Reduced fragmentation of anti-alpha-beta TCR binding polypeptides

Abstract

This invention provides an antibody composition with improved stability, exhibiting reduced fragmentation of the antibody polypeptide chain during manufacturing and subsequent storage. [Solution] This disclosure relates to improved compositions and methods for treating T cell-mediated diseases and disorders (e.g., autoimmune disorders, graft-versus-host diseases, and graft rejection). Provided are antibody-containing anti-αβTCR binding polypeptides comprising at least one amino acid substitution or modification that enhances the stability of the binding polypeptide by reducing fragmentation of the light chain variable region. Methods provided herein generally involve administering an effective amount of a stabilized, humanized binding polypeptide specific to the alpha-beta T cell receptor (αβTCR) to a subject in need thereof.

Description

[Technical Field]

[0001] Related applications This application claims the benefits of U.S. Provisional Application No. 62 / 828,601, filed on 3 April 2019, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on March 30, 2020, is named 704503_SA9-254PC_ST25.txt and has a size of 32,602 bytes. [Background technology]

[0003] Monoclonal antibodies are an important class of conjugated polypeptides and biopharmaceuticals. Generally, polypeptide backbones are highly stable under physiological conditions. Nevertheless, fragmentation of heavy and light chain polypeptides remains a major concern for therapeutic monoclonal antibodies.

[0004] Generally, protein backbones are highly stable under physiological conditions. However, fragmentation can be caused by various mechanisms, such as the disruption of innate covalent bonds that lead to the cleavage of the polypeptide backbone through spontaneous or enzymatic reactions. Furthermore, certain regions and motifs (e.g., Asn-Pro motifs) may be more susceptible to fragmentation due to their amino acid sequence, the flexibility of their two- or three-dimensional polypeptide structure, and unsuitable solvent and environmental conditions (e.g., temperature and pH).

[0005] Fragmentation can occur at any stage during the manufacturing or storage of biological compositions. Since fragmentation can lead to reduced potency or the presence of unwanted and potentially immunogenic species, reducing fragmentation is a critical consideration in the production of any biological therapeutic agent. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Therefore, there is a need in the art for antibody compositions with improved stability that exhibit reduced fragmentation of antibody polypeptide chains during manufacturing and subsequent storage. [Means for solving the problem]

[0007] This disclosure provides improved compositions and methods useful for the treatment of T cell-mediated diseases and disorders. Humanized binding polypeptides that specifically bind to the alpha-beta T cell receptor (αβTCR) are provided. The anti-αβTCR compositions provided herein are improved from known compositions in that the improved compositions include at least one amino acid substitution or modification that enhances the stability of the binding polypeptide by reducing fragmentation of the light chain variable region. Methods for treating T cell-mediated diseases and disorders (e.g., graft-versus-host disease, autoimmune diseases, and graft rejection) using the improved compositions are also provided. The methods provided herein generally involve administering an effective amount of a humanized binding polypeptide that specifically binds to αβTCR to a subject in need.

[0008] Surprisingly, the inventors found that removing the Asn clipping site from the light chain of the anti-human αβTCR antibody VH31 reduced the fragmentation of this antibody. This discovery is particularly surprising because the clipping site occurs within the complementarity-determining region (CDR), and previous attempts to remove the Asn clipping site with an unrelated antibody (i.e., sFLT01) did not prevent antibody fragmentation.

[0009] In one embodiment, a binding polypeptide that specifically binds to the human αβTCR / CD3 complex comprises a heavy chain variable region, a light chain variable region, and a constant region: The light chain variable region includes three complementarity-determining regions (CDRs), LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences described in SEQ ID NOs. 26, 27, and 28, respectively; SEQ ID NO: 28 contains the amino acid sequence Q-Q-W-S-S-X1-X2-L-T (where X1 is an amino acid selected from the group consisting of Q, D, H, S, Y, and A; and X2 is an amino acid selected from the group consisting of P and A); There is provided the binding polypeptide, wherein the constant region is of human origin.

[0010] In certain embodiments, X1 is S.

[0011] In certain embodiments, X2 is P.

[0012] In certain embodiments, X1 is S and X2 is P.

[0013] In certain embodiments, the light chain variable region further comprises the human light chain framework region set forth in SEQ ID NO: 14.

[0014] In certain embodiments, the binding polypeptide has improved stability at pH values greater than 5.0 compared to VH31.

[0015] In certain embodiments, the binding polypeptide has improved stability at temperatures greater than 4°C compared to VH31.

[0016] In certain embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 12, 13, 15, and 16.

[0017] 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.

[0018] In certain embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 16.

[0019] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15.

[0020] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 16.

[0021] In certain embodiments, the constant region comprises an Fc modification having a modified glycosylation pattern that reduces Fcγ receptor binding.

[0022] In certain embodiments, the Fcγ receptor is one or more receptors selected from the group consisting of FcγRIIIa and FcγRI.

[0023] In certain embodiments, the Fc modification is selected from the group consisting of N297Q / S298N / Y300S, S298N / T299A / Y300S, and S298N / Y300S.

[0024] In certain embodiments, the Fc modification is N297Q / S298N / Y300S.

[0025] In certain embodiments, the Fc modification is S298N / T299A / Y300S. In one embodiment, the Fc modification is S298N / Y300S.

[0026] In certain embodiments, the binding polypeptide is humanized.

[0027] In certain embodiments, the binding polypeptide is a monoclonal antibody.

[0028] In certain embodiments, the binding polypeptide is multispecific.

[0029] In certain embodiments, the binding polypeptide is bispecific.

[0030] In another aspect, there is provided a pharmaceutical composition comprising the binding polypeptide described herein and a pharmaceutically acceptable carrier or diluent.

[0031] In another embodiment, a formulation comprising a pharmaceutical composition described herein is provided. In a particular embodiment, the formulation is a liquid formulation. In a particular embodiment, the formulation is a lyophilized formulation.

[0032] In another embodiment, a method is provided for treating a subject for a T cell-mediated disease or disorder, comprising administering to the subject an effective amount of a conjugated polypeptide or pharmaceutical composition described herein so as to achieve treatment. In certain embodiments, the T cell-mediated disease or disorder is 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 mellitus (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 syndrome, Addison's disease, etc. The group is comprised of Egener'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, Behçet'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.

[0033] In another embodiment, nucleic acids encoding the binding polypeptides described herein are provided.

[0034] In another embodiment, a vector comprising the nucleic acid described herein is provided.

[0035] In another embodiment, cells containing nucleic acids described herein are provided. In one embodiment, the cells are mammalian cells. In one embodiment, the mammalian cells are selected from the group consisting of Chinese hamster ovary (CHO) cells and human fetal kidney (HEK) cells.

[0036] The aforementioned and other features and advantages of the present invention will be better understood from the following detailed description of exemplary embodiments, which will be understood in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0037] [Figure 1] This is a photograph of a protein gel showing the emergence of low molecular weight (LMW) fragments of the reference humanized anti-αβTCR antibody VH31 formulation antibody under accelerated conditions including high temperature (45°C) and the indicated pH for 5 weeks. HC: heavy chain; LC: light chain. [Figure 2] This line graph plots the potency of reference VH31 formulation antibodies at various concentrations under accelerated conditions over a 5-week period. The open squares correspond to reference VH31 antibodies stored at 45°C and pH 8.0; the open circles correspond to reference VH31 antibodies stored at 45°C and pH 5.0; and the open diamonds correspond to reference VH31 antibodies stored under control conditions, i.e., ≤0°C and pH 5.0. [Figure 3] This is a photograph of a protein gel showing the identity of the reference VH31 band, which was present under the indicated acceleration conditions, based on its molecular weight and N-terminal sequencing. HC: heavy chain; LC: light chain. [Figure 4] This figure shows the amino acid sequence of the reference VH31 light chain (LC) (SEQ ID NO: 25) and the estimated clipping sites of N93 / P94. [Figure 5-1] Figures 5A–5C are line graphs showing various light chain fragments resulting from clipping at N93 / P94 of the trypsin digest product (amino acids 61–102 or 61–106 according to Kabat numbering) of the reference VH31 light chain. These fragments correspond to amino acid residues 94–102 (Figure 5A), 61–93 (Figure 5B), and 94–106 (Figure 5C) of VH31 LC. The C-terminuses 102 and 106 are due to incomplete trypsin digestion. [Figure 5-2] Continuation of Figure 5-1. [Figure 6] This diagram illustrates a proposed process in which cleavage at Asn93 (N93) can occur in solution. [Figure 7]This figure shows various VH31 LC amino acid mutations designed to enhance LC stability. [Figure 8] These are photographs of a pair of protein gels showing high expression and purity of VH31 antibody containing either mutant (lanes 1-8) or non-mutant LC. 1, N93Q; 2, N93D; 3, N93H; 4, N93S; 5, N93Y; 6, N93A; 8, N93A / P94A. [Figure 9] The images show a pair of photographs of a protein gel (left panel) and three SEC-HPLC plots (right panel) demonstrating that the purity of the antibody containing the N93SLC mutation was comparable to that of the wild-type (WT) and control (VH31) antibodies. [Figure 10] This is a line graph plotting the potency of VH31 antibodies containing the indicated LC mutation. Antibodies containing the LC variant N93S (white diamond symbol) were only slightly less potent than either the reference VH31 (white square symbol) or the wild-type (WT) antibody (white octagon symbol). [Figure 11] The left panel shows a photograph of a protein gel in which the N93S mutant showed significantly reduced degradation compared to either the wild-type (WT) or the VH31 control antibody after 6 weeks of storage at 45°C and pH 8. The right panel shows that the N93S mutant showed significantly less loss of potency under accelerated conditions (3x loss) compared to either the wild-type or the VH31 control antibody (both showing more than 20x loss). [Figure 12] This is a photograph of a protein gel showing fragmentation of the reference VH31 antibody and the N93S mutant after storage at high temperature (45°C) and pH 5.5 for 6 weeks. [Modes for carrying out the invention]

[0038] This disclosure provides improved compositions and methods for treating T cell-mediated disorders (e.g., graft-versus-host disease, autoimmune diseases, and allograft rejection). Methods provided herein generally involve administering an effective amount of a humanized binding polypeptide specific to the alpha-beta T cell receptor (αβTCR) to a subject in need. The anti-αβTCR compositions provided herein are improved compositions that reduce fragmentation of the light chain variable region. This composition is an improvement over known compositions in that it includes one or more amino acid substitutions or modifications that improve the stability of the bound polypeptide.

[0039] I. Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. Any methods and materials similar to or equivalent to those described herein may be used in the methods of this disclosure. All publications referenced herein are incorporated herein by whole by reference for the purpose of describing and disclosing methodologies, reagents, and tools reported in publications that may be used in connection with this disclosure.

[0040] The methods and techniques of this application are generally carried out in accordance with the prior art methods well known in the art, as well as the prior art methods described in the various general and more specific references cited and discussed throughout this specification, unless otherwise specified. For example, Gennaro, AR. (ed.) (1990) Remington's Pharmaceutical. Sciences, 18th edition, Mack Publishing Co.; Hardman, JG, Limbird, LE, and Gilman, AG (eds.) (2001) The Pharmacological Basis of Therapeutics, 10th edition, McGraw-Hill Co.; Colowick, S. et al. (eds.), Methods In Enzymology, Academic Press, Inc.; Weir, DM and Blackwell, CC (eds.) (1986) Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications; Maniatis, T. et al. (eds.) (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (eds.) (1999) Short Protocols in Molecular Biology, 4th edition, John See Wiley & Sons; Ream et al. (eds.) (1998) Molecular Biology Technique: An Intensive Laboratory Course, Academic Press; Newton, CR and Graham, A. (eds.) (1997) PCR (Introduction to Biotechniques Series), 2nd edition, Springer-Verlag.

[0041] The human αβTCR / CD3 complex is a T cell receptor complex presented on the surface of T cells. See Kuhns et al. (2006) Immunity 24: pp. 133-139. This complex is targeted by the mouse monoclonal antibody BMA031 (European Patent Application No. EP0403156, the whole of which is incorporated herein by reference; see Sequence IDs 1 and 2), as well as the humanized and stabilized antibodies disclosed herein.

[0042] The mouse IgG2b monoclonal antibody BMA031 (Borst et al. (1990) Hum.Immunol.29(3):175~88;EP0403156) is specific to common determinants on the alpha-beta TCR / CD3 complex and does not bind to the gamma-delta TCR. BMA031 is highly immunosuppressive and can induce apoptosis of activated T cells via the activation-induced cell death (AICD) mechanism (Wesselborg et al. (1993) J.Immunol.150(10):4338~4345). In vitro, BMA031 inhibits the mixed lymphocyte response and has shown preliminary clinical efficacy in preventing graft rejection in several solid organ transplantation scenarios and in treating acute graft-versus-host disease (Kurrle et al. (1989) Transplant Proc.21(1):1017~1019). BMA031 is found in the majority of human populations. BMA031 does not bind to the 1c gamma receptor (FcγR). Therefore, BMA031 does not induce T cell activation via T cell receptor cross-linking, and thus does not induce T cell activation or related cytokine release. In this respect, the profile of BMA031 is far more favorable than that of OKT3. However, BMA031 is a mouse antibody, and therefore, considering the human anti-mouse antibody (HAMA) response induced by repeated administration, it is not suitable for repeated administration in human subjects.

[0043] Several humanized versions of BMA031 have been described (see, e.g., WO2013 / 037484; EP0403156; also see Shearman et al. (1991) J.Immunol. 147: pp. 4366-4373). As stated in EP0403156, simple CDR transplantation was unsuccessful in retaining antigen binding. One clone with significant "training" framework modifications, EUCIV3, successfully bound to T cells; however, as stated in EP0403156, binding to αβTCR was not as effective as that of the parental BMA031 antibody when determined by flow cytometry competitive assay. Furthermore, EUCIV3 was originally generated with a wild-type human IgG1 or IgG4 scaffold that still retained FcγR binding. These humanized antibodies therefore enabled T cell activation, proliferation, and simultaneous cytokine release, thus differing considerably from the original properties of BMA031.

[0044] The terms “binding protein” or “binding polypeptide” are used, unless otherwise specified, to refer to a polypeptide (e.g., an antibody or its antigen-binding fragment) that contains at least one binding site involved in selective 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 embodiments, the binding polypeptides described herein may contain multiple (e.g., two, three, four, or more) binding sites.

[0045] The term "antibody" is used to refer to the entire antibody and the antigen-binding fragment of such an antibody, unless otherwise specified. For example, the term includes a quadruple-chain IgG molecule and an antibody fragment.

[0046] As used herein, the term “antibody fragment” refers to a portion of an intact, full-length antibody, such as those further described below.

[0047] The antibody may be any class such as IgG, IgA, IgE, IgD, or IgM; and any subclass such as IgG1 or IgG4. Different classes and subclasses of immunoglobulins have different properties that may be advantageous in different applications. For example, IgG4 antibodies have reduced binding to Fc receptors.

[0048] Naturally occurring immunoglobulins have a common core structure in which two identical light chains (approximately 24 kDa) and two identical heavy chains (approximately 55 or 70 kDa) form a tetramer. The amino-terminal region of each chain is known as the variable (V) region and can be distinguished from the remaining, more conserved constant (C) region of each chain.

[0049] Most amino acid sequence mutations in immunoglobulins are localized to three distinct locations within each V region, known as hypervariable regions or complementarity-determining regions (CDRs), which are directly involved in antigen binding. Proceeding from the amino terminus, these regions are named CDR1, CDR2, and CDR3, respectively. The CDRs are held in place by more conserved framework regions (FRs). Proceeding from the amino terminus, these regions are named FR1, FR2, FR3, and FR4, respectively. Proceeding from the amino terminus, these composite regions within the V region are named FR1, CDR1, FR2, CDR2, FR3, CDR3, These regions are named FR4. The locations and numbering systems of the CDR and FR regions are defined by Kabat et al. (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, USD Department of Health and Human Services, US Government Printing Office (1991), and its updates which can be found online). Furthermore, the boundaries of the CDR region are further defined by IMGT nomenclature.

[0050] As used herein, a "humanized monoclonal antibody" is an antibody comprising a human antibody framework into which a complementation-determining region (CDR) derived from a non-human antibody has been transplanted. Modifications to the human acceptor framework may also be made. Procedures for the design and preparation of humanized antibodies are well known in the art and are described, for example, in U.S. Patent Nos. 4,816,397; 4,816,567; and 5,225,539; European Patent Application Nos. 0120694; 0125023; 0194276B1; 0239400; 0519596; and International Patent Application WO86 / 01533. Further details regarding antibodies, humanized antibodies, human-modified antibodies, and methods for preparing them can be found in Kontermann, R. and Dijbel, S. (eds.) (2001, 2010), Antibody Engineering, 2nd edition, Springer-Verlag, New York, NY. The entire contents of each of the patents and patent application publications listed above are incorporated herein by reference.

[0051] The variable regions of the antibodies according to the described embodiments can be obtained, at least in part, by humanizing BMA031, i.e., by introducing the CDR of BMA031 into a human framework, and by further modifying one or more VL CDRs to improve the stability of the binding polypeptide by reducing fragmentation of the light chain variable region. Two series of humanized BMA031 antibodies are described in PCT Publication WO2013 / 037484, which is incorporated herein by reference. These two series are the HEBE1 series, including SEQ ID NOs. 5-7, 12 and 13, and the GL1BM series, which includes the heavy chain variable region shown in SEQ ID NOs. 8, 15 and 16. In both cases, the light chain variable region used is as shown in SEQ ID NO. 14 (GL1BM VK43). The human framework used is IGH3-23 (SEQ ID NO. 17) in the case of HEBE1 and IGHV1-3 in the case of GL1BM. * 01 and IGKV3-11 *These are 01 (sequence numbers 18 and 19, respectively).

[0052] The constant region may be derived from the constant region of any human antibody. The variable region gene may be cloned in frame with the constant region gene into an expression vector to express heavy and light immunoglobulin chains. Such an expression vector can be transfected into antibody-producing host cells for antibody synthesis.

[0053] The variable and constant regions of human antibodies can be obtained from sequence databases. For example, immunoglobulin sequences are available in the IMGT / LIGM database (Giudicelli et al., (2006) Nucleic Acids Res. 34 (suppl. 1): D781~D784) or VBase 30 (vbase.mrc-cpe.cam.ac.uk). Aglycosylated antibodies can have a wide range of modified functionalities; see Boyd et al. (1996) Mol. Immunol. 32: pp. 1311~1318. The "delta ab" or Δab modification, as used herein, is the Fc modification described in Armour et al., (1999) Eur. J. Immunol. 29: pp. 2613~2624. Methods for modifying the glycosylation of the antibody Fc region are known in the art, including chemical, enzymatic and / or mutagenetic means, e.g., CH2 domicile One example is a mutation at position N297 of the gene. Methods for mutating antibody genes to produce aglycosylated IgG molecules are described in Tao and Morrison (1989) J. Immunol. 143: pp. 2595-2601.

[0054] The specificity in association with the antibodies described herein means that the claimed antibodies can selectively bind to their defined homologous antigen, namely the αβTCR / CD3 complex. The antibodies described herein bind to the αβTCR / CD3 complex expressed in cells including T cells.

[0055] The terms “stable,” “stability,” and “stabilized,” as used herein in relation to bound polypeptides, refer to the resistance of a bound polypeptide to thermal decomposition and chemical decomposition or fragmentation under given conditions of manufacture, preparation, transport, and storage. A “stable” composition retains 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% or more of biological activity under given conditions of manufacture, preparation, transport, and storage. The stability of a bound polypeptide can be evaluated, for example, with respect to the degree of decomposition or fragmentation, the level of specific fragments, or the type or size of aggregates, using methods and measurements known to those skilled in the art, compared to a control or to the starting material. Such methods and measurements include, but are not limited to, area under curve (AUC) reduction compared to a reference, size exclusion chromatography (SEC), high-speed (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).

[0056] The term “nucleic acid,” as used herein, includes the DNA molecule encoding the antibody described herein. A preferred DNA molecule encoding the antibody described herein is an expression vector suitable for expressing the antibody gene in a host cell. Expression vectors and host cells for antibody gene expression are known in the art; see, for example, Morrow, KJ Genetic Engineering & Biotechnology News (June 15, 2008) 28(12), and Backliwal, G. et al. (2008) Nucleic Acids Res. 36(15):e96~e96.

[0057] The terms “treat” and “treatment,” as used herein, refer to the care of a patient or subject having a disease, disorder, or condition. Treatment may, but is not limited to, one or any combination of the following: cure of a disease, disorder, or condition; improvement of at least one symptom of a disease, disorder, or condition; and / or preventive or preventive action aimed at preventing or reducing the occurrence of a disease, disorder, or condition. In certain embodiments, treatment may, but is not limited to, cure of a disease, disorder, or condition; or improvement of at least one symptom of a disease, disorder, or condition.

[0058] 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 non-human mammal. In certain embodiments, the subject is a non-human primate. In certain embodiments, the subject is a human.

[0059] II. Antibodies This disclosure comprises a method for administering one or more antigen-binding fragments of the humanized anti-αβTCR antibodies described herein to a subject requiring such fragment. The antibody fragment is αβT They can bind to CR / CD3 complexes. They include Fab, Fab', F(ab')2, and F(v) fragments, or individual light chain or heavy chain variable regions or any portion thereof. Examples of fragments include Fab, Fab', F(ab')2, Fv, scFv, etc. In certain embodiments, the fragments may lack the Fc portion of the intact antibody, disappear more rapidly from circulation, and / or have less nonspecific tissue binding than the intact antibody. In certain embodiments, the fragments may be prepared from the intact antibody by protein cleavage using enzymes such as papain (for producing Fab fragments) or pepsin (for producing F(ab')2 fragments) using well-known methods.

[0060] In certain embodiments, an antibody and / or antibody fragment comprises a single-chain antibody fragment (scFv) that binds to the αβTCR / CD3 complex. In certain embodiments, the scFv comprises an antibody heavy-chain variable region (VH) operably linked to an antibody light-chain variable region (VL), and one or both of the heavy-chain and light-chain variable regions together or individually form a binding site that binds to the αβTCR. The scFv may contain a VH region at the amino terminus and a VL region at the carboxyl terminus. Alternatively, the scFv may contain a VL region at the amino terminus and a VH region at the carboxyl terminus. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombination by a synthetic linker that causes the VL and VH regions to pair up to form a single protein chain (known as single-chain Fv (scFv)) that forms a monovalent molecule. The scFv may optionally further contain a polypeptide linker between the heavy-chain and light-chain variable regions.

[0061] Antibodies and antibody fragments also include domain antibody (dAb) fragments consisting of a VH domain, as described in Ward, ES et al. (1989) Nature 341: pp. 544-546. Antibodies and antibody fragments also include heavy chain antibodies (HCAb). HCAb have been reported to form antigen-binding domains using only the heavy chain variable region, in that these functional antibodies are dimers of only the heavy chain (referred to as "heavy chain antibodies" or "HCAb"). Therefore, in certain embodiments, antibodies and antibody fragments may be HCAb that specifically bind to the αβTCR / CD3 complex. Antibodies and antibody fragments also include antibodies that are αβTCR / CD3 complex-specific small modular immunotherapy (SMIP) or binding domain immunoglobulin fusion proteins. These constructs are single-chain polypeptides containing an antigen-binding domain fused to an immunoglobulin domain necessary to perform antibody effector function (see WO2005 / 017148). Antibodies and antibody fragments also include diabodies. A diabody refers to a bivalent antibody in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to pair the two domains on the same chain. This forces the domains to pair with complementary domains on another chain, thereby creating two antigen-binding sites (see, for example, WO93 / 11161). Diabodies can be bispecific or monospecific.

[0062] In certain embodiments, the antibody or antibody fragment specifically binds to the human αβTCR / CD3 complex. That is, such an antibody or antibody fragment does not cross-react with any target other than the human αβTCR / CD3 complex.

[0063] Antibodies or antibody fragments can be modified to increase their serum half-life by, for example, adding molecules such as PEG or other water-soluble polymers containing polysaccharide polymers to increase the half-life of the antibody or antibody fragment.

[0064] Antibodies and antibody fragments may be multispecific or bispecific. For example, a bispecific antibody or antibody fragment may be a single antibody containing two different binding sites (variable regions) (or The antibody fragment may be similar to a bispecific antibody. Bispecific antibodies can be produced by various methods, such as chemical methods, polydoma methods, or recombinant DNA methods. A bispecific antibody can have binding specificity to at least two different epitopes, at least one of which is the αβTCR / CD3 complex. The other specificity can be selected from any useful or desired specificity, including, for example, specificity to human serum albumin to extend the half-life in vivo.

[0065] The clinical use of bispecific antibodies for oncological applications is now becoming a reality with the trifunctional catumakisomab (REMOVAB®), approved for use in cases of malignant ascites, and the bispecific antibody blinatumomab (BLINCYTO®), approved for use as a second-line treatment for relapsed or refractory acute lymphoblastic leukemia (ALL) with Philadelphia chromosome negative characteristics. These antibodies commonly possess a binding arm that binds to T cells and a second arm that binds to tumor target cells, resulting in T cell-mediated lysis of tumor targets. Also commonly, these molecules recruit T cells via the CD3 protein located on the cell surface. An alternative to CD3-mediated recruitment is the utilization of the αβ T cell receptor (αβ TCR), which is also expressed on the cell surface.

[0066] In certain exemplary embodiments, the antibodies according to this disclosure may be used to develop antitumor antibodies by combining specificity for tumor-associated antigens with specificity for αβ T cell receptors (αβ TCRs).

[0067] III. Anti-αβTCR antibody As described above, two series of such humanized anti-αβTCR antibodies are described in PCT Publication WO2013 / 037484, which is incorporated herein by reference. These two series are based on the mouse anti-αβTCR antibody BMA031 and include the HEBE1 series, which includes SEQ ID NOs. 5-7, 12 and 13, and the GL1BM series, which includes the heavy chain variable region shown in SEQ ID NOs. 8, 15 and 16. In both cases, the light chain variable region used is as shown in SEQ ID NO. 14 (GL1BM VK43). The human framework used is IGH3-23 (SEQ ID NO. 17) in the case of HEBE1, and IGHV1-3 in the case of GL1BM. * 01 (SEQ ID NO: 18) and IGKV3-11 * This is 01 (sequence number 19).

[0068] In the sequences listed in Table 1, the selected CDRs are shown in bold and are as described in sequence numbers 26-28.

[0069] [Table 1] [Table 2] [Table 3]

[0070] A comparison of the LCDR3 of GL1BM VK43 in SEQ ID NO: 14 (QQWSSNPLT (SEQ ID NO: 29)) with the LCDR3 of stabilized GL1BM VK43 (QQWSS-X1-X2-LT (wherein X1 is Q, D, H, S, Y, or A, and X2 is P or A) (SEQ ID NO: 28)) reveals that amino acids X1 and X2 in SEQ ID NO: 28 correspond to N and P in SEQ ID NO: 29, respectively.

[0071] In a particular embodiment, a binding polypeptide that specifically binds to the human αβTCR / CD3 complex comprises a heavy chain variable region, a light chain variable region, and a constant region: The light chain variable region includes three complementarity-determining regions (CDRs), LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences described in SEQ ID NOs. 26, 27, and 28, respectively; Sequence ID No. 28 contains the amino acid sequence QQWSS-X1-X2-LT (wherein X1 is an amino acid selected from the group consisting of Q, D, H, S, Y, and A, and X2 is an amino acid selected from the group consisting of P and A); The conjugated polypeptide is provided, the constant region of which is derived from humans.

[0072] In a particular embodiment, X1 is Q.

[0073] In a particular embodiment, X1 is D.

[0074] In a particular embodiment, X1 is H.

[0075] In a particular embodiment, X1 is S.

[0076] In a particular embodiment, X1 is Y.

[0077] In a particular embodiment, X1 is A.

[0078] In a particular embodiment, X2 is P.

[0079] In a particular embodiment, X2 is A.

[0080] In a particular embodiment, X1 is Q and X2 is P.

[0081] In a particular embodiment, X1 is Q and X2 is A.

[0082] In a particular embodiment, X1 is D and X2 is P.

[0083] In a particular embodiment, X1 is D and X2 is A.

[0084] In a particular embodiment, X1 is H and X2 is P.

[0085] In a particular embodiment, X1 is H and X2 is A.

[0086] In a particular embodiment, X1 is S and X2 is P.

[0087] In a particular embodiment, X1 is S and X2 is A.

[0088] In a particular embodiment, X1 is Y and X2 is P.

[0089] In a particular embodiment, X1 is Y and X2 is A.

[0090] In a particular embodiment, X1 is A and X2 is P.

[0091] In a particular embodiment, X1 is A and X2 is A.

[0092] In a particular embodiment, the light chain variable region further includes the human light chain framework region according to Sequence ID No. 14.

[0093] In certain embodiments, the bound polypeptide exhibits improved stability at pH levels above 5.0 compared to the reference antibody VH31.

[0094] In certain embodiments, the bound polypeptide exhibits improved stability at temperatures above 4°C compared to the reference antibody VH31.

[0095] In a particular embodiment, the heavy chain variable region includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 12, 13, 15, and 16.

[0096] In a particular embodiment, the heavy chain variable region includes an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 13.

[0097] In a particular embodiment, the heavy chain variable region includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 16.

[0098] In a particular embodiment, the heavy chain variable region includes the amino acid sequence described as SEQ ID NO: 15.

[0099] In a particular embodiment, the heavy chain variable region includes the amino acid sequence described as SEQ ID NO: 16.

[0100] IV. Modified anti-αβTCR antibodies The anti-αβTCR antibody may contain one or more modifications. The modified anti-αβTCR antibody according to the present invention can be prepared using any method known in the art.

[0101] i) Reduction of fragmentation When used herein in relation to a conjugated polypeptide composition, the term "fragmentation" refers to the cleavage of a conjugated polypeptide or a first portion thereof into two or more portions, each having a lower molecular weight than the original conjugated polypeptide or a first portion thereof. Such forms of fragmentation include: 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 first full-length heavy chain paired with a second full-length heavy chain and a full-length light chain, and a first full-length heavy chain paired with a second full-length heavy chain and a partial light chain. Examples include, but are not limited to, heavy chains, a first full-length heavy chain paired with a first partial light chain, a second full-length heavy chain paired with a second partial light chain, a first full-length heavy chain paired with a partial heavy chain and a full-length light chain, a first full-length heavy chain paired with a partial heavy chain and two full-length light chains, a first full-length heavy chain paired with a partial heavy chain and a partial light chain, a first partial heavy chain paired with a second partial heavy chain and a full-length light chain, and a first partial heavy chain paired with a second partial heavy chain and a partial light chain.

[0102] 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. In particular, with respect to 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.

[0103] Fragmentation can be caused by various mechanisms, such as the disruption of innate covalent bonds leading to cleavage of the polypeptide backbone through spontaneous reactions (e.g., non-enzymatic reactions) or enzymatic reactions. Generally, protein backbones are highly stable under physiological conditions. However, certain regions and motifs may be more susceptible to fragmentation due to their amino acid sequence, the flexibility of their two- or three-dimensional polypeptide structure, and unsuitable solvent and environmental conditions (e.g., temperature and pH).

[0104] For example, the amino acids Asp(D), Gly(G), Ser(S), Thr(T), Cys(C), and Asn(N) have been shown to be particularly susceptible to cleavage in the polypeptide backbone of antibodies. See, for example, Liu H. et al. J.Am.Soc.Mass Spectrom.2009;20:2258-2264. Furthermore, the Asn-Pro(NP) amide bond is known to undergo complete cleavage in the presence of ammonia. See, for example, Tarelli E. and Corran PHJPeptide. See Res.2003;62:245-251.

[0105] Fragmentation can occur at any stage during the manufacturing or storage of biological compositions. Since fragmentation can lead to reduced potency or the presence of unwanted and potentially immunogenic species, reducing fragmentation is a critical consideration in the production of any biological therapeutic agent.

[0106] In some embodiments, the antibody content is less than 5% (e.g., 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) Fragmentation occurs after storage at 2°C to 8°C for at least one month (e.g., at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty

[0107] Methods for determining the amount of monomer-bound polypeptide and the amounts of monomeric, oligomeric, aggregated, or fragmented forms of the bound polypeptide present in solution are described herein and illustrated in the examples. For example, those skilled in the art can determine the percentage of the entire intermediate, fragmented intermediate, unfolded intermediate, and / or aggregated species present in a given solution using, for example, size exclusion chromatography (SEC-HPLC), static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding, endogenous tryptophan fluorescence, non-reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and differential scanning calorimetry (DSC). In the examples described herein, the inventors particularly illustrate the use of SEC-HPLC and SDS-PAGE to determine the physical state of the bound polypeptide in solution.

[0108] ii) Reduction of immunogenicity In certain exemplary embodiments, deimmunization may be used to reduce the immunogenicity of an antibody or its antigen-binding moiety. As used herein, the term “deimmunization” includes modification of an antibody or its antigen-binding moiety to modify one or more T cell epitopes (see, e.g., WO98 / 52976A1, WO00 / 34317A2). For example, VH and VL sequences derived from a starting antibody may be analyzed, and a human T cell epitope “map” showing the epitope’s location in relation to complementarity-determining regions (CDRs) and other key residues in the sequence may be generated from each V region. Individual T cell epitopes from the T cell epitope map may be analyzed to identify alternative amino acid substitutions that carry a low risk of altering the activity of the final antibody. A series of alternative VH and VL sequences containing combinations of amino acid substitutions may be designed, and these sequences may then be incorporated into a series of anti-αβTCR antibodies or anti-αβTCR antibody fragments for use in the methods disclosed herein, and then tested for function. Complete heavy and light chain genes, including modified V and human C regions, can then be cloned into expression vectors, and the plasmids can subsequently be introduced into cell lines for the production of the entire antibody. The antibodies can then be compared using appropriate biochemical and biological assays to identify the optimal variant.

[0109] iii) Effects function and Fc modification Humanized monoclonal antibodies, or fragments of humanized monoclonal antibodies (e.g., anti-αβTCR antibodies or fragments thereof), may contain an antibody constant region (e.g., an IgG constant region, e.g., a human IgG constant region, e.g., a human IgG1 or IgG4 constant region) that mediates one or more effector functions. For example, binding of the complement C1 component to the antibody constant region can activate the complement system. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. Furthermore, antibodies bind to various cell receptors via their Fc region, through the Fc receptor binding site of the antibody Fc region that binds to Fc receptors (FcRs) on cells. There are many Fc receptors specific to different classes of antibodies, including IgG (gamma receptor, i.e., Fcγ receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). Antibody binding to Fc receptors on the cell surface leads to phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, and lysis of antibody-coated target cells by killer cells (antibody-dependent). It triggers many important and diverse biological responses, including cell-mediated cytotoxicity (known as ADCC), the release of inflammatory mediators, placental cross-transition, and regulation of immunoglobulin production.

[0110] In certain embodiments, the humanized monoclonal antibody, or humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof), binds to the Fcγ receptor. In alternative embodiments, the humanized monoclonal antibody, or humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof), may include a constant region that cannot direct one or more effector functions (e.g., ADCC activity) and / or cannot bind to the Fcγ receptor.

[0111] Certain embodiments described herein provide humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) in which at least one amino acid of one or more constant region domains is deleted or otherwise modified to result in desired biochemical properties compared to an unmodified antibody of substantially the same immunogenicity, such as decreased or enhanced effector function, non-covalent dimerization ability, increased ability to localize to tumor sites, decreased serum half-life, and / or increased serum half-life. For example, certain antibodies or fragments thereof for use in diagnostic and therapeutic methods described herein are domain deletion 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 one of the constant regions of the modified antibody is deleted, for example, all or part of the CH2 domain is deleted.

[0112] In certain other embodiments, the humanized monoclonal antibody, or humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or fragment thereof), comprises constant regions derived from different antibody isotypes (e.g., constant regions derived from two or more human IgG1, IgG2, IgG3, or IgG4). In other embodiments, the humanized monoclonal antibody, or humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or fragment thereof), comprises a chimeric hinge (i.e., a hinge containing hinge portions derived from hinge domains of different antibody isotypes, e.g., an upper hinge domain from the IgG4 molecule and a middle hinge domain from IgG1). In one embodiment, the humanized monoclonal antibody, or humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or fragment thereof), comprises an Fc region or portion derived from the human IgG4 molecule and a Ser228Pro (S228P) mutation (EU numbering) in the core hinge region of the molecule.

[0113] 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, deletion or inactivation of a constant region domain (by point mutation or other means) can reduce the Fc receptor binding of the circulatingly modified antibody, thereby increasing tumor localization. In other cases, constant region modifications consistent with this disclosure will relax complement binding, and therefore reduce serum half-life and nonspecific binding of conjugated cytotoxins. Further modifications of the constant region are used to modify disulfide bond or oligosaccharide moieties, which allow for enhanced localization by increasing antigen specificity or flexibility. The resulting physiological profile, bioavailability, and other biochemical effects of the modifications, such as tumor localization, in vivo distribution, and serum half-life, can be readily measured and quantified using well-known immunological techniques without excessive experimentation.

[0114] In a particular embodiment, 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 Fc This is a variant. As used herein, the term “Fc variant” refers to an Fc domain having at least one amino acid substitution compared to the wild-type Fc domain from which the Fc domain is derived. For example, if the Fc domain is derived from a human IgG1 antibody, the Fc variant of the human IgG1 Fc domain contains at least one amino acid substitution compared to the Fc domain.

[0115] The amino acid substitutions in the Fc variant may be at any position within the Fc domain (i.e., any conventional EU amino acid position). In one embodiment, the Fc variant includes a substitution at an amino acid position located in the hinge domain or a portion thereof. In another embodiment, the Fc variant includes a substitution at an amino acid position located in the CH2 domain or a portion thereof. In yet another embodiment, the Fc variant includes a substitution at an amino acid position located in the CH3 domain or a portion thereof. In yet another embodiment, the Fc variant includes a substitution at an amino acid position located in the CH4 domain or a portion thereof.

[0116] The antibody may use any Fc variant known in the art to provide effector function and / or improved (e.g., reduced or enhanced) FcR binding. Such Fc variants include, for example, the International PCT Publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, and WO00 / 32767A1, each of which is incorporated herein by reference. , WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, WO03 / 074569A2, WO04 / 016750A2, WO04 / 02 9207A2, WO04 / 035752A2, WO04 / 063351A2, WO04 / 074455A2, WO04 / 099249A2, WO05 / 040217A2 , WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 047350A2, and WO06 / 085967A2 or U.S. Patent Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046 Examples include any one of the amino acid substitutions disclosed in patents 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.

[0117] In one exemplary embodiment, the humanized monoclonal antibody, or humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof), comprises an Fc variant with an amino acid substitution at EU position 268 (e.g., H268D or H268E). In another exemplary embodiment, the humanized monoclonal antibody, or humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof), comprises an amino acid substitution at EU position 239 (e.g., S239D or S239E) and / or EU position 332 (e.g., I332D or I332Q).

[0118] In certain embodiments, a humanized monoclonal antibody, or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof), comprises an Fc variant containing amino acid substitutions that alter the antibody's antigen-dependent effector function, particularly its circulating half-life. Such antibodies exhibit either increased or decreased binding to the neonatal Fc receptor (FcRn) compared to antibodies lacking these substitutions, and therefore their serum half-life increases or decreases, 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 of treating mammals where a longer half-life of the administered antibody is desired, for example, to treat chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity have a shorter half-life. Such molecules are expected to have the following properties, and such molecules are also useful, for example, for in vivo imaging or in situations where the starting antibody has toxic side effects when present in circulation for extended periods, such as in mammals where shorter circulation time is advantageous. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta and are therefore useful in the treatment of diseases or disorders in pregnant women. Furthermore, other applications where reduced FcRn binding affinity may be desirable include applications where localization to the brain, kidneys, and / or liver is desired. In one exemplary embodiment, the antibody modification shows reduced passage of the renal glomerular epithelium from the vascular system. In another embodiment, the antibody modification shows reduced passage of the blood-brain barrier (BBB) ​​from the brain to the vascular lumen.

[0119] In one embodiment, an antibody with altered FcRn binding comprises an Fc domain having one or more amino acid substitutions within an "FcRn binding loop" of the Fc domain. The FcRn binding loop consists of amino acid residues 280–299 (according to EU numbering). Exemplary amino acid substitutions that alter FcRn binding activity are disclosed in International PCT Publication WO05 / 047327, which is incorporated herein by reference in its entirety for all purposes. In certain exemplary embodiments, a humanized monoclonal antibody, or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof), comprises an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (according to EU numbering).

[0120] In other embodiments, a humanized monoclonal antibody or humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof) for use in the diagnostic and therapeutic methods described herein has a constant region modified to reduce or eliminate glycosylation, e.g., an IgG1 or IgG4 heavy chain constant region. For example, the antibody may also include an Fc variant having an amino acid substitution that alters 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 includes a reduction in glycosylation of an N-linked glycan typically found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif (e.g., an N-linked glycosylation motif containing the amino acid sequence NXT or NXS). In a particular embodiment, the antibody includes an Fc variant having an amino acid substitution at amino acid position 228 or 299 (EU numbering). In more specific embodiments, the antibody comprises an IgG1 or IgG4 constant region containing the S228P and T299A mutations (EU numbering).

[0121] Exemplary amino acid substitutions that result in reduction or alteration of glycosylation are disclosed in International PCT Publication WO05 / 018572, which is incorporated herein by reference in its entirety for all purposes. In certain embodiments, an antibody, or fragment thereof, is modified to eliminate glycosylation. Such an antibody, or fragment thereof, is referred to as an “agly” antibody, or fragment thereof (e.g., an “agly” antibody fragment). While not bound by scientific theory, it is conceivable that an agly antibody, or fragment thereof, may have an improved in vivo safety and stability profile. An exemplary agly antibody, or agly antibody fragment, contains an aglycosylated Fc region of an IgG4 antibody lacking Fc effector function, thereby eliminating the possibility of Fc-mediated toxicity to normal vital organs. In yet other embodiments, an agly antibody, or agly antibody fragment, contains an altered glycan. For example, an agri antibody or agri antibody fragment may have a reduction in the number of fucose residues of the N-glycan at Asn297 (N297) in the Fc region, i.e., it may be afucosylated. In another embodiment, an agri antibody or agri antibody fragment may have a change in the number of sialic acid residues of the N-glycan at Asn297 in the Fc region.

[0122] iv) covalent bond Humanized monoclonal antibodies, or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof), can be modified, for example, by covalent bonding of molecules to the antibody, such that the covalent bond does not interfere with the antibody's specific binding to its homologous epitope. For example, but not limited to, humanized monoclonal antibodies, or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof), can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage, binding to cell ligands or other proteins, etc. Any of the numerous chemical modifications can be carried out by known methods, including but not limited to specific chemical cleavage, acetylation, and formylation. Furthermore, derivatives may contain one or more non-classical amino acids.

[0123] Humanized monoclonal antibodies, or fragments of humanized monoclonal antibodies (e.g., anti-αβTCR antibodies or fragments thereof), can be further recombinantly fused to heterologous polypeptides at their N-terminus or C-terminus, or chemically conjugated to polypeptides or other compositions (including covalent and non-covalent conjugations). For example, anti-αβTCR antibodies can be recombinantly fused to or conjugated to molecules useful as labels in detection assays, and to effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, for example, PCT Publications WO92 / 08495, WO91 / 14438, and WO89 / 12624; U.S. Patent No. 5,314,995; and EP396,387.

[0124] 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 their in vivo half-life or for use in immunoassays using methods known in the art. For example, in one embodiment, polyethylene glycol (PEG) is conjugated to a humanized monoclonal antibody, or humanized monoclonal antibody fragment (e.g., anti-αβTCR antibodies or fragments thereof) to increase its 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).

[0125] Furthermore, a humanized monoclonal antibody, or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof), can be fused to one or more marker sequences, such as peptides, to facilitate the purification or detection of the humanized monoclonal antibody, or the humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or a fragment thereof). In certain embodiments, the marker amino acid sequence is a hexahistidine peptide, such as a tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), many of which are commercially available. For example, as described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), hexahistidine provides a convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag (Wilson et al., Cell 37:767-778 (1984)) and the "flag" tag, which correspond to epitopes derived from influenza hemagglutinin protein.

[0126] Humanized monoclonal antibodies, or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof), may be used in a non-conjugated form, for example, to improve the therapeutic properties of a molecule, to facilitate target detection, or for imaging or therapy of a target. Humanized monoclonal antibodies, or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof), can be labeled or conjugated either before or after purification, if purification is performed. 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, pharmaceuticals, or PEGs.

[0127] This disclosure further includes humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) conjugated to diagnostic or therapeutic agents. Humanized monoclonal antibodies or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof) can be used diagnostically to monitor the onset or progression of immunocytotoxicity (e.g., CLL) as part of a clinical laboratory procedure to determine, for example, the efficacy of a given therapeutic and / or prophylactic regimen. Detection can be facilitated by coupling the humanized monoclonal antibody or humanized monoclonal antibody fragment (e.g., anti-αβTCR antibodies or fragments thereof) to a detectable substance. 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 techniques, and non-radioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use in the diagnostic methods of this 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, dansilloride, and phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include, 125 I, 131 I, 111 In and 99 Tc was mentioned.

[0128] Humanized monoclonal antibodies, or humanized monoclonal antibody fragments (e.g., anti-αβ TCR antibodies or fragments thereof), for use 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, cell division inhibitors, biotoxins, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceuticals, immunologically active ligands (e.g., lymphokines or other antibodies where the resulting molecule binds to both neoplastic cells and effector cells such as T cells), or PEG.

[0129] In another embodiment, humanized monoclonal antibodies, or humanized monoclonal antibody fragments (e.g., anti-αβ TCR antibodies or fragments thereof), for use in the diagnostic and therapeutic methods disclosed herein can be conjugated to molecules that decrease tumor cell proliferation. In other embodiments, the disclosed compositions may include humanized monoclonal antibodies, or humanized monoclonal antibody fragments (e.g., anti-αβ TCR antibodies or fragments thereof), conjugated to a drug or prodrug. Still other embodiments described herein include the use of antibodies, or fragments thereof, conjugated to specific biotoxins such as ricin, gelonin, Pseudomonas aeruginosa exotoxin or diphtheria toxin, or cytotoxic fragments thereof. The choice of which conjugated or unconjugated antibody to use will depend on the type and stage of the cancer, the use of adjuvant therapies (e.g., chemotherapy or external irradiation), and the condition of the subject. It will be understood that one of ordinary skill in the art can readily make such a choice in light of the teachings herein.

[0130] It will be understood that in previous studies, anti-tumor antibodies labeled with isotopes have been successfully used to destroy tumor cells in animal models and in some instances 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 act by producing ionizing radiation that causes multiple strand breaks in nuclear DNA, leading to cell death. The isotopes used to create therapeutic conjugates typically produce high-energy alpha or beta particles with short pathway lengths. Such radionuclides kill cells in very close proximity, such as neoplasms to which the conjugate binds or invades. The radionuclides have little to no effect on delocalized cells. Radionuclides are inherently non-immunogenic.

[0131] V. Antibody production Antibody production can be carried out by any method known in the art, including transgenic organisms such as goats (see Pollock et al. (1999) J. lmmunol. Methods 231: pp. 147-157), chickens (see Morrow, KJJ (2000) Genet. Eng. News 20: pp. 1-55), mice (see Pollock et al., above), or plants (see Doran, PM (2000) Curr. Opinion Biotechnol. 11: pp. 199-204; Ma. JK-C. (1998) Nat. Med. 4: pp. 601-606; Baez, J. et al. (2000) BioPharm. 13: pp. 50-54; Stoger, E. et al. (2000) Plant Mol. Biol. 42: pp. 583-590). Antibodies can also be produced through chemical synthesis or by the expression of the gene encoding the antibody in the host cell.

[0132] A polynucleotide encoding a humanized monoclonal antibody, or a humanized monoclonal antibody fragment (e.g., an anti-αβTCR antibody or its fragment), is isolated and inserted into a replicable construct or vector, such as a plasmid, for further proliferation or expression in host cells. Constructs or vectors (e.g., expression vectors) suitable for the expression of humanized monoclonal antibodies, or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or their fragments) according to the described embodiments are available in the Art. Various vectors are available, including vectors maintained in a single or multiple copy in host cells, or vectors that become integrated into the chromosomes of host cells. The construct or vector is introduced into suitable host cells, and cells expressing humanized immunoglobulins are produced and maintained in culture. A single or multiple vectors are used for the expression of humanized immunoglobulins.

[0133] Polynucleotides encoding humanized monoclonal antibodies, or fragments of humanized monoclonal antibodies (e.g., anti-αβTCR antibodies or fragments thereof), are readily isolated and sequenced using conventional procedures (e.g., oligonucleotide probes). Vectors that can be used include plasmids, viruses, phages, transposons, and minichromosomes, with plasmids being a typical embodiment. Generally, such vectors further include signal sequences operably linked to the light and / or heavy chain polynucleotides to facilitate expression, an origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. The polynucleotides encoding the light and heavy chains are inserted into separate vectors and introduced simultaneously or sequentially into the same host cell (e.g., by transformation, transfection, electroporation, or transduction), or, if desired, both the heavy and light chains are inserted into the same vector prior to such introduction.

[0134] Promoters are provided for expression in appropriate host cells. Promoters may be homeostatic or inducible. For example, a promoter can be operably ligated to a nucleic acid encoding a humanized immunoglobulin or immunoglobulin chain to direct the expression of the encoded polypeptide. A variety of suitable promoters are available for prokaryotic and eukaryotic hosts. Prokaryotic promoters include the lac, tac, T3, and T7 promoters for E. coli; 3-phosphoglycerate kinases or other glycolytic enzymes, such as enolase, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose 6-phosphate isomerase, 3-phosphoglycerate mutase, and glucokinase. Eukaryotic promoters include inducible yeast promoters such as alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, metallothionein, and enzymes involved in nitrogen metabolism or maltose / galactose utilization; viral promoters such as polyoma, fowlpox and adenovirus (e.g., adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus (especially the pre-early gene promoter), retrovirus, hepatitis B virus, actin, Rous sarcoma virus (RSV) promoter, and early or late Simian virus 40 promoter; and RNA polymerase II promoters, including non-viral promoters such as EF-1 alpha (Mizushima and Nagata (1990) Nucleic Acids Res. 18(17): 5322). Those skilled in the art will be able to select a suitable promoter for expressing a humanized antibody or a portion thereof.

[0135] If necessary, additional enhancer elements may be included, for example, for expression in higher eukaryotic cells, in place of or in conjunction with those found in the promoter described above. Suitable mammalian enhancer sequences include enhancer elements derived from globin, elastase, albumin, fetoprotein, metallothione, and insulin. Alternatively, enhancer elements derived from eukaryotic cell viruses, such as the SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, baculovirus enhancer, or mouse IgG2a locus, may be used (see WO04 / 09823). Such enhancers are often located on the vector upstream of the promoter, but may also be located elsewhere, e.g., within the untranslated region or downstream of the polyadenylation signal. The selection and placement of enhancers may be based on their compatibility with the host cell used for expression.

[0136] Furthermore, vectors (e.g., expression vectors) may include selectable markers for selecting host cells containing the vector, and, in the case of replicable vectors, origins of replication. Genes encoding antibiotic or drug resistance-contributing products are common selectable markers and are used in prokaryotes (e.g., β-lactamase genes (ampicillin resistance), tet genes (tetracycline resistance)) and eukaryotic cells (e.g., neomycin (G418 or Geneticin), gpt (mycophenolic acid), ampicillin, or hygromycin 5 resistance genes). Dihydrofolate reductase marker genes enable selection using methotrexate in various hosts. Genes encoding gene products of host nutritional requirement markers (e.g., LEU2, URA3, HIS3) are often used as selectable markers in yeast. The use of vectors that can be integrated into the genome of host cells, such as viral (e.g., baculovirus) or phage vectors, and retroviral vectors, is also envisioned.

[0137] In eukaryotes, polyadenylation and termination signals are operably linked to polynucleotides encoding the antibodies described herein. Such signals are typically located at 3' of the open reading frame. In mammalian systems, non-limiting examples of polyadenylation / termination signals include growth hormone, elongation factor 1-alpha, and viruses. Examples include those derived from the SV40) gene or retroviral long-terminal repeats. In yeast systems, non-limiting examples of polyadenylation / termination signals include those derived from phosphoglycerate kinase (PGK) and alcohol dehydrogenase 1 (ADH) genes. In prokaryotic systems, polyadenylation signals are typically not required, and instead, it is useful to use shorter, more defined terminator sequences. The selection of polyadenylation / termination sequences may be based on compatibility with the host cells used for expression. In addition to the above, other characteristics used to increase yield include chromatin remodeling elements, introns, and host cell-specific codon modifications. The codon usage frequency of antibodies described herein is modified to correspond to the host cell codon bias in order to increase transcript and / or product yield (e.g., Hoekema, A. et al. (1987) Mol. Cell Biol. 7(8):29 pp. 14-24). The selection of codons may be based on compatibility with the host cells used for expression.

[0138] This disclosure therefore relates to isolated nucleic acid molecules encoding humanized immunoglobulins, or their heavy or light chains. This disclosure also relates to isolated nucleic acid molecules encoding immunoglobulins and the antigen-binding moieties of their chains.

[0139] Humanized monoclonal antibodies, or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibodies or fragments thereof), can be produced, for example, by the expression of one or more recombinant nucleic acids encoding the antibody in a suitable host cell. The host cell can be produced using any suitable method. For example, the expression constructs described herein (e.g., one or more vectors, e.g., mammalian cell expression vectors) are introduced into a suitable host cell, and the resulting cell is maintained under conditions suitable for the expression of the construct or vector (e.g., in culture, animals, or plants). Host cells include prokaryotes such as bacterial cells, e.g., Escherichia coli (e.g., strain DH5α(trademark)) (Invitrogen, Carlsbad, CA), PerC6 (Crucell, Leiden, NL), Bacillus subtilis and / or other suitable bacteria; eukaryotic cells, e.g., fungal or yeast cells (e.g., Pichia pastris, Aspergillus species, budding yeast, fission yeast, Neurospora crassa), or other lower eukaryotic cells; and cells of higher eukaryotes, e.g., insects (e.g., Drosophila schneider S2 cells, Sf9 insect cells) (WO94 / 126087), BTI-TN-5B1-4 (High Five (trademark) insect cells (Invitrogen), mammalian cells (e.g., COS cells, e.g., COS-1 (ATCC accession number CRL-1650) and COS-7 (ATCC accession number CRL-1651), CHO (e.g., ATCC accession number CRL-9096), CHO DG44 (Urlaub, G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA, 77(7):4216-4220), 293 (ATCC accession number CRL-1573), HEK, HeLa (ATCC accession number CCL-2), CVI (ATCC accession number CCL-2), 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 derived from plants (e.g., tobacco, Lemna (duckweed), and algae).See, for example, Ausubel, FM et al., *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 (e.g., a plant or animal), but rather, for example, an isolated host cell or part of a cell culture.

[0140] Host cells are placed in spinner flasks, shaking flasks, roller bottles, wave reactors (e.g., wavebiotech.com System 1000) or hollow fiber systems. While culture can be performed in various ways, for large-scale production, agitated tank reactors or bag reactors (e.g., Wave Biotech, Somerset, New Jersey, USA) are particularly preferred for suspension culture. Agitated tank reactors can be adapted for aeration using, for example, spargers, baffles, or low-shear impellers. For bubble columns and airlift reactors, direct aeration by bubbles or oxygen bubbles is used. When host cells are cultured in serum-free culture medium, cytoprotective agents such as Pluronic F-68 are added to the medium to help prevent cell damage as a result of the aeration process. Depending on the characteristics of the host cells, microcarriers are used as growth substrates for anchorage-dependent cell lines, or the cells are adapted for suspension culture. Culture of host cells, particularly vertebrate host cells, is performed in batch, fed-batch, or repeated batch processes (Drapeau et al. (1994) Cytotechnology). (See pages 15:103-109) Various operating modes, such as extended batch processing or perfusion culture, can be utilized. Recombinant transformed mammalian host cells can be cultured in serum-containing media, such as a medium containing fetal bovine serum (FCS), but such host cells are preferably cultured in serum-free media, such as those disclosed in Keen et al. (1995) Cytotechnology 17:153-163, supplemented with energy sources such as glucose and synthetic growth factors such as recombinant insulin as needed, or in commercially available media such as ProCHO® or UltraCHO® (Cambrex NJ, USA). Serum-free culture of host cells may require that the cells be adapted to growth under serum-free conditions. One adaptation approach involves culturing such host cells in serum-containing medium and repeatedly replacing 80% of the culture medium with serum-free medium to allow the host cells to adapt to serum-free conditions (see, for example, Scharfenberg, K. et al. (1995) Animal Cell Technology: Developments Towards the 21st Century (edited by Beuvery, EC et al.), pp. 619-623, Kluwer Academic publishers).

[0141] Humanized monoclonal antibodies, or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibody or fragments thereof), according to the embodiments described, are secreted into a culture medium and recovered and purified therefrom using various methods that provide a degree of purification suitable for the intended use. For example, the use of humanized monoclonal antibodies, or humanized monoclonal antibody fragments (e.g., anti-αβTCR antibody or fragments thereof), for the treatment of human subjects typically requires at least 95% purity, more typically 98% or 99% purity, compared to the culture medium containing the therapeutic antibody, as determined by reduced SDS-PAGE. In the first example, cell residue is removed from the culture medium using centrifugation, and then the supernatant is clarified using, for example, microfiltration, ultrafiltration, and / or deep filtration. Alternatively, the humanized monoclonal antibody, or humanized monoclonal antibody fragment (e.g., anti-αβTCR antibody or fragments thereof), is recovered by microfiltration, ultrafiltration, or deep filtration without prior centrifugation. Various other methods such as dialysis and gel electrophoresis, as well as chromatographic methods such as hydroxyapatite (HA), affinity chromatography (including, optionally, affinity tag systems such as polyhistidine), and / or hydrophobic interaction chromatography (HIC) (see US5,429,746), are available. In one embodiment, after various clarification steps, a humanized monoclonal antibody, or a fragment of a humanized monoclonal antibody (e.g., anti-αβTCR antibody or a fragment thereof), is captured using protein A or protein G affinity chromatography, followed by further chromatographic steps such as ion exchange and / or HA chromatography, anion or cation exchange, size exclusion chromatography, and ammonium sulfate precipitation. Various virus removal steps are also used (e.g., nanofiltration using a DV-20 filter). After these various steps, a purified preparation containing at least 10 mg / mL, e.g., 100 mg / mL or more of the antibody described herein is provided, thus forming another embodiment described herein. A concentrate to 100 mg / mL or more is provided. It is produced by ultracentrifugation. Such preparations are substantially free of the antibody aggregation forms described herein.

[0142] Bacterial systems are particularly well-suited for the expression of antibody fragments. Such fragments are localized intracellularly or within the periplasm. Insoluble periplasmic proteins can be extracted and refolded to form active proteins by methods known to those skilled in the art. See Sanchez et al. (1999) J. Biotechnol. 72: pp. 13-20; Cupit, PM et al. (1999) Lett. Appl. Microbiol. 29: pp. 273-277.

[0143] This disclosure also relates to cells containing nucleic acids, for example, vectors described herein (e.g., expression vectors). For example, nucleic acids (i.e., one or more nucleic acids) encoding the heavy and light chains of humanized immunoglobulin according to the embodiments described, or constructs containing such nucleic acids (e.g., one or more constructs, e.g., one or more vectors), are introduced into suitable host cells by a method suitable for selected host cells (e.g., transformation, transfection, electroporation, infection), and the nucleic acids are operably ligated to one or more expression regulatory elements (e.g., in a vector incorporated into the host cell genome, in a construct produced by processing in the cell), or become operably ligated to them. The host cells are maintained under conditions suitable for expression (e.g., in the presence of inducing factors, suitable culture medium supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), thereby producing the encoded polypeptide. If desired, the encoded humanized antibody is isolated from, for example, host cells, culture medium, or milk. This process involves expression in host cells (e.g., mammary gland cells) of transgenic animals or plants (e.g., tobacco) (see, for example, WO92 / 03918).

[0144] VI. Methods for treating or preventing T-cell-mediated disorders In some situations where immunosuppression is justified and / or autoimmune conditions arise, suppression of T cell activity is desirable. Therefore, targeting of the αβTCR / CD3 complex in the treatment of diseases involving inappropriate or undesirable immune responses, such as inflammation, autoimmunity, and / or other conditions involving such mechanisms, is indicated. In one embodiment, such diseases or disorders are autoimmune and / or inflammatory diseases. Examples of such autoimmune and / or inflammatory T cell-mediated diseases include: 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 mellitus (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, and Guillain-Barré syndrome. This includes, but is not limited to, the following conditions: Goodpasture 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, Behçet's disease, primary biliary cirrhosis, uveitis, myocarditis, rheumatic fever, ankylosing spondylitis, glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, alopecia areata, and vitiligo.

[0145] In one embodiment, such disease or disorder is SLE. In one embodiment, such disease or disorder is RA. In one embodiment, such disease or disorder is IBD. In one embodiment, such disease or disorder is MS. In one embodiment, such disease or disorder is T1D.

[0146] In another embodiment, such a disease or disorder is xenotransplantation, allotransplantation, xenopregnancy, preeclampsia, or Rh disease.

[0147] In certain embodiments, the antibodies according to the described embodiments are used to suppress the target immune system and aid in transplantation. Such use reduces graft-versus-host disease (GvHD), a common complication after xenografting or allografting (including, but not limited to, transplantation of stem cells, bone marrow, tissues, body parts, and solid organs). Tissues include, but are not limited to, the cornea, sclera, bone, skin, blood vessels, and heart valves. Body parts include, but are not limited to, the face or any part thereof, one or more arms or any part thereof, one or more hands or any part thereof, one or more feet or any part thereof, and scalp or any part thereof. Solid organs include, but are not limited to, the heart, lungs, liver, kidneys, pancreas, stomach, small intestine, large intestine, testes, and ovaries. For a description of existing treatments for graft-versus-host disease, see, for example, Svennilson, Bone Marrow Transplantation (2005) 35: pp. 65-67, and the references cited therein. Advantageously, the antibodies presented in this disclosure are used in combination with other available therapies.

[0148] With regard to the treatment of autoimmune diseases, combination therapy may include administering an antibody described herein together with a drug, the drug comprising an effective amount for preventing or treating such autoimmune disease together with the antibody. If the autoimmune disease is type 1 diabetes, the combination therapy may include one or more drugs such as beta cell proliferation or survival factors or immunomodulatory antibodies that promote the proliferation of pancreatic beta cells or enhance beta cell transplantation. If the autoimmune disease is rheumatoid arthritis, the combination therapy may include one or more 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, additional drugs may be biologics such as anti-TNF agents (e.g., Enbrel®, infliximab (Remicade®), and adalimumab (Humira®)) or rituximab (Rituxan®). If the autoimmune disease is hematopoietic transplant rejection, hematopoietic growth factors (e.g., erythropoietin, G-CSF, GM-CSF, IL-3, IL-11, thrombopoietin, etc.) or antibacterial agents (e.g., antimicrobial agents, antiviral agents, antifungal agents). If the autoimmune disease is psoriasis, additional drugs may be tar and its derivatives. The additional agents may be one or more of the following: phototherapy, corticosteroids, cyclosporine A, vitamin D analogues, methotrexate, p38 mitogen-activated protein kinase (MAPK) inhibitors, and bioagents such as anti-TNF-α agents and rituximab®. If the autoimmune disease is, for example, an inflammatory bowel disease (IBD) such as Crohn's disease or ulcerative colitis, the additional agents may be one or more of the following: aminosalicylic acid, corticosteroids, immunomodulators, antibiotics, or bioagents such as Remicade® and Humira®.

[0149] Combination therapy may be carried out in any manner deemed necessary or convenient by those skilled in the art, and for the purposes of this specification, no restrictions are intended regarding the order, amount, repetition, or relative amount of compounds used in combination. Accordingly, antibodies according to the embodiments described may be formulated into pharmaceutical compositions for use in therapy.

[0150] VII. Pharmaceutical composition and administration method of anti-αβTCR antibody In certain embodiments, a pharmaceutical composition is provided comprising a humanized monoclonal antibody or a fragment of a humanized monoclonal antibody (e.g., an anti-αβTCR antibody or a fragment thereof), or a ligand identifiable by an assay method defined in the embodiments prior to this disclosure. The ligand may be an immunoglobulin, peptide, nucleic acid, or small molecule as discussed herein. They are referred to as “compounds” in the following discussion.

[0151] The pharmaceutical compositions described herein are compositions comprising a compound capable of modulating T cell activity as an active ingredient. The compound may be in any pharmaceutically acceptable salt form, or, for example, an analog, a free base form, a tautomer, an enantiomer, a racemate, or a combination thereof, as needed. The active ingredient of the pharmaceutical compositions described herein is intended to exhibit therapeutic activity when administered in a case-dependent amount, for example, in the treatment of graft-versus-host disease.

[0152] In certain embodiments, the pharmaceutical composition comprises a humanized monoclonal antibody or a humanized monoclonal antibody fragment described herein (e.g., an anti-αβTCR antibody or a fragment thereof), and a pharmaceutically acceptable carrier or diluent.

[0153] In certain embodiments, one or more compounds described herein can be used in combination with any compound known in the art that is suitable for treating a particular indication in order to treat any of the conditions described above. Thus, one or more compounds described herein can be combined with one or more compounds known in the art that is suitable for treating the aforementioned indications, so that a convenient single composition is administered to the subject. The drug regimen is adjusted to produce an optimal therapeutic response.

[0154] For example, several divided doses may be administered daily, or the dose may be reduced in proportion to the urgency of the treatment situation.

[0155] The active ingredient can be administered by a convenient method, such as orally, intravenously (if water-soluble), intramuscularly, subcutaneously, intranasally, intradermally, or via suppository route, or implanted (e.g., using a sustained-release molecule). In transplantation cases, the active ingredient can also be used to treat cells, tissues, or organs to be transplanted into the patient before transplantation. This is done, for example, to prevent or reduce the likelihood of graft-versus-host disease, or to alleviate the symptoms of graft-versus-host disease.

[0156] Depending on the route of administration, the active ingredient may need to be coated with a material that protects it from the action of enzymes, acids, and other natural conditions that could potentially inactivate it.

[0157] To administer the active ingredient by means other than parenteral administration, the active ingredient may be coated with a material that prevents its inactivation, or administered together with such material. For example, the active ingredient may be administered as an adjuvant co-administered with an enzyme inhibitor, or in liposomes. The term "adjuvant" is used most broadly and includes any immunostimulatory compound such as interferon. Adjuvants intended herein include resorcinol, nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Examples of enzyme inhibitors include pancreatic trypsin.

[0158] Liposomes include water-in-oil emulsions and conventional liposomes.

[0159] The active ingredient can also be administered parenterally or intraperitoneally.

[0160] The dispersions are also manufactured in glycerin, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these products contain preservatives to prevent microbial growth.

[0161] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions. Examples include sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and fluid enough to be easily injected. The form must be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerin, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, maintaining the required particle size in the case of dispersions, and using surfactants.

[0162] Prevention of microbial action is achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In certain cases, it may be preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of the injectable composition is achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0163] Sterile injectable solutions are prepared by combining the required amount of the active ingredient, along with some of the other components listed above as needed, in a suitable solvent, and then sterilizing by filtration. Generally, dispersions are prepared by combining the sterilized active ingredient in a sterile vehicle containing a base dispersion medium and other required components from those listed above. For sterile powders for preparing sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying, which yield the powder of the active ingredient plus any additional desired components from a pre-sterilized filtered solution.

[0164] Various other materials may be present as coating agents or to alter the physical form of the dosage unit. Naturally, any materials used in the manufacture of any dosage unit form should be pharmaceutically pure and substantially nontoxic in the amounts used. Furthermore, the active ingredient may be incorporated into sustained-release products and formulations.

[0165] As used herein, “pharmaceutically acceptable carriers and / or diluents” include any solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, isotonic and absorption retardant agents, etc. In certain embodiments, the 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. Their use in therapeutic compositions is intended unless any conventional media or agent is incompatible with the active ingredient. Auxiliary active ingredients may also be incorporated into the composition.

[0166] Formulating parenteral compositions in dosage unit form is particularly advantageous for ease of administration and uniformity of dosage. Where used herein, dosage unit form refers to a physically distinct unit suitable as a single dosage form for the mammalian subject to be treated; each unit contains a predetermined amount of the active substance calculated to produce the desired therapeutic effect in relation to the required pharmaceutically acceptable carrier. The specifications of the novel dosage unit forms described herein are determined and directly depend on (a) the inherent properties of the active substance and the specific therapeutic effect to be achieved, and (b) limitations specific to the field of the compound, such as the active substance for the treatment of diseases in organisms having diseases or conditions that impair the health of the body. The main active ingredient is formulated with a suitable pharmaceutically acceptable carrier in the dosage unit form for convenient and effective administration in an effective amount. In the case of compositions containing auxiliary active ingredients, the dosage is determined with reference to the usual dose and mode of administration of the said ingredient.

[0167] The dosage is not limited to the following, but may include: 0.01 mg / kg, 0 .02mg / kg、0.03mg / kg、0.04mg / kg、0.05mg / kg、0.06mg / kg、0.07mg / kg、0.08mg / kg、0.09mg / kg、0.10mg / kg、0.2mg / kg、0.3mg / kg、0.4mg / kg、0.5mg / kg、0.6mg / kg、0.7mg / kg、0.8mg / kg、0.9mg / kg、1.0mg / kg、1.1mg / kg、1.2mg / kg、1.3mg / kg、1.4mg / kg、1.5mg / kg、1.6mg / kg、1.7mg / kg、1.8mg / kg、1.9mg / kg、2.0mg / kg、2.1mg / kg、2.2mg / kg、2.3mg / kg、2.4mg / kg、2.5mg / kg、2.6mg / kg、2.7mg / kg、2.8mg / kg、2.9mg / kg、3.0mg / kg、3.1mg / kg、3.2mg / kg、3.3mg / kg、3.4mg / kg、3.5mg / kg、3.6mg / kg、3.7mg / kg、3.8mg / kg、3.9mg / kg、4.0mg / kg、4.1mg / kg、4.2mg / kg、4.3mg / kg、4.4mg / kg、4.5mg / kg、4.6mg / kg、4.7mg / kg、4.8mg / kg、4.9mg / kg、5.0mg / kg、5.1mg / kg、5.2mg / kg、5.3mg / kg、5.4mg / kg、5.5mg / kg、5.6mg / kg、5.7mg / kg、5.8mg / kg、5.9mg / kg、6.0mg / kg、6.1mg / kg、6.2mg / kg、6.3mg / kg、6.4mg / kg、6.5mg / kg、6.6mg / kg、6.7mg / kg、6.8mg / kg、6.9mg / kg、7.0mg / kg、7.1mg / kg、7.2mg / kg、7.3mg / kg、7.4mg / kg、7.5mg / kg、7.6mg / kg、7.7mg / kg、7.8mg / kg、7.9mg / kg、8.0mg / kg、8.1mg / kg、8.2mg / kg、8.3mg / kg、8.4mg / kg、8.5mg / kg、8.6mg / kg、8.7mg / kg、8.8mg / kg、8.9mg / kg、9.0mg / kg、9.1mg / kg、9.2mg / kg、9.3mg / kg、9.4mg / kg、9.5mg / kg、9.6mg / kg、9.7mg / 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、10.8m g / 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、1 2.7mg / 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、13.9m g / 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、16.4 mg / 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、1 8.3mg / 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、19.5m g / kg、19.6mg / kg、19.7mg / kg、19.8mg / kg、19.9mg / kgおよび20.0mg / kgを、0.0.1 mg / kg ~ 20 mg. g / kg.

[0168] Dosages are not limited to, but include: approximately 100 ng / kg, 200 ng / kg, 300 ng / kg, 400 ng / kg, 500 ng / kg, 600 ng / kg, 700 ng / kg, 800 ng / kg, 900 ng / kg, 1 microgram / kg, 2 micrograms / kg, 3 micrograms / kg, 4 micrograms / kg, 5 micrograms / kg, 6 micrograms / kg, 7 micrograms / kg, 8 micrograms / kg, 9 micrograms / kg, and 10 micrograms / kg, ranging from approximately 100 ng / kg to approximately 0.01 mg / kg.

[0169] To facilitate the delivery of antibody-containing peptide compounds to cells, peptides are modified to improve their ability to cross the cell membrane. For example, US5,149,782 discloses the use of fusion peptides, ion channel-forming peptides, membrane peptides, long-chain fatty acids, and other membrane additives to increase protein transport across the cell membrane. These and other methods are also described in WO97 / 37016 and US5,108,921, which are incorporated herein by reference.

[0170] In a further embodiment, the active ingredients described herein are provided for use in the treatment of a disease, either alone or in combination with compounds known in the art to be suitable for treating a particular indication. As a result, the use of the active ingredients described herein is provided for the manufacture of pharmaceuticals for treating diseases associated with abnormal immune responses.

[0171] Furthermore, methods are provided for treating conditions associated with abnormal immune responses, including administering a therapeutically effective dose of a ligand identifiable using the assay methods described above.

[0172] The examples provided below are for illustrative purposes only and should not be considered as limitations on the compositions and methods described herein. [Examples]

[0173] Accelerated stability testing of VH31 anti-αβTCR antibody preparation. Accelerated stability tests were performed on various formulations of the VH31 antibody. The reference or control VH31 antibody was a wild-type humanized anti-human αβTCR antibody from the GL1BM series, containing a heavy chain variable domain with the amino acid sequence described as SEQ ID NO: 16 and a light chain variable domain with the amino acid sequence described as SEQ ID NO: 14 (Table 1). No fragmentation was observed in the liquid formulation after 4 months at a refrigerated temperature of 4°C or below. However, fragmentation was detected in the VH31 liquid formulation under accelerated conditions, including a 5-week period of increased temperature and pH (i.e., 45°C; pH 4.0-8.0). It was noted that the presence of two low molecular weight (LMW) bands appeared to correlate with the disappearance of the band corresponding to the light chain (LC) of the VH31 antibody, implicitly indicating light chain fragmentation of the observed LMW source (Figure 1, arrows). [Examples]

[0174] Cell-based assays for evaluating the efficacy of stressed formulations The VH31 anti-αβTCR antibody selectively depletes activated T cells. A cell-based efficacy assay was performed to determine whether VH31 formulations exposed to accelerated conditions retained this ability. The efficacy assay was based on the activation of T cell receptors and subsequent binding of the anti-αβTCR antibody, which stimulates the apoptotic pathway.

[0175] Jarcut T lymphocytes express T cell receptor complexes. The efficacy assay is Lucifer We utilized a Jurcutt T cell line containing a nuclear factor-of-transaction (NFAT) response element linked to the luciferase receptor of activated T cells. Using this modified cell line, we investigated T cell activation and subsequent inhibition of the activation signal by an anti-αβTCR antibody. Activation of the T cell receptor complex stimulates the NFAT pathway, leading to luciferase production. Luciferase production was then measured using a luciferase substrate (e.g., luciferin) and a luminescence detector. The level of activation is proportional to luciferase production. Immobilized anti-CD3 activates Jurcutt T cells, as measured by induction of the NFAT pathway and subsequent luciferase production in NFAT-luciferase Jurcutt cells. Activation is dose-dependently inhibited by an anti-αβTCR antibody. This is a qualitative measure of anti-αβTCR efficacy.

[0176] A qualitative efficacy assay was performed in a 96-well plate format. The 96-well plates were coated with anti-CD3 1 μg / mL overnight at 2-8°C. The plates were washed, and NFAT-luciferase jar-cut T lymphocytes (5 × 10⁶) were collected. 4 Cells (per well) were added to anti-CD3 coated plates. To activate the T cell receptor complex, cells were incubated with anti-CD3 at 37°C and 5% CO2 for 5 hours. Each cell condition was then incubated for a further 18 hours with multiple levels (0.003–200 μg / mL) of anti-αβTCR (control and sample). Negative anti-CD3 (0 μg / mL) control and anti-αβTCR (0 μg / mL) control were included in each condition. After the second incubation step, the anti-αβTCR diluent was removed and cells were lysed with cell culture lysis reagent. Luciferase production of the lysates was measured using luciferase substrate and a luminescence plate reader. Higher doses of anti-αβTCR antibody were expected to result in lower luminescence signals indicating inhibition of the activation response. Dose-response data were plotted using Softmax Pro software, and sample dose-response curves were visually compared with control dose-response curves.

[0177] The graph in Figure 2 shows data from cell-based efficacy assays. VH31 antibody preparations exposed to accelerated conditions (i.e., 45°C; pH 8.0) for 5 weeks (white square symbols) showed a significant loss of efficacy compared to VH31 preparations exposed to less severe conditions (i.e., 45°C; pH 5.0) (white circle symbols) or control conditions (i.e., ≤0°C; pH 5.0; white diamond symbols) for 5 weeks. This study demonstrated a relationship between LMW species appearance and loss of antibody efficacy, which may be due to light chain fragmentation. [Examples]

[0178] Analysis of antibody fragments The antibody fragments were further analyzed by molecular weight estimation of the observed bands, as well as N-terminal sequencing and peptide mapping (Figure 3). Chemical and enzymatic cleavage of polypeptides is known to occur between Asn(N) and Pro(P) residues. Therefore, based on sequencing and mapping, light chain amino acid residues N93 and P94 were identified as the estimated site of light chain clipping and the source of the identified fragments (Figure 4). To further confirm this, the VH31 light chain (P7) was subjected to trypsin digestion at pH 4.0–8.0. The graph in Figure 5 shows various light chain fragments that would be expected if enzymatic clipping occurred at N93 / P94. These fragments correspond to amino acid residues 94–102 (Figure 5A), 61–93 (Figure 5B), and 94–106 (Figure 5C). The two C-terminal products corresponding to amino acids 94–102 and 94–106 were due to incomplete trypsin digestion. As expected, the increase in pH was associated with the predicted increase in fragments.

[0179] Asn(N) is known to be convertible to Asp(D) in solution, for example, by deamidation of the Asn side chain (Figure 6). To better understand the mechanism of LC fragmentation, we analyzed the LC fragments to determine whether residue 93 is N or D. The isotopic distribution revealed that most of the 61-93 peptide fragments ended in Asn. No separate peak corresponding to D93 was observed, indicating that the conversion from Asn to Asp and subsequent Asp / Pro cleavage are related to VH31 LC This demonstrated that it was not the primary cause of fragmentation. For these reasons, subsequent modification stability tests focused on residues N93 / P94. [Examples]

[0180] Mutagenesis of light chain (LC) variants A mutagenesis approach was employed to modify the VH31 light chain variant to improve stability and resistance to fragmentation. Previous attempts to remove the Asn clipping site of an unrelated antibody (i.e., sFLT01) did not prevent antibody fragmentation. Further complicating matters, it was noted that amino acid residues N93 and P94 are located within the light chain complementarity-determining region 3 (LC CDR3). Considering the above, a light chain variant was rationally designed with the aim of simultaneously maintaining antigen-binding affinity and antibody potency while enhancing light chain stability.

[0181] The amino acid Pro(P) has very little freedom in the folded protein structure. Therefore, it was assumed that replacing Pro at residue 94 without destabilizing the conformation of LC CDR3 might be difficult. Ala(A) is commonly used in mutagenesis due to its physical properties. As a starting point, residues N93 and P94 were replaced with A individually or simultaneously. Furthermore, several other substitutions were made at residue N93 based on amino acid properties that were considered compatible, such as charge, size, H bond, polarity vs. nonpolarity, etc. Figure 7 shows the various amino acid substitutions that were made and tested.

[0182] A wild-type anti-αβTCR VH31 expression vector was used as both a template and expression control for mutagenesis. Eight primer sets were designed, and PCR mutagenesis was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). Mutant DNA was then confirmed by DNA sequencing. [Examples]

[0183] Expression and analysis of LC variants Mutant and wild-type (WT) DNA were transfected into an Expi293 transient expression system. The conditioned medium was collected 4 days after transfection, and expression levels were measured by the Octet Protein A assay. The results (shown in Table 2) showed good expression for αβTCR wild-type (114 μg / mL medium) and 7 out of 8 mutants (>45 μg / mL medium). One mutant (N93A) was expressed at a relatively low level (16 μg / mL). Transfection was scaled up to 30 mL for all 8 mutants. The conditioned medium was collected for protein A purification.

[0184] The conditioned medium was passed through a 1 mL HiTrap Protein A HP(GE) column for purification. The column was equilibrated with 5 column volumes of PBS pH 7.2 (Gibco). The medium was packed at a flow rate of 0.5 mL / min, and then eluted with 20 column volumes (CV) of PBS before elution. The samples were washed with pH 7.2. The antibody was eluted in 10 mM succinate, pH 3.75, and immediately after elution, the pH was adjusted to 5.5 with 0.2 M sodium hydroxide. All samples were filtered through a 0.2 μm low-protein bound membrane equipped with a syringe filter (recovery rate 50%~80%), and the samples were concentrated using Amicon YM30 (recovery rate 100%). The results are summarized in Table 2.

[0185] [Table 4]

[0186] The purified antibody was treated with Mini-PROTEAN TGX Stain-Free Gels (Bio-Rad) at a load of 1.5 μg per well. The results showed that all tested mutants were comparable to wild-type mutants and possessed good purity (Figure 8). The purified mutants were further characterized by stability and functional assays.

[0187] Transfection of the αβTCR N93S mutant was performed on a 60 ml scale using the Expi293 transient expression system. The conditioned medium was collected 4 days after transfection. The expression level was measured by the Octet Protein A assay and determined to be 37 μg / mL.

[0188] A HiTrap Protein A HP(GE) column (1 mL) was used for purification. The column was equilibrated with 5 CV of PBS pH 7.2 (Gibco). The acclimatization medium sample was packed at a flow rate of 0.5 mL / min and then washed with 20 CV of PBS pH 7.2 before elution. The antibody was eluted in 5 CV of 10 mM succinate, pH 3.75 (total 5 mL). The yield was nearly 100%. The eluted antibody was adjusted to pH 5.5 with 0.2 M sodium hydroxide after elution. The solution became slightly turbid at pH 5.5 and was filtered through a 50 mL tube-top filter (0.2 μm CA, low protein binding). The filtration recovery was 77% (total 1.7 mg), probably due to relatively large volume of loss. The purified antibody and αβTCR VH31 control were treated with Mini-PROTEAN TGX Stain-Free Gels (2 μg load per well). The results showed that all tested mutants were comparable to the VH31 control. Purified N93S and pre-purified WT and VH31 controls were treated with SEC-HPLC, and the profiles are shown in Figure 9. The SEC profiles for both the WT and N93S mutants appeared to be comparable to those of the VH31 control. [Examples]

[0189] Assay for evaluating the efficacy of LC variants The qualitative efficacy assay described in Example 2 was repeated with an antibody containing the variant light chain. The wild-type and control antibodies contained the same VH31 amino acid sequence but were prepared from different preparations. The wild-type VH31 antibody was prepared together with the LC variant antibody, while the control antibody was prepared from a separate large-scale preparation. As shown in Figure 10, the antibody containing the LC variant N93S... The antibodies showed only slight decreased potency compared to either wild-type VH31 or the control antibody. In contrast, antibodies containing LC variants N93Q, N93A, and N93A / P94A all showed significantly decreased potency compared to any of the aforementioned antibodies. Based on these results, the N93S variant was selected as a candidate for accelerated stability testing. [Examples]

[0190] Accelerated stability testing of LC variants Antibodies containing the LC variant N93S were subjected to elevated temperature (i.e., 45°C) and pH (i.e., pH 8.0) for 6 weeks to determine whether the N93S amino acid substitution stabilized LC and prevented fragmentation. After 6 weeks at elevated temperature and pH 8.0, fragmentation was observed in wild-type, reference VH31, and N93S antibodies. However, the N93S variant showed significantly reduced degradation compared to either the wild-type or VH31 control antibody (Figure 11). In particular, the N93S variant showed significantly reduced loss of potency under accelerated conditions (3-fold loss) compared to either the wild-type or VH31 control antibody (both showing more than 20-fold loss). At pH 5.5, which is closer to the pH of the hypothetical antibody drug formulation than pH 8.0, the extent of fragmentation was significantly less at 6 weeks (Figure 12).

[0191] The results described above showed that the N93S variant exhibited improved LC stability and efficacy compared to the VH31αβTCR antibody under storage conditions including elevated temperature and elevated pH. These results were surprising, as previous attempts to remove the Asn clipping site from an unrelated antibody (i.e., sFLT01) did not prevent antibody fragmentation.

Claims

1. A binding polypeptide that specifically binds to the human αβTCR / CD3 complex, comprising a heavy chain variable region, a light chain variable region, and a constant region: The light chain variable region includes three complementarity-determining regions (CDRs), LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences described in SEQ ID NOs: 26, 27, and 28, respectively; Sequence ID 28 has the amino acid sequence Q-Q-W-S-S-X １ -X ２ -Includes L-T (where X １ is an amino acid selected from the group consisting of Q, D, H, S, Y, and A, and X ２ ( is an amino acid selected from the group consisting of P and A); The conjugated polypeptide has a constant region of human origin.

2. X １ The bound polypeptide according to claim 1, wherein is S.

3. X ２ The conjugated polypeptide according to claim 1, wherein is P.

4. X １ is S, X ２ The conjugated polypeptide according to claim 1, wherein is P.

5. The conjugated polypeptide according to any one of claims 1 to 4, wherein the light chain variable region further comprises the human light chain framework region described in Sequence ID No.

14.

6. The bound polypeptide is the bound polypeptide according to any one of claims 1 to 5, wherein the bound polypeptide exhibits improved stability at a pH above 5.0 compared to VH31.

7. The bound polypeptide is the bound polypeptide according to any one of claims 1 to 6, wherein the bound polypeptide exhibits improved stability at temperatures above 4°C compared to VH31.

8. The conjugated polypeptide according to any one of claims 1 to 7, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 12, 13, 15, and 16.

9. The conjugated polypeptide according to any one of claims 1 to 7, wherein 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.

10. The conjugated polypeptide according to any one of claims 1 to 7, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO:

16.

11. The conjugated polypeptide according to any one of claims 1 to 7, wherein the heavy chain variable region includes the amino acid sequence described as SEQ ID NO:

15.

12. The conjugated polypeptide according to any one of claims 1 to 7, wherein the heavy chain variable region includes the amino acid sequence described as SEQ ID NO:

16.

13. The binding polypeptide according to any one of claims 1 to 12, wherein the constant region comprises an Fc modification having a modified glycosylation pattern that reduces Fcγ receptor binding.

14. The conjugated polypeptide according to claim 13, wherein the Fcγ receptor is selected from the group consisting of FcγRIIIa and FcγRI.

15. The Fc modification is selected from the group consisting of N297Q / S298N / Y300S, S298N / T299A / Y300S, and S298N / Y300S, as described in claim 13. Synthetic polypeptide.

16. The conjugated polypeptide according to claim 13, wherein the Fc modification is N297Q / S298N / Y300S.

17. The conjugated polypeptide according to claim 13, wherein the Fc modification is S298N / T299A / Y300S.

18. The conjugated polypeptide according to claim 13, wherein the Fc modification is S298N / Y300S.

19. The conjugated polypeptide according to claim 1, wherein the conjugated polypeptide is humanized.

20. The conjugated polypeptide according to any one of claims 1 to 19, wherein the conjugated polypeptide is a monoclonal antibody.

21. The conjugated polypeptide according to any one of claims 1 to 19, wherein the conjugated polypeptide is multispecific.

22. The conjugated polypeptide according to claim 21, wherein the conjugated polypeptide is bispecific.

23. A pharmaceutical composition comprising a bound polypeptide according to any one of claims 1 to 22 and a pharmaceutically acceptable carrier or diluent.

24. A formulation comprising the pharmaceutical composition described in claim 23, wherein the formulation is selected from the group consisting of lyophilized formulations and liquid formulations.

25. A method for treating a subject for a T cell-mediated disease or disorder, comprising administering an effective amount of the conjugated polypeptide described in any one of claims 1 to 22 to the subject.

26. T-cell-mediated diseases or disorders include 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 syndrome, Addison's disease, Wegener's granulomatosis, and primary The method according to claim 25, selected from the group consisting of biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, polymyalgia rheumatica, Raynaud's phenomenon, temporal arteritis, giant cell arteritis, autoimmune hemolytic anemia, pernicious anemia, polyarteritis nodosa, Behçet'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.

27. A nucleic acid encoding a conjugated polypeptide according to any one of claims 1 to 22.

28. A vector comprising the nucleic acid described in claim 27.

29. A cell expressing the nucleic acid described in claim 27.

30. The cell according to claim 29, wherein the cell is a mammalian cell.

31. The mammalian cells according to claim 30, wherein the mammalian cells are selected from the group consisting of Chinese hamster ovary (CHO) cells and human fetal kidney (HEK) cells.

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