described herein, the inventors exemplify the use of SEC-HPLC and SDS-PAGE, etc., to determine the physical state of binding polypeptides in solution.

[0135] ii) Reduce immunogenicity

[0136] In certain exemplary embodiments, deimmunization can be used to reduce the immunogenicity of an antibody or its antigen-binding portion. As used herein, the term "deimmunization" includes changing an antibody or its antigen-binding portion to modify one or more T cell epitopes (see, e.g., WO 98 / 52976 A1, WO 00 / 34317 A2). For example, the VH and VL sequences from the starting antibody can be analyzed, and a human T cell epitope "map" can be generated from each V region, the map showing the position of the epitope associated with the complementary determining region (CDR) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map can be analyzed to identify alternative amino acid substitutions with a low risk of changing the activity of the final antibody. A series of alternative VH and VL sequences can be designed, the sequences including a variety of combinations of amino acid substitutions, and these sequences can then be incorporated into a series of anti-αβTCR antibodies or anti-αβTCR antibody fragments for use in the methods disclosed herein, and then the function of the antibodies or fragments is tested. The complete heavy and light chain genes containing the modified V and human C regions are then cloned into expression vectors, and the subsequent plasmids can be introduced into cell lines to produce complete antibodies. The antibodies can then be compared in appropriate biochemical and biological assays, and the best variants can be identified.

[0137] iii) Effector function and Fc modification

[0138] Humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) may include antibody constant regions (e.g., IgG constant regions, such as human IgG constant regions, such as human IgG1 or IgG4 constant regions) that mediate one or more effector functions. For example, the binding of the C1 component of complement to the antibody constant region can activate the complement system. The activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates inflammatory responses and may also participate in autoimmune hypersensitivity reactions. In addition, antibodies bind to receptors on a variety of cells via the Fc region, wherein the Fc receptor binding site on the antibody Fc region binds to the Fc receptor (FcR) on the cell. There are many Fc receptors, and the Fc receptors are specific for antibodies of different classes, including IgG (γ receptors, i.e., Fcγ receptors), IgE (ε receptors), IgA (α receptors), and IgM (μ receptors). Binding of antibodies to Fc receptors on the cell surface triggers a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (termed antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[0139] In certain embodiments, a humanized monoclonal antibody or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof) binds to an Fcγ receptor. In alternative embodiments, a humanized monoclonal antibody or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof) may include a constant region that is unable to direct one or more effector functions (e.g., ADCC activity) and / or is unable to bind to an Fcγ receptor.

[0140] Certain embodiments described herein provide humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof), wherein at least one amino acid in one or more constant region domains has been deleted or otherwise altered to provide desired biochemical characteristics, such as reduced or enhanced effector function, ability to non-covalently dimerize, improved ability to localize at tumor sites, reduced serum half-life, and / or increased serum half-life when compared to an intact, unaltered antibody having approximately the same immunogenicity. For example, certain antibodies or fragments thereof used in the diagnostic and therapeutic methods described herein are domain-deleted antibodies that contain a polypeptide chain similar to an immunoglobulin heavy chain, but lack at least a portion of one or more heavy chain domains. For example, in certain antibodies, an entire domain of the constant region of the modified antibody will be deleted, for example, all or part of the CH2 domain will be deleted.

[0141] In certain other embodiments, the humanized monoclonal antibody or humanized monoclonal antibody fragment (e.g., anti-αβTCR antibody or fragment thereof) comprises a constant region derived from a different antibody isotype (e.g., a constant region from two or more of human IgG1, IgG2, IgG3, or IgG4). In other embodiments, the humanized monoclonal antibody or humanized monoclonal antibody fragment (e.g., anti-αβTCR antibody or fragment thereof) comprises a chimeric hinge (i.e., a hinge comprising a hinge portion of a hinge domain derived from a different antibody isotype, such as an upper hinge domain from an IgG4 molecule and an IgG1 middle hinge domain). In one embodiment, the humanized monoclonal antibody or humanized monoclonal antibody fragment (e.g., anti-αβTCR antibody or fragment thereof) comprises an Fc region or portion thereof from a human IgG4 molecule, and a Ser228Pro (S228P) mutation (EU numbering) in the core hinge region of the molecule.

[0142] In certain exemplary embodiments, the Fc portion of a humanized monoclonal antibody or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof) can be mutated using techniques known in the art to increase or decrease effector function. For example, the deletion or inactivation of a constant region domain (by point mutation or other means) can reduce the Fc receptor binding of a circulating modified antibody, thereby increasing tumor localization. In other cases, it may be that constant region modifications consistent with the present disclosure weaken complement binding, thereby reducing serum half-life and nonspecific association of conjugated cytotoxins. Other modifications of the constant region can be used to modify disulfide bonds or oligosaccharide moieties, which allow enhanced localization due to increased antigenic specificity or flexibility. The resulting physiological characteristics, bioavailability, and other biochemical effects of modification (such as tumor localization, biodistribution, and serum half-life) can be easily measured and quantified using well-known immunological techniques without excessive experiments.

[0143] In certain embodiments, the Fc domain used in a humanized monoclonal antibody or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof) is an Fc variant. As used herein, the term "Fc variant" refers to an Fc domain having at least one amino acid substitution relative to a wild-type Fc domain from which the Fc domain is derived. For example, wherein the Fc domain is derived from a human IgG1 antibody, the Fc variant of the human IgG1 Fc domain comprises at least one amino acid substitution relative to the Fc domain.

[0144] The one or more amino acid substitutions of the Fc variant can be located at any position (i.e., any EU customary amino acid position) within the Fc domain. In one embodiment, the Fc variant comprises a substitution at an amino acid position located in the hinge domain or a portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in the CH2 domain or a portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in the CH3 domain or a portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in the CH4 domain or a portion thereof.

[0145] The antibodies may employ any art-recognized Fc variant known to confer improved (eg, reduced or enhanced) effector function and / or FcR binding. The Fc variants can include, for example, International PCT Publications WO 88 / 07089A1, WO 96 / 14339A1, WO 98 / 05787A1, WO 98 / 23289A1, WO 99 / 51642A1, WO 99 / 58572A1, WO 00 / 09560A2, WO 00 / 32767A1, WO 00 / 42072A2, WO 02 / 44215A2, WO 02 / 060919A2, WO 03 / 074569A2, WO 04 / 016750A2, WO 04 / 029207A2, WO 04 / 035752A2, WO 04 / 063351A2, WO 04 / 074455A2, WO 04 / 099249A2, WO 05 / 040217A2, WO05 / 070963A1, WO 05 / 077981A2, WO 05 / 092925A2, WO 05 / 123780A2, WO 06 / 019447A1, WO 06 / 047350A2 and WO Any one of the amino acid substitutions disclosed in 06 / 085967A2 or U.S. Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784, each of which is incorporated herein by reference.

[0146] In an exemplary embodiment, a humanized monoclonal antibody or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof) may include an Fc variant comprising an amino acid substitution (e.g., H268D or H268E) at EU position 268. In another exemplary embodiment, a humanized monoclonal antibody or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof) may include an amino acid substitution at EU position 239 (e.g., S239D or S239E) and / or EU position 332 (e.g., I332D or I332Q).

[0147] In certain embodiments, a humanized monoclonal antibody or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof) may include an Fc variant comprising an amino acid substitution that changes the antigen-independent effector function of the antibody, particularly the circulating half-life of the antibody. When compared to antibodies lacking these substitutions, such antibodies exhibit increased or decreased binding to neonatal Fc receptors (FcRn), and therefore have an increased or decreased half-life in serum, respectively. Fc variants with improved affinity for FcRn are expected to have a longer serum half-life, and such molecules have useful applications in methods for treating mammals, where the administered antibodies are required to have a long half-life, for example, to treat chronic diseases or disorders. On the contrary, Fc variants with reduced FcRn binding affinity are expected to have a shorter half-life, and such molecules may also be used, for example, for administration to mammals, where shortened circulation times may be advantageous, for example, for in vivo diagnostic imaging or when the initial antibody is present in the circulation for a long time with toxic side effects. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta and therefore may also be used to treat diseases or disorders in pregnant women. In addition, other applications that may require reduced FcRn binding affinity include those that require localization to the brain, kidney and / or liver. In an exemplary embodiment, the altered antibody exhibits reduced transport from the vasculature through the glomerular epithelium. In another embodiment, the altered antibody exhibits reduced transport from the brain through the blood-brain barrier (BBB) ​​into the vascular space.

[0148] In one embodiment, the antibody with altered FcRn binding comprises an Fc domain with one or more amino acid substitutions within the "FcRn binding loop" of the Fc domain. The FcRn binding loop is composed of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions that change FcRn binding activity are disclosed in International PCT Publication No. WO 05 / 047327, which is incorporated herein by reference in its entirety for all purposes. In certain exemplary embodiments, a humanized monoclonal antibody or humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof) comprises an Fc domain with one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0149] In other embodiments, the humanized monoclonal antibody or humanized monoclonal antibody fragment (e.g., anti-αβTCR antibody or fragment thereof) used in the diagnostic and therapeutic methods described herein has a constant region that is changed to reduce or eliminate glycosylation, such as an IgG1 or IgG4 heavy chain constant region. For example, the antibody may also include an Fc variant comprising an amino acid substitution that changes the glycosylation of the antibody. For example, the Fc variant may have reduced glycosylation (e.g., N- or O-linked glycosylation). In an exemplary embodiment, the Fc variant comprises reduced glycosylation of N-linked glycans typically found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution within or near a glycosylation motif (e.g., an N-linked glycosylation motif containing an amino acid sequence NXT or NXS). In a specific embodiment, the antibody includes an Fc variant having an amino acid substitution at amino acid position 228 or 299 (EU numbering). In a more specific embodiment, the antibody comprises an IgG1 or IgG4 constant region containing S228P and T299A mutations (EU numbering).

[0150] In international PCT publication number WO 05 / 018572, exemplary amino acid substitutions that confer reduced or altered glycosylation are disclosed, and the document is incorporated herein by reference in its entirety for all purposes. In certain embodiments, the antibody or its fragment is modified to eliminate glycosylation. Such antibodies or their fragments may be referred to as "agly" antibodies or their fragments (e.g., "agly" antibody fragments). Although it is not intended to be bound by scientific theory, it is believed that agly antibodies or their fragments may have improved safety and stability characteristics in vivo. Exemplary agly antibodies or agly antibody fragments include deglycosylated Fc regions of IgG4 antibodies lacking Fc effector functions, thereby eliminating the possibility of Fc-mediated toxicity to normal vital organs. In yet other embodiments, agly antibodies or agly antibody fragments include altered polysaccharides. For example, agly antibodies or agly antibody fragments may have a reduced number of fucose residues on the N- polysaccharides at Asn297 (N297) in the Fc region, i.e., defucosylation. In another embodiment, the agly antibody or agly antibody fragment may have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.

[0151] iv) Covalent attachment

[0152] A humanized monoclonal antibody or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof) can be modified, for example, by covalently attaching a molecule to the antibody so that the covalent attachment does not prevent the antibody from specifically binding to its cognate epitope. For example, but without limitation, a humanized monoclonal antibody or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof) can be modified by: glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, etc. In addition, the derivative may contain one or more non-classical amino acids.

[0153] Humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) can be further recombinantly fused to heterologous polypeptides at the N-terminus or C-terminus or chemically conjugated (including covalent and non-covalent conjugation) to polypeptides or other compositions. For example, anti-αβTCR antibodies can be recombinantly fused or conjugated to molecules that can be used as labels in detection assays and effector molecules (such as heterologous polypeptides, drugs, radionuclides, or toxins). See, for example, PCT Publications WO 92 / 08495, WO 91 / 14438, and WO 89 / 12624; U.S. Pat. No. 5,314,995; and EP 396,387.

[0154] Humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) can be fused to one or more heterologous polypeptides to increase the in vivo half-life or used in immunoassays using methods known in the art. For example, in one embodiment, polyethylene glycol (PEG) can be conjugated to humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) to increase their in vivo half-life. Leong, SR, et al., Cytokine 16:106 (2001); Chapman AP, Adv. Drug Deliv. Rev. 54:531 (2002); or Weir et al., Biochem. Soc. Transactions 30:512 (2002).

[0155] In addition, humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) can be fused to one or more marker sequences (e.g., peptides) to facilitate purification or detection of humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof). In certain embodiments, the marker amino acid sequence is a six-histidine peptide, particularly a tag such as provided in a pQE vector (QIAGEN, Inc., 9259Eton Avenue, Chatsworth, Calif., 91311), many of which are commercially available. As described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), for example, six-histidine allows for convenient purification of fusion proteins. Other peptide tags useful for purification include, but are not limited to, the "HA" tag corresponding to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767-778 (1984)), and the "flag" tag.

[0156] Humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) can be used in non-conjugated form or can be conjugated to at least one of a variety of molecules, for example to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging or treatment of a subject. When purified, humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) can be labeled or conjugated before or after purification. In particular, humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) can be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents or PEG.

[0157] The present disclosure also includes humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) conjugated to diagnostic agents or therapeutic agents. Humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) can be used in diagnosis to, for example, monitor the occurrence or progression of immune cell disorders (e.g., CLL), as part of a clinical testing program to, for example, determine the efficacy of a given treatment and / or prevention regimen. Detection can be facilitated by coupling humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) to detectable substances. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use as diagnostic agents according to the present disclosure, see, for example, U.S. Patent No. 4,741,900. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125 I. 131 I. 111 In and 99 Tc.

[0158] The humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) used in the diagnostic and therapeutic methods disclosed herein can be conjugated to one or more cytotoxic (e.g., radioisotopes, cytotoxic drugs, or toxins) therapeutic agents, cytostatic agents, biotoxins, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, immunologically active ligands (e.g., lymphokines or other antibodies, where the resulting molecules bind to both tumor cells and effector cells (e.g., T cells)), or PEG.

[0159] In another embodiment, the humanized monoclonal antibody or humanized monoclonal antibody fragment (e.g., anti-αβTCR antibody or fragment thereof) used in the diagnostic and therapeutic methods disclosed herein can be conjugated to a molecule that reduces tumor cell growth. In other embodiments, the disclosed compositions may comprise a humanized monoclonal antibody or humanized monoclonal antibody fragment (e.g., anti-αβTCR antibody or fragment thereof) coupled to a drug or prodrug. Still other embodiments described herein include the use of antibodies or fragments thereof conjugated to specific biotoxins or cytotoxic fragments thereof such as ricin, polyanthin, Pseudomonas exotoxin, or diphtheria toxin. The choice of which conjugated or unconjugated antibody to use will depend on the type and stage of cancer, the use of adjuvant therapy (e.g., chemotherapy or external radiation), and the condition of the subject. It should be understood that such a choice can be easily made by those skilled in the art in view of the teachings herein.

[0160] It should be understood that in previous studies, isotope-labeled anti-tumor antibodies have been successfully used to destroy tumor cells in animal models and in some cases in humans. Exemplary radioisotopes include, but are not limited to: 67 Cu, 67 Ga, 90 Y. 105 Rh, 111 In, 123 I. 125 I. 131 I. 153 Sm, 166 Ho, 177 Lu, 186 Re, and 188 Re. Radionuclides work by producing ionizing radiation, causing multiple strand breaks in nuclear DNA, leading to cell death. Isotopes used to produce therapeutic conjugates generally produce high-energy alpha or beta particles with short path lengths. Such radionuclides kill cells to which they are in close proximity, such as tumor cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells. Radionuclides are essentially non-immunogenic.

[0161] V. Antibody Production

[0162] Antibody production can be performed by any technique known in the art, including in transgenic organisms such as goats (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 antibodies in host cells.

[0163] The polynucleotide encoding humanized monoclonal antibody or humanized monoclonal antibody fragment (e.g., anti-αβTCR antibody or its fragment) is separated and inserted into a reproducible construct or vector (such as a plasmid) for further propagation or expression in a host cell. Constructs or vectors (e.g., expression vectors) suitable for expressing humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibody or its fragment) according to the embodiment described are available in the art. A variety of vectors are available, including vectors that are maintained as single copies or multiple copies in a host cell, or vectors that are integrated into one or more chromosomes of a host cell. Constructs or vectors can be introduced into suitable host cells, and cells expressing humanized immunoglobulins can be produced and maintained in culture. Humanized immunoglobulins can be expressed using a single vector or multiple vectors.

[0164] Polynucleotides encoding humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) are easily separated and sequenced using conventional procedures (e.g., oligonucleotide probes). Useful vectors include plasmids, viruses, bacteriophages, transposons, mini-chromosomes, wherein plasmids are typical embodiments. Typically, such vectors also include signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences, which are operably connected to light chain and / or heavy chain polynucleotides to promote expression. The polynucleotides encoding light and heavy chains can be inserted into separate vectors and introduced (e.g., by transformation, transfection, electroporation, or transduction) into the same host cell in parallel or sequentially, or if desired, both heavy and light chains can be inserted into the same vector and then introduced.

[0165] A promoter can be provided for expression in a suitable host cell. The promoter can be constitutive or inducible. For example, a 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 are available. Prokaryotic promoters include lac, tac, T3, T7 promoters for Escherichia coli; 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, 6-phosphoglucose isomerase, 3-phosphoglycerate mutase and glucokinase. Eukaryotic promoters include inducible yeast promoters, such as alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, metallothionein and enzymes responsible for nitrogen metabolism or maltose / galactose utilization; RNA polymerase II promoters, including viral promoters, such as polyoma virus, fowlpox virus and adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (particularly the immediate early gene promoter), retrovirus, hepatitis B virus, actin, Rous sarcoma virus (RSV) promoter and early or late simian virus 40 and non-viral promoters, such as EF-1α (Mizushima and Nagata (1990) Nucleic Acids Res. 18(17):5322). Those skilled in the art will be able to select an appropriate promoter to express a humanized antibody or portion thereof.

[0166] If suitable for, for example, expression in higher eukaryotic cells, other enhancer elements may be included to replace the enhancer found to be located in the above-mentioned promoter, or to coexist with the enhancer. Suitable mammalian enhancer sequences include enhancer elements from globulin, elastase, albumin, fetoprotein, metallothionein, and insulin. Alternatively, enhancer elements from eukaryotic cell viruses may be used, such as SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, baculovirus enhancer, or mouse IgG2a locus (see WO 04 / 09823). The enhancer is typically located at a promoter upstream site on the vector, and it may also be located at other positions, such as in a non-translational region or downstream of a polyadenylation signal. The selection and positioning of enhancers may be based on the compatibility with the host cell for expression.

[0167] In addition, the vector (e.g., expression vector) may comprise a selective marker for selecting a host cell carrying the vector, and an origin of replication in the case of a replicative vector. Genes encoding products that confer antibiotic or drug resistance are commonly used selective 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 5 resistance genes). The dihydrofolate reductase marker gene allows selection with methotrexate in a variety of hosts. Genes encoding the gene products of the host's auxotrophic marker (e.g., LEU2, URA3, HIS3) are commonly used as selective markers in yeast. The use of viral (e.g., baculovirus) or phage vectors and vectors (such as retroviral vectors) that can be integrated into the host cell genome are also contemplated.

[0168] In eukaryotic systems, polyadenylation and termination signals are operably connected to the polynucleotides encoding the antibodies described herein. The signals are generally located 3' of the open reading frame. In mammalian systems, non-limiting examples of polyadenylation / termination signals include signals derived from growth hormone, elongation factor-1α and viral (e.g., SV40) genes or retroviral long terminal repeats. In yeast systems, non-limiting examples of polyadenylation / termination signals include signals derived from phosphoglycerate kinase (PGK) and alcohol dehydrogenase 1 (ADH) genes. In prokaryotic systems, polyadenylation signals are generally not required, and are generally replaced by shorter and more definite terminator sequences. The selection of polyadenylation / termination sequences can be based on compatibility with the host cell used for expression. In addition to the above, other features that can be used to improve yield include chromosome remodeling elements, introns, and host cell-specific codon modifications. The codon usage of the antibodies described herein can be modified to accommodate the codon bias 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). The choice of codons can be based on compatibility with the host cell used for expression.

[0169] Thus, the present disclosure relates to isolated nucleic acid molecules encoding humanized immunoglobulins or heavy or light chains thereof.The present disclosure also relates to isolated nucleic acid molecules encoding immunoglobulins and antigen binding portions of their chains.

[0170] Humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) can be produced, for example, by expressing one or more recombinant nucleic acids encoding the antibodies in suitable host cells. Host cells can be produced using any suitable method. For example, the expression constructs described herein (e.g., one or more vectors, such as mammalian cell expression vectors) can be introduced into suitable host cells, and the resulting cells can be maintained (e.g., in culture, in animals, in plants) under conditions suitable for expressing one or more constructs or one or more vectors. The host cell can be a prokaryotic cell, including a bacterial cell, such as Escherichia coli (e.g., strain DH5α TM ) (Invitrogen, Carlsbad, CA), PerC6 (Crucell, Leiden, The Netherlands), Bacillus subtilis and / or other suitable bacteria; eukaryotic cells, such as fungal or yeast cells (e.g., Pichia pastoris, Aspergillus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa) or other lower eukaryotic cells, and higher eukaryotic cells: such as those from insects (e.g., Schneider's Drosophila S2 cells, Sf9 insect cells) (WO 94 / 126087), 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), HEK, 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), NS0 cells, SP2 / 0 cells, HuT 78 cells, etc., or plants (e.g., tobacco, duckweed (lemma), and algae). See, e.g., Ausubel, FM et al., ed. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc. (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 part of a cell culture.

[0171] Host cells can be cultivated in the following devices: spinner flasks, oscillation flasks, roller bottles, wave reactors (e.g., System 1000 from wavebiotech.com) or hollow fiber systems, but for large-scale production, preferably stirred tank reactors or bag reactors (e.g., Wave Biotech, Somerset, New Jersey, USA) are particularly used for suspension culture. Stirred tank reactors can be suitable for ventilation using, for example, sprinklers, baffles or low shear impellers. For bubble towers and airlift reactors, direct ventilation with air or oxygen bubbles can be used. If host cells are cultivated in serum-free medium, cell protectants can be supplemented in the culture medium, such as Pluronic F-68 to help prevent cells from being damaged by the ventilation process. According to the characteristics of the host cell, microcarriers can be used as the growth substrate of the anchorage-dependent cell line, or the cell can be suitable for 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 procedures, or perfusion culture. Although recombinantly transformed mammalian host cells can be cultured in serum-containing media (e.g., media containing fetal calf serum (FCS)), it is preferred that such host cells be cultured in serum-free media, such as disclosed in Keen et al. (1995) Cytotechnology 17: 153-163, or commercially available media, such as ProCHO TM or UltraCHO TM (Cambrex, New Jersey, USA), the medium is supplemented with energy sources such as glucose and synthetic growth factors such as recombinant insulin when necessary. Serum-free culture of host cells may require that those cells are adapted to grow under serum-free conditions. One adaptation method is to culture such host cells in serum-containing medium and repeatedly replace 80% of the medium with serum-free medium so that the host cells learn to adapt to serum-free conditions (see, for example, Scharfenberg, K. et al. (1995) Animal Cell Technology: Developments Towards the 21st Century (Beuvery, EC et al., eds.), pp. 619-623, Kluwer Academic publishers).

[0172] According to the described embodiments, humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) can be secreted into the culture medium and recovered and purified therefrom using a variety of techniques to provide a certain degree of purification suitable for the intended use. For example, when compared to a culture medium containing therapeutic antibodies, the use of humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) to treat human subjects generally requires at least 95% purity as determined by reducing SDS-PAGE, more typically 98% or 99% purity. In the first case, cell debris can be removed from the culture medium by centrifugation, followed by a clarification step of the supernatant using, for example, microfiltration, ultrafiltration, and / or deep filtration. Alternatively, humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) can be harvested by microfiltration, ultrafiltration, or deep filtration without prior 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, humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) are captured in the following manner: using protein A or protein G affinity chromatography, followed by further chromatography steps, such as 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) can also be used. After these different steps, a purified preparation containing at least 10 mg / mL or more, such as 100 mg / mL or more of the antibodies described herein is provided, and thus another embodiment described herein is formed. Ultracentrifugation can produce a concentration of 100 mg / mL or more. Such preparations are substantially free of aggregated forms of antibodies described herein.

[0173] Bacterial systems are particularly suitable for expressing antibody fragments. Such 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.

[0174] The disclosure also relates to cells comprising nucleic acid as described herein such as vectors (for example, expression vectors).For example, nucleic acid (that is, one or more nucleic acids) of the heavy chain and light chain of the humanized immunoglobulin (i.e., one or more nucleic acids) of coding according to the embodiment or constructs (for example, one or more constructs, for example, one or more vectors) comprising such one or more nucleic acids can be introduced into suitable host cells by methods applicable to selected host cells (for example, conversion, transfection, electroporation, infection), wherein the one or more nucleic acids are operably connected or become operably connected to one or more expression control elements (for example, in the vector, constructs that are integrated into the host cell genome produced by the method in cells).Host cells can be maintained under conditions suitable for expression (for example, in the presence of an inducing agent, a suitable culture medium supplemented with suitable salts, growth factors, antibiotics, nutritional supplements, etc.), thus producing one or more polypeptides encoded.If desired, the humanized antibodies encoded can be separated from, for example, host cells, culture medium or milk.This process is included in the host cell (for example, mammary cell) of transgenic animals or plants (for example, tobacco) and expressed (see, for example, WO 92 / 03918).

[0175] VI. Methods of Treating or Preventing T Cell-Mediated Disorders

[0176] In many cases where immunosuppression is required and / or autoimmune disorders occur, it is necessary to inhibit T cell activity. Therefore, it is indicated that targeting the αβTCR / CD3 complex can treat diseases involving inappropriate or unwanted immune responses, such as inflammation, autoimmunity, and / or other disorders involving such mechanisms. In one embodiment, such a disease or disorder is an autoimmune disease and / or an inflammatory disease. Examples of such T cell-mediated autoimmune and / or inflammatory diseases include, but are not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD) (including ulcerative colitis (UC) and Crohn's disease (CD)), multiple sclerosis (MS), scleroderma, type 1 diabetes (T1D), and other diseases and disorders such as pemphigus vulgaris (PV), psoriasis, atopic dermatitis, celiac disease, chronic obstructive pulmonary disease, Hashimoto's thyroiditis, Graves' disease (thyroid), Sjögren's syndrome, Guillain-Barré syndrome, Goodpasture's syndrome, Addison's disease, Wegener's granulomatosis, primary biliary sclerosis, 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.

[0177] In one embodiment, the disease or disorder is SLE. In one embodiment, the disease or disorder is RA. In one embodiment, the disease or disorder is IBD. In one embodiment, the disease or disorder is MS. In one embodiment, the disease or disorder is T1D.

[0178] In another embodiment, the disease or disorder is xenotransplantation, allogeneic transplantation, xenogeneic pregnancy, preeclampsia, or Rh disease.

[0179] In a specific embodiment, the antibody according to the embodiment is used to help transplantation by immunosuppressing the subject. This use alleviates graft-versus-host disease (GvHD), which is a common complication after xenotransplantation or allogeneic transplantation (including but not limited to: transplantation of stem cells, bone marrow, tissue, body parts and solid organs). Tissues may include but are not limited to: cornea, sclera, bone, skin, blood vessels and heart valves. Body parts may include but are not limited to: face or part thereof, one or more arms or parts thereof, one or more hands or parts thereof, one or more legs or parts thereof, and scalp or part thereof. Solid organs may include but are not limited to: heart, lungs, liver, kidneys, pancreas, stomach, small intestine, large intestine, testicles and ovaries. For descriptions 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 provided in the present disclosure can be used in combination with other available therapies.

[0180] Regarding the treatment of autoimmune diseases, the combination therapy may include administering the antibodies described herein together with a drug that together with the antibody constitutes an effective amount for preventing or treating such autoimmune diseases. When the autoimmune disease is type 1 diabetes, the combination therapy may include one or more agents that promote pancreatic beta cell growth or enhance beta cell transplantation, such as beta cell growth or survival factors or immunomodulatory antibodies. When the autoimmune disease is rheumatoid arthritis, the combination therapy may include one or more of the following: methotrexate, anti-TNF-α antibodies, TNF-α 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, the additional agent may be a biologic, such as an anti-TNF agent (e.g., Infliximab Adalimumab or rituximab When the autoimmune disease is hematopoietic cell transplant rejection, one or more hematopoietic growth factors (such as erythropoietin, G-CSF, GM-CSF, IL-3, IL-11, thrombopoietin, etc.) or one or more antimicrobial agents (such as antibacterial, antiviral, antifungal drugs) can be administered. When the autoimmune disease is psoriasis, the additional agent can be one or more of the following: tar and its derivatives, phototherapy, corticosteroids, cyclosporin A, vitamin D analogs, methotrexate, p38 mitogen-activated protein kinase (MAPK) inhibitors and biological agents (such as anti-TNF-α agents and When the autoimmune disease is inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis, the additional agent may be one or more of the following: an aminosalicylate, a corticosteroid, an immunomodulator, an antibiotic, or a biological agent (such as and ).

[0181] Combination therapy can be performed in any manner deemed necessary or convenient by a person skilled in the art, and for the purposes of this specification, no limitation is considered to be imposed on the order, amount, repetition or relative amounts of the compounds used. Thus, the antibodies according to the described embodiments can be formulated into pharmaceutical compositions for treatment.

[0182] VII. Pharmaceutical compositions and administration methods of anti-αβTCR antibodies

[0183] In certain embodiments, a pharmaceutical composition is provided, comprising a humanized monoclonal antibody or a humanized monoclonal antibody fragment as described herein (e.g., an anti-αβTCR antibody or a fragment thereof), or one or more ligands that can be identified by an assay method as defined in the foregoing aspects of the present disclosure. As discussed herein, a ligand can be an immunoglobulin, a peptide, a nucleic acid, or a small molecule. In the following discussion, they are referred to as "compounds".

[0184] The pharmaceutical compositions described herein are material compositions comprising one or more compounds capable of modulating T cell activity as active ingredients. The compound is in the form of any pharmaceutically acceptable salt, or, for example, where appropriate, an analog, a free base form, a tautomer, an enantiomeric racemate, or a combination thereof. It is contemplated that the active ingredients of the pharmaceutical compositions comprising the active ingredients described herein exhibit therapeutic activity when administered in an amount depending on the circumstances, for example, in the treatment of graft-versus-host disease.

[0185] In certain embodiments, a pharmaceutical composition comprises a humanized monoclonal antibody or a humanized monoclonal antibody fragment described herein (eg, an anti-αβ TCR antibody or a fragment thereof), and a pharmaceutically acceptable carrier or diluent.

[0186] In certain embodiments, in treating any of the above-mentioned conditions, one or more compounds described in the present disclosure may be used in combination with any art-recognized compound known to be suitable for treating a particular indication. Thus, one or more compounds described herein may be combined with one or more art-recognized compounds known to be suitable for treating the above-mentioned 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.

[0187] 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.

[0188] The active ingredient may be administered in a convenient manner, such as by oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implantation (e.g., using slow release molecules). In the case of transplantation, the active ingredient may also be used to treat cells, tissues or organs to be transplanted into the patient prior to transplantation. This may be done to prevent, reduce the likelihood of, or alleviate the symptoms of, for example, graft versus host disease.

[0189] Depending on the route of administration, the active ingredient may need to be coated in a material to protect the ingredient from the action of enzymes, acids, and other natural conditions that may inactivate the ingredient.

[0190] In order to apply the active ingredient by a mode other than parenteral administration, the active ingredient is coated with a material or applied together with a material to prevent its inactivation. For example, the active ingredient can be applied in an adjuvant, co-administered with an enzyme inhibitor, or applied in a liposome. Adjuvants are used in their broadest sense and include any immunostimulatory compound, such as interferon. Adjuvants considered herein include resorcinol, nonionic surfactants (such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether). Enzyme inhibitors include pancreatic trypsin.

[0191] Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes.

[0192] The active ingredient may also be administered parenterally or intraperitoneally.

[0193] 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.

[0194] The pharmaceutical form suitable for injection includes sterile aqueous solution (water-soluble) or dispersion, and sterile powder for temporary preparation of sterile injectable solution or dispersion. In all cases, the form must be sterile and must have the fluidity to reach the degree of easy injection. It must be stable under manufacturing and storage conditions and must resist the contamination of microorganisms (such as bacteria and fungi) and preserve. The carrier can be a solvent or dispersion medium, and the solvent or dispersion medium contains, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), its suitable mixture and vegetable oil. For example, by using coating (such as lecithin), by maintaining required particle size in the case of dispersion and by using a surfactant, suitable fluidity can be maintained.

[0195] The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like). In some cases, it may be preferred to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using in the compositions agents that delay absorption, for example, aluminum monostearate and gelatin.

[0196] Sterile injectable solutions are prepared by incorporating the active ingredient in the desired amount into an appropriate solvent optionally with a number of the other ingredients listed above, followed by sterilization filtration. In general, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional required ingredients from a previously sterile filtered solution.

[0197] Various other materials may be present as coatings or otherwise modify the physical form of the dosage unit. Of course, any material used to prepare any dosage unit form should be pharmaceutically pure and substantially nontoxic in the amounts employed. In addition, the active ingredient may be incorporated into sustained release formulations and preparations.

[0198] 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. In certain embodiments, a pharmaceutically acceptable carrier or diluent is an aqueous fluid. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, their use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the composition.

[0199] It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for mammalian subjects to be treated; each unit contains a predetermined amount of active substance calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier. The specifications of the novel dosage unit form described herein depend on and are directly dependent on (a) the unique characteristics of the active substance and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the field of synthesizing such active substances for treating diseases of living subjects (whose physical health is impaired) suffering from diseases or disorders. The main active ingredient is mixed in an effective amount with a suitable pharmaceutically acceptable carrier in a dosage unit form for convenient and effective administration. In the case of a composition containing a supplementary active ingredient, the dosage is determined with reference to the usual dose and mode of administration of the ingredient.

[0200] The dosage may include but is not limited to 0.01 mg / kg to 20 mg / kg, including: 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.10 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 3.0 mg / kg, 3.1 mg / kg, 3.2 mg / kg, 3.3 mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 mg / kg, 4.0 mg / kg, 4.1 mg / kg, 4.2 mg / kg, 4.3 mg / kg, 4.4 mg / kg, 4.5 mg / kg, 4.6 mg / kg, 4.7 mg / kg, 4.8 mg / kg, 4.9 mg / kg, 5.0 mg / kg, 5.1 mg / kg, 5.2 mg / kg, 5.3 mg / kg, 5.4 mg / kg, 5.5 mg / kg, 5.6 mg / kg, 5.7 mg / kg, 5.8 mg / kg, 5.9 mg / kg, 6.0 mg / kg, 6.1 mg / kg, 6.2 mg / kg, 6.3 mg / kg, 6.4 mg / kg, 6.5 mg / kg, 6.6 mg / kg, 6.7 mg / kg, 6.8 mg / kg, 6.9 mg / kg, 7.0 mg / kg, 7.1 mg / kg, 7.2 mg / kg, 7.3 mg / kg, 7.4 mg / kg, 7.5 mg / kg, 7.6 mg / kg, 7.7 mg / kg, 7.8 mg / kg, 7.9 mg / kg, 8.0 mg / kg, 8.1 mg / kg, 8.2 mg / kg, 8.3 mg / kg, 8.4 mg / kg, 8.5 mg / kg, 8.6 mg / kg, 8.7 mg / kg, 8.8 mg / kg, 8.9 mg / kg, 9.0 mg / kg, 9.1 mg / kg, 9.2 mg / kg, 9.3 mg / kg, 9.4 mg / kg, 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8mg / kg、9.9mg / kg、10.0mg / kg、10.1mg / kg、10.2mg / kg、10.3mg / kg、10.4mg / kg、10.5mg / kg、10.6mg / kg、10.7mg / kg、10.8mg / kg、10.9mg / kg、11.0mg / kg、11.1mg / kg、11.2mg / kg、11.3mg / kg、11.4mg / kg、11.5mg / kg、11.6mg / kg、11.7mg / kg、11.8mg / kg、11.9mg / kg、12.0mg / kg、12.1mg / kg、12.2mg / kg、12.3mg / kg、12.4mg / kg、12.5mg / kg、12.6mg / kg、12.7mg / kg、12.8mg / kg、12.9mg / kg、13.0mg / kg、13.1mg / kg、13.2mg / kg、13.3mg / kg、13.4mg / kg、13.5mg / kg、13.6mg / kg、13.7mg / kg、13.8mg / kg、13.9mg / kg、14.0mg / kg、14.1mg / kg、14.2mg / kg、14.3mg / kg、14.4mg / kg、14.5mg / kg、14.6mg / kg、14.7mg / kg、14.8mg / kg、14.9mg / kg、15.0mg / kg、15.1mg / kg、15.2mg / kg、15.3mg / kg、15.4mg / kg、15.5mg / kg、15.6mg / kg、15.7mg / kg、15.8mg / kg、15.9mg / kg、16.0mg / kg、16.1mg / kg、16.2mg / kg、16.3mg / kg、16.4mg / kg、16.5mg / kg、16.6mg / kg、16.7mg / kg、16.8mg / kg、16.9mg / kg、17.0mg / kg、17.1mg / kg、17.2mg / kg、17.3mg / kg、17.4mg / kg、17.5mg / kg、17.6mg / kg、17.7mg / kg、17.8mg / kg、17.9mg / kg、18.0mg / kg、18.1mg / kg、18.2mg / kg、18.3mg / kg、18.4mg / kg、18.5mg / kg、18.6mg / kg、18.7mg / kg、18.8mg / kg、18.9mg / kg、19.0mg / kg、19.1mg / kg、19.2mg / kg、19.3mg / kg、19.4mg / kg、19.5mg / kg、19.6mg / kg、19.7mg / kg、19.8mg / kg, 19.9mg / kg, and 20.0mg / kg. .

[0201] The dosage may also include, but is not limited to, about 100 ng / kg to about 0.01 mg / kg, including about 100 ng / kg, about 200 ng / kg, about 300 ng / kg, about 400 ng / kg, about 500 ng / kg, about 600 ng / kg, about 700 ng / kg, about 800 ng / kg, about 900 ng / kg, about 1 microgram / kg, about 2 micrograms / kg, about 3 micrograms / kg, about 4 micrograms / kg, about 5 micrograms / kg, about 6 micrograms / kg, about 7 micrograms / kg, about 8 micrograms / kg, about 9 micrograms / kg, and about 10 micrograms / kg.

[0202] In order to promote the 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 blending agents to increase the transport of proteins 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.

[0203] In another aspect, there is provided an active ingredient as described herein, which is used alone or in combination with a known art-recognized compound suitable for treating a specific indication for the treatment of a disease. Thus, there is provided the use of an active ingredient as described herein in the preparation of a medicament for treating a disease associated with an abnormal immune response.

[0204] Additionally, provided is a method of treating a disorder associated with an aberrant immune response, the method comprising administering to a subject a therapeutically effective amount of a ligand identifiable using an assay as described above.

[0205] The examples provided below are for illustrative purposes only and are not to be construed as limitations of the compositions and methods described herein.

[0206] Example

[0207] Example 1: Accelerated stability study of VH31 anti-αβTCR antibody formulation

[0208] Accelerated stability studies were performed on various formulations of the VH31 antibody. The reference or control VH31 antibody was a wild-type humanized anti-human αβTCR antibody in the GL1BM series, comprising a heavy chain variable domain having an amino acid sequence as shown in SEQ ID NO: 16 and a light chain variable domain having an amino acid sequence as shown in SEQ ID NO: 14 (Table 1). No breaks were observed in the liquid formulations after four months at refrigerated temperatures of 4°C or less. However, fragments were detected in VH31 liquid formulations subjected to accelerated conditions including elevated temperature and pH (i.e., 45°C; pH 4.0-8.0) for five weeks. Note that the presence of two low molecular weight (LMW) bands appeared to be associated with the disappearance of the band corresponding to the VH31 antibody light chain (LC) ( Figure 1 , arrow), which means that the source of the observed LMW species has undergone light chain fragmentation.

[0209] Example 2: Cell-based assay to evaluate the efficacy of stress formulations

[0210] The VH31 anti-αβTCR antibody selectively depletes activated T cells. To determine whether the VH31 formulations exposed to accelerated conditions retained this ability, a cell-based potency assay was performed. The potency assay is based on activation of the T cell receptor and subsequent binding of the anti-αβTCR antibody (to stimulate the apoptotic pathway).

[0211] Jurkat T lymphocytes express T cell receptor complexes. The potency assay utilizes Jurkat T cell lines, which contain a nuclear factor of activated T cells (NFAT) response element connected to a luciferase reporter gene. The engineered cell line is used to examine T cell activation and subsequent inhibition of activation signals by anti-αβTCR antibodies. The activation of the T cell receptor complex stimulates the NFAT pathway, leading to the production of luciferase. The production of luciferase is then measured using a luciferase substrate (e.g., luciferin) and a luminescence detector. The activation level is proportional to the production of luciferase. Immobilized anti-CD3 activates Jurkat T cells, such as by inducing NFAT pathways and subsequent luciferase production in NFAT-luciferase Jurkat cells to measure. Anti-αβTCR antibodies inhibit activation in a dose-dependent manner, which is a qualitative measure of anti-αβTCR potency.

[0212] The qualitative potency assay was run in a 96-well plate format. The 96-well plates were coated with 1 μg / mL of anti-CD3 overnight at 2-8°C. The plates were washed and NFAT-luciferase Jurkat T lymphocytes (5x10 4 Cells were incubated at 37°C and 5% CO 2The cells were incubated for five hours at 4 °C to activate the T cell receptor complex. Each cell condition was then incubated for another eighteen hours with anti-αβTCR (control and sample) at multiple levels (0.003 to 200 μg / mL). Each condition included negative anti-CD3 (0 μg / mL) and anti-αβTCR (0 μg / mL) controls. After the second incubation step, the anti-αβTCR diluent was removed and the cells were lysed with a cell culture lysis reagent. Luciferase production of the lysate was measured using a luciferase substrate and a luminescent plate reader. It is expected that a higher dose of anti-αβTCR antibody will result in a lower luminescent signal, indicating that the activation reaction is inhibited. Dose-response data were plotted using Softmax Pro software, and the sample dose-response curve was visually compared with the control dose-response curve.

[0213] Figure 2 Figures in show data from cell-based potency assays. VH31 antibody formulations exposed to accelerated conditions (i.e., 45°C; pH 8.0) for five weeks showed considerable potency loss compared to VH31 formulations exposed to less harsh conditions (i.e., 45°C; pH 5.0) for five weeks (open circle symbols) or control conditions (i.e., ≤0°C; pH 5.0; open diamond symbols). This study demonstrates the relationship between the appearance of LMW species and antibody potency loss, both of which may be due to light chain breakage.

[0214] Example 3: Analysis of Antibody Fragments

[0215] The antibody fragments were further analyzed by molecular weight estimation of the observed bands and by N-terminal sequencing and peptide mapping studies. Figure 3 ). It is known that chemical and enzymatic cleavage of polypeptides occurs between Asn (N) and Pro (P) residues. Therefore, based on sequencing and mapping studies, light chain amino acid residues N93 and P94 were identified as possible locations of light chain cleavage and the source of the identified fragments ( Figure 4 ). To further confirm this, the VH31 light chain (P7) was trypsinized at pH 4.0 to 8.0. The diagram in Figure 5 shows the various light chain fragments that would be expected if enzymatic cleavage occurred at N93 / P94. These fragments correspond to amino acid residues 94-102 ( Figure 5A ), residues 61-93( Figure 5B ) and residues 94-106 ( Figure 5C ). The two C-terminal products corresponding to amino acids 94-102 and 94-106 are due to incomplete trypsin digestion. As expected, an increase in pH was associated with an increase in the predicted fragments.

[0216] It is known that Asn(N) can be converted to Asp(D) in solution, for example, by deamidation of the Asn side chain ( Figure 6). To better understand the mechanism of LC fragmentation, the LC fragments were analyzed to determine whether residue 93 was N or D. Isotopic distribution revealed that most of the 61-93 peptide fragments terminated with Asn. No separate peak corresponding to D93 was observed, indicating that conversion of Asn to Asp followed by Asp / Pro cleavage was not the primary cause of VH31 LC fragmentation. For these reasons, subsequent engineering stability studies focused on residues N93 / P94.

[0217] Example 4: Engineering light chain (LC) variants by mutagenesis

[0218] A mutagenesis approach was used to engineer VH31 light chain variants with improved stability and resistance to breakage. Previous attempts to remove the Asn cleavage site in an unrelated antibody (i.e., sFLT01) did not prevent antibody breakage. Further complicating matters, it was noted that amino acid residues N93 and P94 are located within the light chain complementary determining region 3 (LC CDR3). With the above in mind, light chain variants were rationally designed with the goal of enhancing light chain stability while also maintaining antigen binding affinity and antibody potency.

[0219] The amino acid Pro (P) provides almost no degrees of freedom in the folded protein structure. Therefore, it is assumed that it may be difficult to replace Pro at residue 94 without disrupting the conformation of LCCDR3. Ala (A) is commonly used for mutagenesis due to its physical characteristics. As a starting point, residues N93 and P94 were replaced by A alone or concomitantly. In addition, several other substitutions were made at residue N93 based on amino acid characteristics that were considered compatible, such as charge, size, hydrogen bonding, polarity and non-polarity, etc. Figure 7 Various amino acid substitutions made and tested are shown.

[0220] The wild-type anti-αβTCR VH31 expression vector was used as a template for mutagenesis and expression control. Eight pairs of primers were designed and PCR mutagenesis was performed using the QuikChange Lightning site-directed mutagenesis kit (Agilent). Mutant DNA was then confirmed by DNA sequencing.

[0221] Example 5: Expression and analysis of LC variants

[0222] Mutant and wild type (WT) DNA were transfected into the Expi293 transient expression system. Conditioned medium was harvested four days after transfection, and expression levels were measured by Octet protein A assay. Results (shown in Table 2) show that αβTCR wild type (114 μg / mL culture medium) and 7 of 8 mutants (>45 μg / mL culture medium) expressed well. The expression level of a mutant (N93A) was relatively low (16 μg / mL). For all 8 mutants, the transfection scale was expanded to 30 mL. Conditioned medium was collected for protein A purification.

[0223] Conditioned medium is passed through 1mL HiTrap protein A HP (GE) post so that purification. Described post is balanced 5 column volumes with PBS pH 7.2 (Gibco).Culture medium is loaded with the flow velocity of 0.5mL / min, and then washed 20 column volumes (CV) with PBS pH 7.2, eluted afterwards.Antibody is eluted in 10mM succinate (pH 3.75), and is adjusted to pH 5.5 with 0.2M sodium hydroxide immediately after elution.All samples are all filtered (recovery rate 50%-80%) with 0.2 μ m low protein binding membrane and syringe filter, and use Amicon YM30 concentrated sample (recovery rate 100%).The results are summarized in Table 2.

[0224] Table 2

[0225]

[0226]

[0227] The purified antibodies were run on Mini-PROTEAN TGX stain-free gels (Bio-Rad) with 1.5 μg loading per well. The results showed that all tested mutants were comparable to WT and had good purity ( Figure 8 ). The purified mutants were further characterized by stability and functional assays.

[0228] Sixty milliliter scale transfection of αβTCR N93S mutant was performed in the Expi293 transient expression system. Conditioned media was harvested four days after transfection. Expression levels were measured by Octet Protein A assay and determined to be 37 μg / mL.

[0229] HiTrap protein A HP (GE) column (1mL) was used for purification. The column was balanced for 5 CVs with PBS pH 7.2 (Gibco). The conditioned medium sample was loaded at a flow rate of 0.5mL / min, and then washed with PBS pH 7.2 for 20 CVs, followed by elution. The antibody was eluted into 10mM succinate (pH 3.75) for a total of 5 CVs (5mL in total), with a yield of almost 100%. After elution, the eluted antibody was adjusted to pH 5.5 with 0.2M sodium hydroxide. The solution became slightly turbid at pH 5.5 and was filtered with a 50mL tube top filter (0.2μm CA, low protein binding). The filtration recovery was 77% (1.7mg in total), which may be due to a relatively large volume loss. The purified antibody and αβTCR VH31 controls were run on Mini-PROTEAN TGX stain-free gel (2μg loading per well). The results showed that all tested mutants were comparable to the VH31 control. The purified N93S and previously purified WT and VH31 controls were run on SEC-HPLC and the profiles are shown in Fig. 9 Middle. SEC profiles of both WT and N93S mutant appeared comparable to the VH31 control.

[0230] Example 6: Assays to evaluate the potency of LC variants

[0231] The qualitative potency assay described in Example 2 was repeated with antibodies comprising variant light chains. The wild-type and control antibodies comprised the same VH31 amino acid sequence but were produced from different preparations. The wild-type VH31 antibody was produced together with the LC variant antibody, while the control antibody was produced from a separate large-scale preparation. Fig.10 As shown, the potency of the antibody comprising the LC variant N93S was only slightly lower than that of the wild-type VH31 or the control antibody. In contrast, the potency of the antibodies comprising the LC variants N93Q, N93A and N93A / P94A were all significantly lower than that of any of the above antibodies. Based on these results, the N93S variant was selected as a candidate for accelerated stability studies.

[0232] Example 7: Accelerated stability studies of LC variants

[0233] The antibodies comprising the LC variant N93S were placed at elevated temperature (i.e., 45°C) and pH (i.e., pH 8.0) for six weeks to determine whether the N93S amino acid substitution stabilizes the LC and prevents fragmentation. After six weeks at elevated temperature and pH 8.0, fragmentation was observed in the wild-type, reference VH31, and N93S antibodies. However, the N93S mutant showed significantly reduced degradation compared to the wild-type or VH31 control antibodies ( Fig.11Notably, the N93S mutant showed significantly less potency loss under accelerated conditions (3-fold loss) compared to either the wild-type or VH31 control antibodies (both greater than 20-fold loss). At pH 5.5 (closer to the pH of a hypothetical antibody drug product formulation than pH 8.0), breakage was significantly less prevalent at six weeks ( Fig.12 ).

[0234] The above results show that the N93S mutant exhibits improved LC stability and potency compared to the VH31αβTCR antibody under storage conditions, including at elevated temperature and elevated pH. These results were unexpected because previous attempts to remove the Asn cleavage site in an unrelated antibody (i.e., sFLT01) did not prevent antibody fragmentation. Sequence Listing <110> New Company <120> Anti-αβTCR binding polypeptides with reduced fragmentation <130> 704503: SA9-254PC <150> US 62 / 828,601 <151> 2019-04-03 <160> 29 <170> PatentIn Version 3.5 <210> 1 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 1 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val His Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 2 <211> 106 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 2 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Thr Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 3 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Met Thr Cys Ser Ala Thr Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ile Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 5 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 5 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val His Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Met Thr Cys Ser Ala Thr Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ile Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 7 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 7 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val His Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Ile Thr Arg Asp Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 9 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Thr Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 10 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 10 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120 125 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 130 135 140 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 145 150 155 160 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 165 170 175 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 180 185 190 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 225 230 235 240 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 245 250 255 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 260 265 270 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280 285 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 290 295 300 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 305 310 315 320 Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 <210> 11 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 11 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 12 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 12 Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 13 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Val Arg Leu Ser Cys Ala Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 106 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Thr Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 15 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 15 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 98 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 17 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys <210> 18 <211> 98 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 19 <211> 95 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 19 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro 85 90 95 <210> 20 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 20 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 21 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 21 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Thr Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 22 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 22 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Thr Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Met Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 23 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 24 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 24 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 25 <211> 213 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 25 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Thr Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 26 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 26 Ser Ala Thr Ser Ser Val Ser Tyr Met His 1 5 10 <210> 27 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 27 Asp Thr Ser Lys Leu Ala Ser 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> Features not yet classified <222> (6) <223> Gln, Asp, His, Ser, Tyr, or Ala <220> <221> Features not yet classified <222> (7) <223> Pro or Ala <400> 28 Gln Gln Trp Ser Ser Xaa Xaa Leu Thr 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 29 Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5

Claims

1. A binding polypeptide that specifically binds to a human αβTCR / CD3 complex, comprising a heavy chain variable region, a light chain variable region and a constant region, wherein: The light chain variable region comprises three complementary determining regions LCDR1, LCDR2 and LCDR3 consisting of the amino acid sequences shown in SEQ ID NOs: 26, 27 and 28, respectively; The amino acid sequence of SEQ ID NO: 28 is QQWSS-X1-X2-LT, wherein X1 is serine, and X2 is proline; wherein the heavy chain variable region comprises any one amino acid sequence selected from SEQ ID NO: 7, 12, 13, 15 and 16; and The constant regions are of human origin.

2. The binding polypeptide according to claim 1, wherein the light chain variable region further comprises the human light chain framework region in the human light chain variable region shown in SEQ ID NO:

14.

3. The binding polypeptide according to claim 1 or 2, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

16.

4. The binding polypeptide of claim 1 or 2, wherein the binding polypeptide has increased stability at a pH greater than 5.0 compared to VH31, wherein VH31 comprises a heavy chain variable region of SEQ ID NO: 16 and a light chain variable region of SEQ ID NO:

14.

5. The binding polypeptide according to claim 1 or 2, wherein the binding polypeptide has increased stability at a temperature greater than 4°C compared to VH31, wherein VH31 comprises a heavy chain variable region of SEQ ID NO: 16 and a light chain variable region of SEQ ID NO:

14.

6. The binding polypeptide according to claim 1 or 2, wherein the heavy chain variable region comprises any one amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 12 and SEQ ID NO:

13.

7. The binding polypeptide according to claim 1 or 2, wherein the heavy chain variable region comprises any one amino acid sequence selected from SEQ ID NO: 15 and SEQ ID NO:

16.

8. The binding polypeptide according to claim 1 or 2, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

15.

9. The binding polypeptide of claim 1 or 2, wherein the constant region comprises an Fc modification having a modified glycosylation pattern that reduces Fcγ receptor binding, wherein the Fc modification is any one selected from the group consisting of N297Q / S298N / Y300S, S298N / T299A / Y300S, and S298N / Y300S, wherein the amino acid positions are numbered according to EU numbering.

10. The binding polypeptide of claim 9, wherein the Fc modification is N297Q / S298N / Y300S, wherein the amino acid positions are numbered according to EU numbering.

11. The binding polypeptide of claim 9, wherein the Fc modification is S298N / T299A / Y300S, wherein the amino acid positions are numbered according to EU numbering.

12. The binding polypeptide of claim 9, wherein the Fc modification is S298N / Y300S, wherein the amino acid positions are numbered according to EU numbering.

13. The binding polypeptide of claim 1, wherein the binding polypeptide is humanized.

14. The binding polypeptide of claim 1 or 2, wherein the binding polypeptide is a monoclonal antibody.

15. A pharmaceutical composition comprising the binding polypeptide according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising the binding polypeptide according to any one of claims 1 to 14 and a pharmaceutically acceptable diluent.

17. A formulation comprising the pharmaceutical composition according to claim 15 or 16, wherein the formulation is a lyophilized formulation or a liquid formulation.

18. A nucleic acid encoding the binding polypeptide of any one of claims 1-14.

19. A vector comprising the nucleic acid according to claim 18.

20. A cell expressing the nucleic acid of claim 18.

21. The cell of claim 20, wherein the cell is a mammalian cell.

22. The cell of claim 21, wherein the mammalian cell is a Chinese hamster ovary cell or a human embryonic kidney cell.

Images