CD3-targeting antibodies or their antigen-binding fragments and their applications

Abstract

This invention relates to an anti-CD3 antibody or its antigen-binding fragment that specifically binds to CD3 in humans and non-human primates. The anti-CD3 antibody or its antigen-binding fragment has enhanced or reduced affinity, enabling improved efficacy or reduced toxicity. Furthermore, it allows for selective adaptation to the target when constructing a bispecific antibody, providing flexible adaptation design in drug development. The invention also relates to conjugates, fusions, bispecific antibodies, or pharmaceutical compositions containing the above-mentioned anti-CD3 antibody or its antigen-binding fragment. The invention also relates to nucleic acids encoding the antibody, host cells containing the nucleic acid, and methods for producing the antibody. Furthermore, the invention relates to the use of these CD3-binding antibodies for the prevention or treatment of cancer, infectious diseases, or autoimmune diseases in subjects.

Description

[Technical Field]

[0001] The present invention relates to the field of antibody drugs. More specifically, the present invention relates to an anti-CD3 antibody that specifically binds to human and non-human primate CD3 and activates T cells with reduced activating activity, as well as a conjugate, fusion, or bispecific antibody containing the anti-CD3 antibody or its antigen-binding fragment. Furthermore, the present invention relates to a nucleic acid encoding the anti-CD3 antibody and a host cell containing the nucleic acid, as well as a method for producing the antibody. The present invention also relates to a pharmaceutical composition containing the anti-CD3 antibody and the medical use of the anti-CD3 antibody. [Background technology]

[0002] The body's immune system is composed of central lymphoid organs, peripheral lymphoid organs, immune cells, and immune molecules. The process of the immune response depends on intercellular interactions within the immune system, namely direct contact and interactions mediated by cytokines and other mediators.

[0003] Immune cells, particularly T cells, play a central role in the entire immune response. The surface of T cells contains numerous CD molecules, including CD3, CD4, CD8, and CD28, which are broadly involved in all immune effects, such as antigen recognition, activation, proliferation, unresponsiveness, apoptosis, removal of foreign antigens, and tolerance to self-antigens.

[0004] Therefore, attempts have been made to improve the body's immunity by specifically binding monoclonal antibodies to CD molecules and regulating the function and state of T cells.

[0005] Among these CD molecules, the CD3 molecule is particularly important. The CD3 molecule is a protein complex that is normally expressed on the surface of mature T cells and is composed of four types of polypeptide chains: γ, δ, ε, and ζ. They form three parts: an extracellular region, a transmembrane region, and a cytoplasmic region. Among them, the N-terminal extracellular domain contains two heterodimeric structural domains, CD3εγ and CD3εδ. On the other hand, the transmembrane domain is a single homodimer CD3ζζ, which non-covalently binds to the α / β chains of the T cell receptor (TCR) via salt bridges to form the TCR-CD3 complex. This complex is responsible for the function of the signal transduction cascade in T cell activation.

[0006] Many therapeutic strategies aim to regulate T cell immunity by targeting TCR signal transduction. Particularly in immunosuppressive therapy, anti-human CD3 monoclonal antibodies (mAbs) are widely used clinically.

[0007] The specific mouse anti-human CD3 monoclonal antibody OKT3 that recognizes the ε chain of the human CD3 molecule is the first monoclonal antibody permitted for use in humans (Sgro, Toxicology 105 (in 1995), 23-29). OKT3 is widely used clinically as an immunosuppressant (Chatenoud, Clin. Transplant 7 (in 1993), 422-430; Chatenoud, Nat. Rev. Immunol. 3 (in 2003), 123-132; Kumar, Transplant. Proc. 30 (in 1998), 1351-1352).

[0008] According to the literature reports, OKT3 can effectively promote the mitosis of T cells (Van Wauve, J. Immunol. 124 (1980), 2708-2718), and can also induce the cytotoxic function of T cells (Wong, Transplantation 50 (1990), 683-689). OKT3 shows a dual activity of activation or inhibition on T cells in a time-dependent manner. Stimulation by OKT3 at the initial stage of T cells causes T cell activation accompanied by cytokine release, but subsequent administration blocks all known T cell functions. Due to such an inhibitory effect on T cell functions in the later stage, OKT3 is widely applied as an immunosuppressant and is used to reduce or eliminate rejection reactions in xenotransplantation. CD3 antibodies can be used not only as immunosuppressants but also as T cell agonists to overcome the immune escape of tumor cells. This is also an important reason why a large number of CD3 bispecific antibodies are currently being developed as anti-tumor drugs.

[0009] However, studies have revealed that many CD3 antibodies have species specificity and recognize only a single site or epitope on the target CD3 molecule.

[0010] For example, OKT-3 reacts with chimpanzee CD3 but does not react with the CD3 homolog of rhesus monkeys, which are also primates. Also, the anti-CD3 monoclonal antibody UCHT-1 reacts with chimpanzee CD3 but does not react with rhesus monkey CD3. Furthermore, it has been reported that the monoclonal antibody FN-18 recognizes only the rhesus monkey CD3 antigen and does not react with the human CD3 counterpart (Uda et al., J. Med. Primatol. 30(2001), 141-147). KLINGER et al. discovered the CD3 monoclonal antibody H2C (WO2008119567A2). This antibody can bind to both human and non-chimpanzee CD3ε chains and possesses a binding domain that exhibits cross-species specificity. This indicates that the antibody can be used in both preclinical animal studies and human clinical studies. The study found that the epitope to which the monoclonal antibody H2C binds is a polypeptide fragment consisting of amino acid residues 1-27 of the N-terminus of the CD3ε extracellular domain. Unlike other CD3ε epitopes known in this field, this epitope retains its three-dimensional structure.

[0011] Currently, CD3 + More than 100 bispecific antibodies targeting T cells are currently in clinical trials. While bispecific antibodies have shown significant progress in terms of safety and efficacy, they also face numerous challenges.

[0012] For example, an anti-CD3 antibody, which is a component of a bispecific antibody, is a CD3 antibody. + Excessive activation of T cells can lead to abnormal cytokine secretion and potentially induce cytokine release syndrome, resulting in CD3 bispecific antibodies exhibiting high toxicity and side effects.

[0013] Therefore, in this field, there is a strong demand for anti-CD3 antibodies with different properties, which can enhance the efficacy or reduce the toxicity of bispecific antibodies. [Overview of the project]

[0014] Through their research, the inventors obtained a group of interspecies anti-CD3 antibodies that activate T cells while reducing their activating activity. These antibodies possess one or more of the following properties: (i) When measured by SEC-HPLC, the purity of the monomer is 90% or higher, preferably 92%, 94%, 96%, or 97% or higher. (ii) When measured by reduced or non-reduced SDS-PAGE, the purity is 90% or higher, preferably 91%, 92%, or 93% or higher. (iii) It specifically binds to CD3 expressed on the cell surface. In firstly, the present invention provides an antibody or antigen-binding fragment that specifically binds to CD3.

[0015] The antibody or fragment contains one of the following: (a) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:28 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:29; (b) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:1 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:31; (c) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:32 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:33; (d) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:1 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:34; (e) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:28 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:36; (f) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:39 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:40; or (g) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:28 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:43.

[0016] In some embodiments, the CD3-specific binding antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region. (a) The heavy chain variable region contains the following sequence based on the AbM number: HCDR1:GFTFSKYAMN(SEQ ID NO:51) HCDR2:RIRSKYNNYATY(SEQ ID NO:46) HCDR3:HGNFGNSYISYWAY(SEQ ID NO:47) The light chain variable region contains the following sequence based on the AbM number: LCDR1:GSSTGAVTSGYYPN(SEQ ID NO:48) LCDR2:GTKFLAP (SEQ ID NO:49) LCDR3:ALWYSNRWV(SEQ ID NO:50)

[0017] (b) The heavy chain variable region contains the following sequence based on the AbM number: HCDR1:GFTFNKYAMN(SEQ ID NO:45) HCDR2:RIRSKYNNYATY(SEQ ID NO:46) HCDR3:HGNFGNSYISYWAY(SEQ ID NO:47) The light chain variable region contains the following sequence based on the AbM number: LCDR1:GSSTGAVTSGYYPN(SEQ ID NO:48) LCDR2:GTKFLAP (SEQ ID NO:49) LCDR3:ALWVDNRWV(SEQ ID NO:55)

[0018] (c) The heavy chain variable region includes HCDR1, indicated by GFTFPKYAMN (SEQ ID NO: 52), HCDR2, indicated by RIRSKYNNYETY (SEQ ID NO: 53), and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47), based on AbM number; the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48), LCDR2, indicated by GTHFLAP (SEQ ID NO: 54), and LCDR3, indicated by ALWYSNRWV (SEQ ID NO: 50), based on AbM number;

[0019] (d) The heavy chain variable region includes HCDR1, indicated by GFTFNKYAMN (SEQ ID NO: 45), HCDR2, indicated by RIRSKYNNYATY (SEQ ID NO: 46), and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47), based on AbM number; the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48), LCDR2, indicated by GTKFLAP (SEQ ID NO: 49), and LCDR3, indicated by ALWQENRWV (SEQ ID NO: 56), based on AbM number;

[0020] (e) The heavy chain variable region includes HCDR1, indicated by GFTFSKYAMN (SEQ ID NO: 51), HCDR2, indicated by RIRSKYNNYATY (SEQ ID NO: 46), and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47), based on AbM number; the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48), LCDR2, indicated by GTKFLAP (SEQ ID NO: 49), and LCDR3, indicated by ALWVDNRWV (SEQ ID NO: 55), based on AbM number;

[0021] (f) The heavy chain variable region includes HCDR1, indicated by GFTFSKYAMN (SEQ ID NO: 51), HCDR2, indicated by RIRSKYNNYETY (SEQ ID NO: 53), and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47), based on AbM number; the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48), LCDR2, indicated by GTHFLAP (SEQ ID NO: 54), and LCDR3, indicated by ALWYSNRWV (SEQ ID NO: 50), based on AbM number; or

[0022] (g) The heavy chain variable region includes HCDR1, indicated by GFTFSKYAMN (SEQ ID NO: 51), HCDR2, indicated by RIRSKYNNYATY (SEQ ID NO: 46), and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47), based on AbM number; the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48), LCDR2, indicated by GTKFLAP (SEQ ID NO: 49), and LCDR3, indicated by ALWQENRWV (SEQ ID NO: 56), based on AbM number.

[0023] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CD3 of the present invention comprises a heavy chain variable region and a light chain variable region, and the antibody or antigen-binding fragment comprises the following: (a) Heavy chain variable region shown in SEQ ID NO:26 and light chain variable region shown in SEQ ID NO:27; (b) Heavy chain variable region shown in SEQ ID NO:28 and light chain variable region shown in SEQ ID NO:29; (c) Heavy chain variable region shown in SEQ ID NO:30 and light chain variable region shown in SEQ ID NO:2; (d) Heavy chain variable region shown in SEQ ID NO:1 and light chain variable region shown in SEQ ID NO:31; (e) Heavy chain variable region shown in SEQ ID NO:32 and light chain variable region shown in SEQ ID NO:33; (f) Heavy chain variable region shown in SEQ ID NO:1 and light chain variable region shown in SEQ ID NO:34; (g) Heavy chain variable region shown in SEQ ID NO:30 and light chain variable region shown in SEQ ID NO:27; (h) Heavy chain variable region shown in SEQ ID NO:35 and light chain variable region shown in SEQ ID NO:29; (i) Heavy chain variable region shown in SEQ ID NO:30 and light chain variable region shown in SEQ ID NO:29; (j) Heavy chain variable region shown in SEQ ID NO:28 and light chain variable region shown in SEQ ID NO:36; (k) Heavy chain variable region shown in SEQ ID NO:30 and light chain variable region shown in SEQ ID NO:31; (l) Heavy chain variable region shown in SEQ ID NO:37 and light chain variable region shown in SEQ ID NO:38; (m) Heavy chain variable region shown in SEQ ID NO:39 and light chain variable region shown in SEQ ID NO:40; (n) Heavy chain variable region shown in SEQ ID NO:41 and light chain variable region shown in SEQ ID NO:33; (o) Heavy chain variable region shown in SEQ ID NO:26 and light chain variable region shown in SEQ ID NO:42; (p) Heavy chain variable region shown in SEQ ID NO:28 and light chain variable region shown in SEQ ID NO:43; or (q) The heavy chain variable region shown in SEQ ID NO:30 and the light chain variable region shown in SEQ ID NO:44.

[0024] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CD3 in the present invention is an IgG1, IgG2, IgG3, or IgG4 antibody, optionally an IgG1 or IgG4 antibody, and even more optionally an IgG1 antibody. In some embodiments, the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, single-chain Fv, single-chain Fab, or diabody.

[0025] In a second aspect, the present invention provides a nucleic acid encoding an antibody according to a first aspect of the present invention, a vector containing the nucleic acid encoding the antibody, a host cell containing the nucleic acid molecule or vector, and a method for producing the antibody. The method includes culturing a host cell into which an expression vector containing the nucleic acid has been introduced under conditions capable of expressing the nucleic acid encoding an antibody or antigen-binding fragment that specifically binds to the CD3 molecule as described in the first aspect of the present invention. Optionally, the method further includes recovering the antibody or antigen-binding fragment that specifically binds to the CD3 molecule from the host cell culture. Preferably, the host cell is a prokaryote or a eukaryote, more preferably selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for producing the antibody or its antigen-binding fragment, most preferably the host cell is a HEK293 cell or a CHO cell.

[0026] In a third aspect, the present invention relates to a conjugate, fusion protein, or bispecific antibody comprising an anti-CD3 antibody or its antigen-binding fragment according to the first aspect of the present invention.

[0027] In a fourth aspect, the present invention relates to a pharmaceutical composition comprising an anti-CD3 antibody or its antigen-binding fragment according to the first aspect of the present invention, or a conjugate, fusion protein, or bispecific antibody according to the third aspect of the present invention, and optionally further comprising a pharmaceutically acceptable carrier.

[0028] In a fifth aspect, the present invention relates to the use of an anti-CD3 antibody or its antigen-binding fragment according to the first aspect of the present invention, a conjugate, fusion protein or bispecific antibody according to the third aspect of the present invention, or a pharmaceutical composition according to the fourth aspect of the present invention, and is used in the manufacture of pharmaceuticals for the prevention or treatment of cancer, infectious diseases or autoimmune diseases in subjects.

[0029] The advantageous effect of the present invention is to provide anti-CD3 antibodies with different affinities, thereby enabling enhanced drug efficacy or reduced toxicity. Furthermore, when constructing bispecific antibodies, selective adaptation according to the target is possible, providing flexible design options in drug development. [Brief explanation of the drawing]

[0030] By reading the following drawings together with the preferred embodiments of the present invention, the understanding of the present invention, which is described in detail below, will become even clearer. For the purpose of illustrating the present invention, the drawings show currently preferred embodiments. However, it should be understood that the present invention is not limited to the exact configuration and means of the embodiments shown in the drawings. [Figure 1] Figure 1 shows that the antibody H2C(IgG1) binds to the antigen huCD3E-AA1-27-huFc constructed and expressed in Example 1. [Figure 2]Figures 2A to 2D show the results of FACS binding of antibodies to huCD3D-CD3E-CHO-K1 cells or Jurkat cells. Figure 2A shows the results of binding of P1-3-scFv-LH, P2-3-scFv-LH, 63-scFv-LH, 78-scFv-LH, and 79-scFv-LH to huCD3D-CD3E-CHO-K1 cells. Figure 2B shows the results of binding of P3-1-scFv-LH to huCD3D-CD3E-CHO-K1 cells. Figure 2C shows the results of binding of P1-3-3-scFv-LH, P2-3-3-scFv-LH, P2-3-5-scFv-LH, 63-P2-3-scFv-LH, 63-P3-1-scFv-LH, and 78-P1-3-scFv-LH to Jurkat cells. Figure 2D shows the results of binding of 78-P2-3-scFv-LH, 78-P3-1-scFv-LH, 79-P1-3-scFv-LH, and 79-P2-3-scFv-LH to Jurkat cells. [Figure 3] Figure 3 shows the results of FACS-based binding of antibodies 63-P2-3-scFv-LH, 63-P3-1-scFv-LH, 78-P1-3-scFv-LH, 78-P2-3-scFv-LH, 78-P3-1-scFv-LH, 79-P1-3-scFv-LH, and 79-P2-3-scFv-LH to CynoCD3E-HEK293 cells. [Figure 4] Figures 4A to 4Q are based on BLI and represent 63-scFv-LH, 78-scFv-LH, 79-scFv-LH, P1-3-scFv-LH, P2-3-scFv-LH, P3-1-scFv-LH, P1-3-3-scFv-LH, P2-3-3-scFv-LH, P2-3-5-scFv-LH, and 63-P2-3-scFv-LH, respectively. The affinity results for 63-P3-1-scFv-LH, 78-P1-3-scFv-LH, 78-P2-3-scFv-LH, 78-P3-1-scFv-LH, 79-P1-3-scFv-LH, 79-P2-3-scFv-LH, and 79-P3-1-scFv-LH against the huCD3E-AA1-27-huFc antigen protein are shown. [Figure 5]Figures 5A to 5H show the affinity results for the huCD3E-AA1-27-huFc antigen protein of 63-P2-3-scFv-LH, 63-P3-1-scFv-LH, 78-P1-3-scFv-LH, 78-P2-3-scFv-LH, 78-P3-1-scFv-LH, 79-P1-3-scFv-LH, 79-P2-3-scFv-LH, and H2C-scFv-LH, respectively, based on SPR. [Figure 6] Figures 6A to 6F show the activation results of antibodies in T cells based on the reporter gene method. Figure 6A shows the T cell activation results for P1-3-scFv-LH and P2-3-scFv-LH. Figure 6B shows the T cell activation results for P3-1-scFv-LH. Figure 6C shows the T cell activation results for 63-scFv-LH, 78-scFv-LH, and 79-scFv-LH. Figure 6D shows the T cell activation results for P1-3-3-scFv-LH, P2-3-3-scFv-LH, and P2-3-5-scFv-LH. Figure 6E shows the T cell activation results for 63-P2-3-scFv-LH, 63-P3-1-scFv-LH, 78-P1-3-scFv-LH, and 78-P2-3-scFv-LH. Figure 6F shows the T cell activation results for 78-P3-1-scFv-LH, 79-P1-3-scFv-LH, and 79-P2-3-scFv-LH. [Figure 7] Figures 7A to 7E show the results of detecting the ability of antibodies to induce the secretion of the cytokine IFN-γ using the ELISA method. Figure 7A shows that 63-scFv-LH and 78-scFv-LH can induce the secretion of cytokine IFN-γ. Figure 7B shows that 79-scFv-LH and P1-3-scFv-LH can induce the secretion of cytokine IFN-γ. Figure 7C shows that P2-3-scFv-LH and P3-1-scFv-LH can induce the secretion of cytokine IFN-γ. Figure 7D shows that 78-P1-3-scFv-LH and 78-P2-3-scFv-LH can induce the secretion of cytokine IFN-γ. Figure 7E shows that 79-P1-3-scFv-LH can induce the secretion of cytokine IFN-γ. [Modes for carrying out the invention]

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the invention pertains. All documents, patent applications, patents, and other references cited herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are for illustrative purposes only and are not intended to be limiting. Other features, purposes, and advantages of the invention will become apparent from this specification, the drawings, and the claims set forth below.

[0032] I. Definition For the purposes of this specification, the following definitions are used. Wherever a term is used in the singular form, it is assumed to include the plural form, and vice versa. It should be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to be limiting.

[0033] When used with a number, the term "approximately" means that the value includes a range of ±5% of the specified number.

[0034] In this specification, the term "and / or" means one or more of the options.

[0035] In this specification, where the terms “includes” or “inclusive” are used, unless otherwise specified, they also include embodiments comprising the elements, integers, or processes described herein.

[0036] For example, when it says "antibody variable region containing a specific sequence," it is intended to include antibody variable regions composed solely of that specific sequence.

[0037] As used herein, "T cell activating antigen" refers to an antigenic determinant expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, that has the ability to activate T cells by interacting with antigen-binding molecules.

[0038] Specifically, the interaction between antigen-binding molecules and T cell-activating antigens can trigger T cell activation by inducing a signaling cascade of the T cell receptor complex.

[0039] In one embodiment, the T cell activating antigen is CD3.

[0040] As used herein, "T cell activation" refers to one or more cellular responses in T lymphocytes, particularly cytotoxic T lymphocytes, the responses being selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Assays for measuring T cell activation are well known in the art.

[0041] As used in this specification, the terms "CD3 antibody," "antibody against CD3," "antibody that specifically binds to CD3," "antibody that specifically targets CD3," and "antibody that specifically recognizes CD3" are used interchangeably and refer to antibodies that specifically bind to CD3.

[0042] The term "antibody" is used in its broadest sense herein, referring to a protein containing an antigen-binding site, encompassing a diverse range of structures of both natural and artificial antibodies. This includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (e.g., bispecific antibodies), single-chain antibodies, complete antibodies, and antibody fragments.

[0043] The terms “antibody fragment” and “antigen-binding fragment” are used interchangeably herein and refer to molecules distinct from a complete antibody, which include a portion of a complete antibody and bind to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv(scFv), single-chain Fab, or diabody.

[0044] The term "scFv" refers to an antibody fragment containing a light chain variable region and a heavy chain variable region, which are sequentially linked by an optional, flexible, short peptide linker and can be expressed as a single-chain polypeptide. The scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, in this specification, the variable regions VL and VH may be in any order (e.g., the order relative to the N-terminus and C-terminus of the polypeptide), and the scFv may contain VL-linker-VH or VH-linker-VL. An antibody exhibiting equivalent or similar binding affinity and / or specificity to a reference antibody means an antibody having at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding affinity and / or specificity of the reference antibody. This can be measured by any binding affinity and / or specificity measurement method known in the art. The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in antigen binding. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FRs) and three highly variable regions (HVRs). HVRs typically contain amino acid residues derived from highly variable loops and / or complementarity-determining regions (CDRs).

[0045] "Framework" or "FR" refers to variable domain residues other than highly variable region (HVR) residues. Variable domain FRs typically consist of four FR domains: FR1, FR2, FR3, and FR4.

[0046] A "complementarity-determining region," "CDR region," or "CDR" refers to a region within the antibody variable domain that is sequence-highly variable, structurally forms a specific loop ("hypervariable loop"), or contains an antigen contact residue ("antigen contact site"). CDRs are primarily involved in binding to the antigen epitope. Heavy chain CDRs are usually called CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The precise amino acid sequence boundaries of each CDR in a specific heavy chain variable region amino acid sequence can be determined by one or a combination of many known antibody CDR assignment systems. These assignment systems include, for example, Chothia's (Chothia et al., Nature 342: 877-883 (1989); Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on the three-dimensional structure of antibodies and the topology of the CDR loop; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)) based on the variability of antibody sequences; AbM (University of Bath); Contact (University College London); the International ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ); and North's CDR definition based on affinity propagation clustering using numerous crystal structures.

[0047] Unless otherwise specified, the terms "CDR" or "CDR sequence" in this invention include CDR sequences determined by any of the methods described above. CDRs can also be determined based on the same AbM numbering position as a reference CDR sequence (e.g., any CDR in the embodiments of the present invention). In the present invention, when the antibody variable region and a specific CDR sequence (including heavy chain variable region residues) are referred to, it means that they are based on the position in the AbM numbering system.

[0048] While CDRs differ among antibodies, only a limited number of amino acid positions within the CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, the minimum overlapping region can be identified, thereby providing the "minimum binding unit" necessary for antigen binding. The minimum binding unit may be part of the CDR. As those skilled in the art will understand, the remaining residues of the CDR sequence can be determined based on the antibody structure and protein folding. Therefore, the present invention also encompasses variants of any CDR described herein. For example, in some CDR variants, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues, as defined by Kabat, Chothia, or AbM, may be substituted with conserved amino acid residues.

[0049] A "humanized antibody" refers to a chimeric antibody containing amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, all or substantially all of the CDRs of the humanized antibody correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to the humanized antibody.

[0050] "Human antibody" refers to an antibody of non-human origin that is produced by a human or human cell, or that utilizes a human antibody library or other human antibody coding sequence, and that has an amino acid sequence corresponding to the amino acid sequence of a human antibody. This definition explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0051] As used herein, the term “Fc region” defines the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes the Fc region of the natural sequence and its variants. In some embodiments, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise noted, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) (see Kabat et al., *Sequences of Proteins of Immunological Interest*, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0052] In one embodiment, the Fc region of the immunoglobulin includes two constant domains, namely CH2 and CH3. In another embodiment, the Fc region of the immunoglobulin includes three constant domains, namely CH2, CH3, and CH4.

[0053] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in antigen binding. The variable domains of the heavy and light chains of natural antibodies usually have similar structures, and each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs) (see, for example, Kindt et al., *Kuby Immunology*, 6th edition, WH Freeman and Co., p. 91 (2007)). A single VH or VL domain can also confer antigen-binding specificity.

[0054] As used herein, the terms “binding” or “specific binding” mean that the binding interaction is selective to the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antibody to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), biolayer interference (BLI) techniques, or other binding assays known in the art.

[0055] As used herein, the term “monospecific” antibody refers to an antibody having one or more antigen-binding sites, where each antigen-binding site binds to the same epitope of the same antigen.

[0056] As used herein, the term “multispecific” antibody means an antibody having at least two antigen-binding sites, where each of the at least two antigen-binding sites binds to a different epitope of the same antigen, or to a different epitope of different antigens. The antibodies disclosed herein are typically multispecific antibodies, such as bispecific antibodies. In one embodiment, this specification provides a bispecific antibody having binding specificity to CD3 and other antigens (e.g., tumor antigens).

[0057] The terms "flexible linker peptide" or "linker peptide" refer to linker peptides composed of amino acids, and include, for example, the use of glycine and / or serine residues alone or in combination to link variable domains in an antibody. In one embodiment, the flexible linker peptide is a Gly / Ser linker, and the amino acid sequence (Gly4Ser) n (where n is an integer greater than or equal to 1, e.g., an integer from 1 to 7). In one embodiment, the flexible linker peptide is (Gly4Ser)3 (SEQ ID NO: 70).

[0058] A "conjugate" refers to an antibody that has been bound to one or more other substances (including, but not limited to, cytotoxic agents and labels).

[0059] The terms “individual” and “subject” are used interchangeably and include mammals. Mammals include, but are not limited to, humans and non-human primates (e.g., monkeys). In particular, the individual or subject is human.

[0060] The terms “tumor” and “cancer” are used interchangeably herein and encompass both solid tumors and humoral tumors.

[0061] The terms "cancer" and "cancerous" refer to physiological diseases in mammals in which cell proliferation becomes uncontrolled.

[0062] The term “tumor” refers to all neoplastic cell proliferation and proliferative disorders, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” and “tumor” as used herein are not mutually exclusive.

[0063] An "isolated" antibody refers to an antibody separated from its natural environment. In some embodiments, CD3 antibodies are purified to a purity of 90% or higher. This is confirmed, for example, by electrophoresis (SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (ion exchange or reverse-phase HPLC, etc.). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0064] "Isolated" nucleic acids refer to nucleic acid molecules separated from the components of their natural environment. Isolated nucleic acids include those normally found in the cell containing the nucleic acid molecule, but where the nucleic acid molecule is located outside the chromosome or at a location different from its natural chromosomal location. "Isolated CD3 antibody-encoding nucleic acid" refers to one or more nucleic acid molecules encoding a CD3 antibody chain or fragment thereof, including those present in a single vector, separate vectors, or at one or more locations within a host cell.

[0065] The following describes how to calculate sequence identity between sequences. To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned to obtain the best possible comparison (for example, gaps can be introduced in either the first or second amino acid sequence or nucleic acid sequence, or both, or non-homologous regions can be excluded for comparison to achieve the best alignment). In one embodiment, the length of the reference sequence used for comparison is at least 30%, preferably 40%, more preferably 50%, 60%, and even more preferably 70%, 80%, 90%, or 100% of the reference sequence length. Then, residues at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are considered identical at that position.

[0066] Mathematical algorithms can be used to perform sequence comparisons between two sequences and calculate the percentage of identity. In a preferred embodiment, the Needleman and Wunsch algorithm (Needleman and Wunsch, (1970) J. Mol. Biol. 48:444-453) (available at http: / / www.gcg.com) within the GAP program integrated into the GCG software package is used, with a Blossum 62 matrix or PAM250 matrix, and the percentage of identity between two amino acid sequences is determined with a gap penalty of 16, 14, 12, 10, 8, 6, or 4 and a length penalty of 1, 2, 3, 4, 5, or 6. In a more preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, along with the NWSgapdna.CMP matrix, to determine the identity percentage between two nucleic acid sequences with a gap penalty of 40, 50, 60, 70, or 80 and a length penalty of 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (and parameter set to be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap-open penalty of 12, a gap-extension penalty of 4, and a frameshift-gap penalty of 5.

[0067] Furthermore, the identity percentage between two amino acid sequences or nucleic acid sequences can be determined using the ALIGN program (version 2.0), which incorporates the algorithm by E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) using the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4.

[0068] Furthermore, or alternatively, the nucleic acid and protein sequences described herein can be used as "query sequences" to search public databases and, for example, to identify other family member sequences or related sequences.

[0069] The terms "amino acid change" and "amino acid modification" are used interchangeably and refer to the addition, deletion, substitution, and other modifications of amino acids. Any combination of amino acid addition, deletion, substitution, and other modifications can be performed under conditions to obtain a final polypeptide sequence with the desired properties.

[0070] "Conservative sequence modification" or "conservative sequence change" refers to an amino acid modification or change that does not substantially affect or alter the binding properties of an antibody or antibody fragment containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. These modifications can be introduced into the antibody or antibody fragment of the present invention using standard techniques known in the art, such as site-directed mutagenesis or PCR-mediated mutagenesis. A conservative substitution is the substitution of an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are defined in this art. These groups include amino acids with basic side chains (lysine, arginine, histidine, etc.), acidic side chains (aspartic acid, glutamic acid, etc.), non-charged side chains (glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan, etc.), nonpolar side chains (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, etc.), β-side chains (threonine, valine, isoleucine, etc.), and aromatic side chains (tyrosine, phenylalanine, tryptophan, histidine, etc.).

[0071] A "pharmaceutical composition" refers to a composition in which the contained active ingredients exist in a form that effectively exerts biological activity, and which does not contain any components that are unacceptably toxic to the subject to which the composition is administered.

[0072] The term "pharmaceutical carrier" refers to diluents, adjuvants (such as complete or incomplete Freund's adjuvants), excipients, buffers, or stabilizers administered together with the active ingredient.

[0073] Size exclusion high-performance liquid chromatography (SEC-HPLC) is a method used for standardization and quality control of antibodies. This method separates molecules primarily based on differences in molecular size or hydrodynamic radius. SEC-HPLC separates antibodies into three main forms: high molecular weight molecules (HMMs), major peaks (primarily antibody monomers), and low molecular weight molecules (LMMs). Antibody purity can be calculated as the percentage of the major peak area on the chromatogram relative to the total peak area. SEC-HPLC can also be used to measure the proportion of antibody monomers in a formulation and to obtain information on the presence of soluble aggregates and degradation products. For further details on the SEC-HPLC method, see, for example, J. Pharm. Sci., 83:1645-1650(1994); Pharm. Res., 11:485(1994); J. Pharm. Biomed. Anal., 15:1928(1997); J. Pharm. Biomed. Anal., 14:1133-1140(1986).

[0074] As used herein, “treatment” means delaying, interrupting, preventing, alleviating, suppressing, reducing, or reversing the progression or severity of an existing symptom, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, reducing symptoms, reducing the direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and improving the prognosis. In one embodiment, the antibody molecule of the present invention is used to slow or decelerate the progression of a disease.

[0075] As used herein, “prevention” includes the suppression of a disease or symptom, or the onset or progression of a particular disease or symptom. In one embodiment, a subject with a family history of cancer is a candidate for a prophylactic administration program. Generally, in the context of cancer, the term “prevention” refers to administering a drug before the clinical signs or symptoms of cancer appear, particularly to subjects at risk of cancer, before the onset of cancer symptoms.

[0076] The term "effective dose" refers to the amount or dosage of the antibody or composition of the present invention that, with a single or multiple administrations, produces the expected effect in a patient requiring treatment or prevention. The effective dose can be readily determined by a skilled physician in the art by considering several factors, including: mammalian species, body weight, age, general health status, type of disease, degree or severity of the disease, individual patient response, specific antibody to be administered, administration pattern, bioavailability of the formulation, selected administration schedule, and presence or absence of concomitant therapy.

[0077] The "therapeutic dose" refers to the amount that effectively achieves the desired therapeutic effect at the required dosage and duration. The therapeutic dose of an antibody or antibody fragment, or its composition, can vary depending on various factors, including disease state, age, sex, and weight of the individual, as well as the ability of the antibody or antibody fragment to induce the desired response within the individual. The therapeutic dose is also the amount at which the toxicity or adverse effects of the antibody or antibody fragment, or its composition, do not outweigh the beneficial effects of the treatment. Compared to an untreated subject, the "therapeutic dose" preferably suppresses a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and even more preferably at least about 80% or 90%. The ability of an antibody or antibody fragment, or its composition, to suppress a measurable parameter (e.g., cancer) can be evaluated in animal model systems that predict efficacy in human tumors.

[0078] The "prophylactic effective dose" refers to the amount of medication needed to effectively achieve the desired preventive effect over the required dosage and duration. Generally, prophylactic administration is used before or during the early stages of the disease, so the prophylactic effective dose is smaller than the therapeutic effective dose.

[0079] In this specification, the term “vector” as used to refer to nucleic acids refers to a nucleic acid molecule capable of replicating other nucleic acids to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors integrated into the genome of a host cell to which they are introduced. Some vectors can direct the expression of nucleic acids to which they are functionally ligated. Such vectors are referred to herein as “expression vectors.”

[0080] The term “host cell” refers to a cell into which an exogenous polynucleotide has been introduced, and includes the offspring of such cells. Host cells include “transforms” and “transformed cells,” which include primary transformed cells and their derived offspring, regardless of passage number. Offspring do not need to be exactly identical to the parent cells in terms of nucleic acid content and may contain mutations. This specification also includes mutant offspring with identical function or biological activity that have been screened or selected from the initially transformed cells. Host cells are any type of cell line available for producing the antibody molecules of the present invention, and include eukaryotic cells (e.g., mammalian cells, insect cells, yeast cells) and prokaryotic cells (e.g., Escherichia coli cells). Host cells include not only cultured cells but also cells in transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0081] II. Antibodies that specifically bind to the CD3 molecule of the present invention This invention provides a group of anti-CD3 antibodies effective across species, including heavy chain and light chain variable regions. Among them: (a) The heavy chain variable region includes HCDR1, indicated by GFTFSKYAMN (SEQ ID NO: 51), HCDR2, indicated by RIRSKYNNYATY (SEQ ID NO: 46), and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47), based on AbM numbers; and the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48), LCDR2, indicated by GTKFLAP (SEQ ID NO: 49), and LCDR3, indicated by ALWYSNRWV (SEQ ID NO: 50), based on AbM numbers; (b) The heavy chain variable region includes HCDR1, indicated by GFTFNKYAMN (SEQ ID NO: 45), HCDR2, indicated by RIRSKYNNYATY (SEQ ID NO: 46), and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47), based on AbM numbers; and the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48), LCDR2, indicated by GTKFLAP (SEQ ID NO: 49), and LCDR3, indicated by ALWVDNRWV (SEQ ID NO: 55), based on AbM numbers; (c) The heavy chain variable region includes HCDR1, indicated by GFTFPKYAMN (SEQ ID NO: 52), HCDR2, indicated by RIRSKYNNYETY (SEQ ID NO: 53), and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47), based on AbM number; and the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48), LCDR2, indicated by GTHFLAP (SEQ ID NO: 54), and LCDR3, indicated by ALWYSNRWV (SEQ ID NO: 50), based on AbM number; (d) The heavy chain variable region includes HCDR1, indicated by GFTFNKYAMN (SEQ ID NO: 45), HCDR2, indicated by RIRSKYNNYATY (SEQ ID NO: 46), and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47), based on AbM number; and the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48), LCDR2, indicated by GTKFLAP (SEQ ID NO: 49), and LCDR3, indicated by ALWQENRWV (SEQ ID NO: 56), based on AbM number; (e) The heavy chain variable region includes HCDR1, indicated by GFTFSKYAMN (SEQ ID NO: 51), HCDR2, indicated by RIRSKYNNYATY (SEQ ID NO: 46), and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47), based on AbM number; and the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48), LCDR2, indicated by GTKFLAP (SEQ ID NO: 49), and LCDR3, indicated by ALWVDNRWV (SEQ ID NO: 55), based on AbM number; (f) The heavy chain variable region includes HCDR1, indicated by GFTFSKYAMN (SEQ ID NO: 51), HCDR2, indicated by RIRSKYNNYETY (SEQ ID NO: 53), and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47), based on AbM number; and the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48), LCDR2, indicated by GTHFLAP (SEQ ID NO: 54), and LCDR3, indicated by ALWYSNRWV (SEQ ID NO: 50), based on AbM number; or (g) The heavy chain variable region includes HCDR1 shown in GFTFSKYAMN (SEQ ID NO: 51) based on AbM number, HCDR2 shown in RIRSKYNNYATY (SEQ ID NO: 46), and HCDR3 shown in HGNFGNSYISYWAY (SEQ ID NO: 47), and the light chain variable region includes LCDR1 shown in GSSTGAVTSGYYPN (SEQ ID NO: 48) based on AbM number, LCDR2 shown in GTKFLAP (SEQ ID NO: 49), and LCDR3 shown in ALWQENRWV (SEQ ID NO: 56).

[0082] In some embodiments, the antibodies that bind to the CD3 molecule of the present invention bind to human and non-human primate CD3.

[0083] The aforementioned non-human primates include New World monkeys (marmosets, common marmosets (Callithrix jacchus), cotton-top tamarins (Saguinus oedipus), and squirrel monkeys (Saimiri sciureus)) and Old World monkeys (crab-eating macaques (Macaca fascicularis), also known as cynomolgus monkeys; or rhesus monkeys (Macaca mulatta), also known as rhesus monkeys). Sequence analysis confirmed the existence of sequence segments with high homology at the N-terminus of the CD3ε extracellular domain in humans and non-human primates.

[0084] In some embodiments, the antibody that binds to the CD3 molecule of the present invention includes a heavy chain variable region and a light chain variable region that specifically bind to CD3.

[0085] (a) Heavy chain variable region shown in SEQ ID NO:26 and light chain variable region shown in SEQ ID NO:27; (b) Heavy chain variable region shown in SEQ ID NO:28 and light chain variable region shown in SEQ ID NO:29; (c) Heavy chain variable region shown in SEQ ID NO:30 and light chain variable region shown in SEQ ID NO:2; (d) Heavy chain variable region shown in SEQ ID NO:1 and light chain variable region shown in SEQ ID NO:31; (e) Heavy chain variable region shown in SEQ ID NO:32 and light chain variable region shown in SEQ ID NO:33; (f) Heavy chain variable region shown in SEQ ID NO:1 and light chain variable region shown in SEQ ID NO:34; (g) Heavy chain variable region shown in SEQ ID NO:30 and light chain variable region shown in SEQ ID NO:27; (h) Heavy chain variable region shown in SEQ ID NO:35 and light chain variable region shown in SEQ ID NO:29; (i) Heavy chain variable region shown in SEQ ID NO:30 and light chain variable region shown in SEQ ID NO:29; (j) Heavy chain variable region shown in SEQ ID NO:28 and light chain variable region shown in SEQ ID NO:36; (k) Heavy chain variable region shown in SEQ ID NO:30 and light chain variable region shown in SEQ ID NO:31; (l) Heavy chain variable region shown in SEQ ID NO:37 and light chain variable region shown in SEQ ID NO:38; (m) Heavy chain variable region shown in SEQ ID NO:39 and light chain variable region shown in SEQ ID NO:40; (n) Heavy chain variable region shown in SEQ ID NO:41 and light chain variable region shown in SEQ ID NO:33; (o) Heavy chain variable region shown in SEQ ID NO:26 and light chain variable region shown in SEQ ID NO:42; (p) Heavy chain variable region shown in SEQ ID NO:28 and light chain variable region shown in SEQ ID NO:43; or (q) The heavy chain variable region shown in SEQ ID NO:30 and the light chain variable region shown in SEQ ID NO:44.

[0086] In some embodiments, the antibody that binds to the CD3 molecule of the present invention is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0087] Preferably, it is an IgG1 or IgG4 antibody, and more preferably an IgG1 antibody, such as a human IgG1 antibody.

[0088] In some embodiments, the antibody that binds to the CD3 molecule of the present invention has one or more of the following properties.

[0089] (i) When measured by SEC-HPLC, the purity of the monomer is 90% or higher, preferably 92%, 94%, 96%, or 97% or higher; (ii) In measurement by reduced or non-reduced SDS-PAGE method, the purity is 90% or higher, preferably 91%, 92%, or 93% or higher; (iii) It specifically binds to CD3 expressed on the cell surface.

[0090] In some embodiments, the present invention provides nucleic acids encoding antibodies or fragments thereof that bind to any of the above-mentioned CD3 molecules, or any chain thereof.

[0091] In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the nucleic acid or vector is provided. In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells) or other cells suitable for the production of antibodies or their antigen-binding fragments. In yet another embodiment, the host cell is a prokaryotic cell.

[0092] For example, the nucleic acids of the present invention include nucleic acids encoding antibodies that bind to the CD3 molecule of the present invention. In some embodiments, one or more vectors comprising the nucleic acids are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. The vector includes, but is not limited to, viruses, plasmids, cosmids, λ phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is a pcDNA3.4 expression vector.

[0093] After constructing an expression vector or DNA sequence, it can be transfected or introduced into a suitable host cell. Many techniques are available for this purpose, including, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retrovirus-mediated transfection, viral infection, gene guns, lipid-mediated transfection, and other known techniques. In the case of protoplast fusion, cells are cultured in a culture medium and screened for those with appropriate activity. Methods and conditions used for culturing the resulting transfected cells and recovering the obtained antibody molecules are known to those skilled in the art and can be modified or optimized according to the specific expression vector and mammalian host cell used, based on this specification and known techniques.

[0094] Furthermore, one or more markers can be introduced to select cells in which DNA has been stably incorporated into the chromosome. These markers may provide nutritional supplementation to nutrient-deficient hosts, resistance to biocides (e.g., antibiotics), or resistance to heavy metals (e.g., copper). The selection marker gene may be introduced by direct ligation to the DNA sequence to be expressed, or by co-transfection into the same cells. Additional elements may be required to enable optimal mRNA synthesis. These elements may include splice signals, transcription promoters, enhancers, and termination signals.

[0095] In one embodiment, a host cell containing the polynucleotide of the present invention is provided.

[0096] In some embodiments, host cells containing the expression vector of the present invention are provided.

[0097] In some embodiments, the host cells are selected from yeast cells, mammalian cells, or other cells suitable for antibody production.

[0098] Suitable host cells include prokaryotic microorganisms (e.g., Escherichia coli).

[0099] Furthermore, the host cell may be a eukaryotic microorganism (e.g., a filamentous fungus or yeast) or various eukaryotic cells (e.g., insect cells).

[0100] Furthermore, vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension culture can be used. Examples of useful mammalian host cell lines include the SV40-converted monkey kidney CV1 line (COS-7),

[0101] Examples include human fetal kidney cell lines (HEK293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (HepG2), Chinese hamster ovary cells (CHO cells), CHO-S cells, NS0 cells, and myeloma cell lines (Y0, NS0, P3X63, Sp2 / 0, etc.).

[0102] For a review of mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0103] In a preferred embodiment, the host cells are CHO cells or HEK293 cells.

[0104] In one embodiment, the present invention provides a method for producing an antibody that binds to a CD3 molecule.

[0105] This method includes the step of culturing host cells containing a nucleic acid encoding an antibody that binds to the CD3 molecule, or an expression vector containing such nucleic acid, under conditions suitable for the expression of the nucleic acid.

[0106] The process optionally further includes a step of separating antibodies that bind to CD3 molecules.

[0107] In one embodiment, the process also includes recovering a CD3-binding antibody from host cells or their culture supernatant.

[0108] The CD3-binding antibody of the present invention can be purified using known techniques.

[0109] Examples include high-performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography (SEC).

[0110] The conditions for purifying specific proteins depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art.

[0111] The purity of the CD3-binding antibody of the present invention can be confirmed by any known analytical method.

[0112] This includes size exclusion chromatography, gel electrophoresis, and high-performance liquid chromatography.

[0113] The CD3-binding antibodies provided in this invention can be identified, screened, and evaluated using a variety of assays known to those skilled in the art for their physical / chemical properties and / or biological activity.

[0114] As an example, the antigen-binding activity of the CD3-binding antibody of the present invention can be measured by known methods such as FACS, ELISA, or Western blotting. Binding to CD3 can be evaluated by methods known to those skilled in the art, and an example of such evaluation is shown in this document.

[0115] In some embodiments, FACS is used to measure the binding of the CD3-binding antibody of the present invention to **cell surface CD3 (e.g., human CD3)**.

[0116] The present invention further provides an assay method for identifying biologically active CD3-binding antibodies.

[0117] Biological activity includes, for example, activation of the NF-κB signaling pathway and secretion of the cytokine IFN-γ.

[0118] The cells used in any of the above in vitro assays include cells that naturally express CD3 or cell lines that have been modified to express CD3.

[0119] This modified cell line refers to a cell line in which cells that do not normally express CD3 are transfected with DNA that encodes CD3, thereby inducing CD3 expression.

[0120] III. Fusions, Conjugates, and Bispecific Antibodies This invention provides fusions, conjugates, and bispecific antibodies containing the antibody of the present invention. Fusions or conjugates can be generated by fusing or conjugating the antibody of the present invention with heterologous molecules.

[0121] Furthermore, by fusing the antibody of the present invention with another antibody capable of binding to a target cell antigen, a bispecific antibody can be generated.

[0122] In some embodiments, the antibody polypeptide of the present invention may be fused or conjugated with one or more heterologous molecules.

[0123] The aforementioned heterologous molecules include, but are not limited to, proteins / polypeptides / peptides, labeled substances, drugs, cytotoxic agents, and the like.

[0124] Methods for fusing or conjugating proteins, polypeptides, peptides, or chemical molecules with antibodies are well known to those skilled in the art. (See, for example, U.S. Patents No. 5,336,603, 5,622,929, and European Patent No. EP 367,166.)

[0125] In one embodiment, the antibody of the present invention undergoes recombinant fusion with a heterologous protein or polypeptide or peptide to form a fusion protein.

[0126] In yet another embodiment, the antibody of the present invention is conjugated with a protein molecule or a non-protein molecule to produce a conjugate.

[0127] In some embodiments, the antibodies of the present invention may be fused or conjugated with heterologous molecules in the form of full-length antibodies or antibody fragments.

[0128] Linkers can be used to covalently link different components within the fusion and / or conjugate body of the present invention.

[0129] Linkers include chemical linkers or single-chain peptide linkers.

[0130] In some embodiments, the antibody of the present invention is fused to another peptide or protein via a peptide linker.

[0131] Furthermore, in some embodiments, the antibody of the present invention is conjugated to other molecules such as labeling products or drug molecules via a chemical linker.

[0132] The peptide linker of the present invention is a peptide composed of amino acid residues.

[0133] Such linker peptides are typically flexible, allowing the antigen-binding site to move independently.

[0134] The length of the linker peptide can be easily determined by those skilled in the art, depending on the actual circumstances.

[0135] For example, it may consist of at least 4 to 15 amino acid residues, or longer, approximately 20 to 25 amino acid residues.

[0136] In some embodiments, the anti-CD3 antibody of the present invention is fused with another antibody capable of binding to a target cell antigen to produce a bispecific antibody.

[0137] In some embodiments, antibodies that bind to target cell antigens are directed towards antigens associated with a pathological condition (e.g., antigens present on tumor cells or virus-infected cells).

[0138] Appropriate antigens are cell surface antigens, which include, but are not limited to, cell surface receptors.

[0139] In certain embodiments, the antigen is a human antigen.

[0140] In one specific embodiment, the target cell antigen is selected from the following: Folate receptor 1 (FolR1), mucin-1 (MUC1), B cell maturation antigen (BCMA), CD19, CD33, EGFR, EpCAM, HER2, CEA, EphA2, ROR1.

[0141] IV. Methods and compositions for treatment This invention provides a method for treating a disease. This method involves administering an effective amount of the anti-CD3 antibody of the present invention or its antigen-binding fragment, or the fusion, conjugate, or bispecific antibody of the present invention to a subject.

[0142] The anti-CD3 antibody or its antigen-binding fragment of the present invention may be administered to cells being cultured in vitro or ex vivo, or to a subject in vivo, to treat or prevent conditions such as cancer, inflammatory diseases, and autoimmune diseases.

[0143] In some embodiments, the present invention is The present invention provides a method for treating or preventing cancer using an anti-CD3 antibody or its antigen-binding fragment.

[0144] The anti-CD3 antibody or its antigen-binding fragment of the present invention exerts a therapeutic or preventive effect by appropriately activating T cells.

[0145] This method involves administering a therapeutically effective or prophylactically effective amount of the anti-CD3 antibody or its antigen-binding fragment of the present invention, or a pharmaceutical composition containing the same, to a subject requiring such treatment.

[0146] In some embodiments, the present invention is This invention provides a method for treating or preventing cancer using bispecific antibodies that specifically bind to CD3 and cancer-related antigens.

[0147] This bispecific antibody comprises the anti-CD3 antibody of the present invention or its antigen-binding fragment, which specifically binds to CD3.

[0148] The other portion of a bispecific antibody specifically binds to cancer-related antigens.

[0149] Examples of cancer-related antigens include: Examples include folate receptor 1 (FolR1), mucin-1 (MUC1), B cell maturation antigen (BCMA), CD19, CD33, EGFR, EpCAM, HER2, CEA, EphA2, and ROR1.

[0150] In some embodiments, the cancers treated or prevented by this bispecific antibody are, for example: These include breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, stomach cancer, colorectal cancer, cervical cancer, pancreatic cancer, testicular cancer, melanoma, soft tissue tumors (e.g., synovial sarcoma), esophageal cancer and head and neck squamous cell carcinoma, B-cell lymphoma, chronic lymphocytic leukemia, or acute lymphoblastic leukemia.

[0151] The CD3 antibody, fusion, conjugate, and bispecific antibody of the present invention can be administered in combination with other therapeutic forms and can be used to treat the above-mentioned diseases.

[0152] Other forms of treatment mentioned above include therapeutic agents, radiation therapy, chemotherapy, transplantation, and immunotherapy.

[0153] In some embodiments, the CD3 antibody, fusion, conjugate, and bispecific antibody of the present invention are used in combination with other therapeutic agents.

[0154] Typical therapeutic agents include chemotherapeutic agents, radiotherapy agents, other therapeutic antibodies, other active substances, and adjuvants (e.g., antitumor drugs).

[0155] The following examples are provided to aid in understanding the present invention.

[0156] This does not limit the scope of protection of the present invention, nor should it be interpreted as such. [Examples]

[0157] Example 1: Preparation and identification of parental antibody protein and antigen protein 1.1 Preparation of parental antibody protein Preparation of control antibody: In this application, the anti-CD3E AA1-27 antibody H2C was used as the positive control antibody. Based on the sequence disclosed in Patent Publication No. WO2008119567A2 (the amino acid sequences of the heavy chain variable region and light chain variable region of the H2C monoclonal antibody are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively), The target fragment was synthesized using JPEG2026511221000001.jpg6170, and the nucleotide sequences of the heavy chain variable region and light chain variable region of the H2C antibody were synthesized. The obtained DNA fragment was constructed in the eukaryotic expression vector pcDNA3.4-TOPO(Invitrogen) by homologous recombination. The constructed recombinant protein expression vector was transformed into E. coli DH5α and cultured overnight at 37°C.

[0158] Subsequently, the plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain a vector expressing the target H2C monoclonal antibody. The resulting expression vector is used in ExpiFectamine TM HEK293 cells (ATCC) were transfected using the CHO transfection kit (Thermo Fisher, A29129). (R) CRL-1573 TM The cells were transfected. After 7 days of culture, the cell culture supernatant was collected and centrifuged at 15,000 g for 10 minutes. The obtained supernatant was filtered through a 0.22 μm filter, and the antibody was affinity-purified using a Protein A / G affinity column. The target antibody was eluted with 100 mM glycine salt buffer (pH 3.0), and the resulting antibody was recovered by replacing the solution with PBS buffer using an ultrafiltration concentrator tube (Millipore, UFC901096).

[0159] 1.2 Preparation and identification of antigen proteins Antigen protein preparation: By gene-level manipulation, a human IgG1 Fc tag (SEQ ID NO: 4) was added to the C-terminus of the human CD3E protein CD3E AA1-27 (WO2008119565A2, SEQ ID NO: 3). The resulting nucleotide sequence was constructed in a pcDNA3.4-TOPO vector, transformed into E. coli DH5α, and cultured overnight at 37°C.

[0160] Plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The obtained plasmids were transiently transfected into HEK293 cells (ATCC TM using the ExpiFectamine TM 293 Transfection Kit (Gibco (R) CRL-1573 TM ).

[0161] After 7 days of expression, the culture supernatant was collected and purified using a Protein A / G affinity column. After purification, the target protein was eluted with 100 mM glycine salt (pH 3.0), concentrated and buffer-exchanged to finally obtain the antigen protein (huCD3E-AA1-27-huFc).

[0162] Identification of antigen: The prepared antigen (huCD3E-AA1-27-huFc) was detected using the quality-tested antibody H2C (IgG1) obtained in item 1.1 above.

[0163] The specific procedure is as follows: Coat ELISA plates with 2 μg / mL of huCD3E-AA1-27-huFc and leave them standing overnight at 4°C. After washing the plates three times, block them with 5% skim milk prepared in PBS for 1 hour at room temperature. After washing again three times, add antibody H2C serially diluted in PBS and incubate for 1 hour at room temperature. After washing, add the secondary antibody Anti-human-IgG-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted 1:6000 in PBS and react for 1 hour at room temperature. After washing six times, develop the color with TMB substrate for 5 - 20 minutes and stop the reaction. Measure OD45*0* with an enzyme plate reader and process and graph the data using GraphPad Prism. The results are as shown in Figure 1. Antibody H2C (IgG1) bound well to the antigen huCD3E-AA1-27-huFc constructed and expressed in Example 1.2, showing an antigen-binding activity with EC50 = 0.01 μg / mL.

[0164] 1.3 Construction of a huCD3D-CD3E-CHO-K1 overexpression cell line The encoding nucleotide sequences of human CD3D (Uniprot ID: P04234, SEQ ID NO: 5) and CD3E (Uniprot ID: P07766, SEQ ID NO: 6) were constructed on pLVX-puro plasmids (Clontech, Cat#632164), respectively. The resulting plasmids were electroporated (Invitrogen, Neon). TM CHO-K1 cells (ATCC) were electroporated using a Transfection System (MP922947). After electroporation, the resulting cells were transferred to F-12K medium (Gibco) containing 10% FBS (Gibco, 15140-141) and free of antibiotics, seeded in 10 × 10 cm culture dishes, and cultured for 48 hours. Subsequently, an average of 0.5 × 10⁶ cells were cultured. 4 Cells were dispensed into 96-well plates at a density of cells / well, and selective pressure was applied by adding puromycin (Gibco, A111138-03) at a final concentration of 8 μg / mL. Colony formation was observed approximately two weeks later, and the formed clonal cell lines were picked and identified.

[0165] Identification of huCD3D-CD3E-CHO-K1 cells by flow cytometry: The above cell lines, which were in the logarithmic growth phase, were digested with trypsin and seeded into 96-well plates. After washing with FACS buffer (1×PBS containing 2% FBS), primary antibody (H2C) serially diluted in PBS was added and incubated at 4°C for 30 minutes. After washing, fluorescently labeled secondary antibody Anti-human IgG Fc (abcam, 98596) was added and reacted at 4°C for 30 minutes. Finally, measurements were performed using a flow cytometer (Beckman, CytoFLEX AOO-1-1102). The analysis confirmed the acquisition of a huCD3D-CD3E-CHO-K1 cell line that highly expresses human CD3D and CD3E on the cell surface.

[0166] 1.4 Construction of NF-κB-Jurkat overexpressing cell lines pGL4.30 plasmid (Promega, #E8481) containing the NF-κB-re nucleotide sequence was introduced into Jurkat cells (ATCC). (R) NF-κB-Jurkat cells were obtained by electroporation of TIB-152. After electrotransmission, the cells were transferred to 1640 medium (Thermo Fisher, 11875093) and cultured in a 37°C incubator for 48 hours. Subsequently, the cells were seeded at a density of 1,000 cells / well into 96-well plates, and selective culture was performed by adding puromycin at a final concentration of 2 μg / mL. Single cell clones grown in the 96-well plates were selected and expanded, and the fluorescence signal was measured after adding a luciferase substrate to identify the cells, thereby establishing the NF-κB-Jurkat cell line.

[0167] 1.5 Construction of a cell line overexpressing huCD3E-AA1-27-HEK293 The encoding nucleotide sequence of human CD3E AA1-27 (SEQ ID NO:3) was constructed in a pLVX-puro plasmid (Clontech, Cat#632164). The obtained plasmid is subjected to electroporation (Invitrogen, Neon TM HEK293 cells (ATCC(R) CRL-1573) using the Transfection System (MP922947) TM The cells were electroporated. After electroporation, the obtained cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) and free of antibiotics. Subsequently, the cells were transferred to 10 × 10 cm culture dishes and cultured for 48 hours. Then, an average of 0.51 × 10⁻⁶ cells were cultured. 4 Cells were seeded at a density of cells / well into a 96-well plate, and selective pressure was applied by adding puromycin at a final concentration of 2 μg / mL. Clones formed approximately two weeks later were picked and the cell line was identified. Identification of huCD3E-AA1-27-HEK293 overexpressing cells by flow cytometry: The above cell lines in the logarithmic growth phase were digested with trypsin and seeded into 96-well plates. After washing with FACS buffer (1×PBS containing 2% FBS), the primary antibody (H2C) obtained in Example 1.1, serially diluted with PBS, was added and incubated at 4°C for 30 minutes. After washing, the fluorescently labeled secondary antibody Anti-human IgG Fc (abcam, 98596) was added and reacted at 4°C for 30 minutes. Finally, analysis was performed using a flow cytometer (Beckman, CytoFLEX AOO-1-1102). As a result, the huCD3E-AA1-27-HEK293 cell line, which highly expresses human CD3E AA1-27 on the cell surface, was obtained.

[0168] 1.6 Construction of CynoCD3E-HEK293 overexpression cell lines The encoding nucleotide sequence of monkey CD3E (Uniprot ID: Q95LI5, SEQ ID NO: 81) was constructed on a pLVX-puro plasmid (Clontech, Cat#632164). The resulting plasmid was electroporated (Invitrogen, Neon). TM HEK293 cells (ATCC) using the Transfection System (MP922947) (R) CRL-1573 TM The cells were electroporated. After electroporation, the obtained cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) and free of antibiotics. Subsequently, the cells were transferred to 10 × 10 cm culture dishes and cultured for 48 hours. Then, an average of 0.5 × 10⁻⁶ cells were cultured. 4 Cells were seeded at a density of cells / well into 96-well plates, and selective pressure was applied by adding puromycin at a final concentration of 2 μg / mL. Clones formed approximately two weeks later were picked, and the cell lines were identified.

[0169] Identification of CynoCD3E-HEK293 overexpressing cells by flow cytometry: The above cell lines were digested with trypsin during the logarithmic growth phase and seeded in 96-well plates. After washing with FACS buffer (1×PBS containing 2% FBS), the primary antibody (H2C) obtained in Example 1.1, serially diluted with PBS, was added and incubated at 4°C for 30 minutes. After washing, the fluorescently labeled secondary antibody Anti-human IgG Fc (abcam, 98596) was added and reacted at 4°C for 30 minutes. Finally, the cells were measured using a flow cytometer (Beckman, CytoFLEX AOO-1-1102). As a result, it was confirmed that a CynoCD3E-HEK293 cell line, which highly expresses monkey CD3E on the cell surface, was obtained.

[0170] Example 2: Antibody engineering modification of anti-CD3 monoclonal antibody 2.1 Humanization Modification In this example, the antibody H2C scFv (H2C-scFv, SEQ ID NO: 7) was modified for humanization.

[0171] The light chain variable region (VL) and heavy chain variable region (VH) of H2C are linked by a linker peptide (SEQ ID NO: 70). Based on this H2C-scFv, the aim was to obtain candidate antibodies with a higher degree of humanization and different affinities.

[0172] Humanization modifications were performed using M13 phage display technology, by constructing a back mutation library using degenerate primers.

[0173] The specific method for building the library is as follows: First, we synthesized a primer containing point mutations (Jin Weizhi Biotechnology Co., Ltd.). Next, the target antibody (also called the parent antibody), H2C-scFv, was used as a PCR amplification template to amplify the sequence containing the designed mutation in the FR region. Using bridge PCR, fragments containing different FR region mutations were ligated, and the antibody sequences with point mutations were ligated to a phage display vector by double enzyme cleavage (Hind III and Not I) and sticky-end ligation. Finally, the obtained mutant antibody sequence was introduced into E. coli SS320 by electroporation. The SS320 bacterial suspension after introduction was diluted with a concentration gradient and spread onto an ampicillin-resistant 2×YT solid medium plate to obtain single colonies, thereby preparing a monoclonal bacterial suspension.

[0174] Using monoclonal bacterial suspension, ELISA screening and sequencing analysis were performed on the antigen protein huCD3E AA1-27-huFc, resulting in the acquisition of modified molecules P1-3-scFv (SEQ ID NO: 8), P2-3-scFv (SEQ ID NO: 9), P3-1-scFv (SEQ ID NO: 10), P1-1-scFv (SEQ ID NO: 71), P2-1-scFv (SEQ ID NO: 72), P2-2-scFv (SEQ ID NO: 73), and P3-4-scFv (SEQ ID NO: 74).

[0175] 2.2 Affinity Maturation Modification In this example, affinity maturation modification was performed on the antibody H2C-scFv to obtain candidate antibodies with different affinities. Based on M13 phage display technology, mutations were introduced into the CDR region using codon-based primers (each codon was configured in an NNK configuration during primer synthesis), and four types of phage display libraries were constructed. Library 1 and Library 2 are single-point combination mutations; Library 1 is a combination mutation of LCDR1, LCDR3, and HCDR3, and Library 2 is a combination mutation of LCDR2, HCDR1, and HCDR2. In addition, Library 3 and Library 4 are two-point saturation mutations; Library 3 is a two-point saturation mutation of LCDR3, and Library 4 is a two-point saturation mutation of HCDR3.

[0176] The specific method for building the library is as follows: First, we synthesized a primer containing point mutations (Jin Weizhi Biotechnology Co., Ltd.). Next, the maternal antibody (H2C-scFv) was used as a template for PCR amplification to amplify sequences containing the designed mutation in the CDR region. Using bridge PCR, fragments containing different CDR mutations were combined, and the antibody sequences, into which point mutations were introduced, were ligated to a phage display vector by double enzyme cleavage (Hind III and Not I) and sticky-end ligation. Finally, the obtained mutant antibody sequence was introduced into E. coli SS320 by electroporation.

[0177] Library volume calculation, phage display library preparation, and screening procedures were performed according to Example 2.1. The obtained SS320 bacterial suspension was diluted with a concentration gradient and spread onto ampicillin-resistant 2×YT solid medium plates to obtain single colonies and prepare monoclonal bacterial suspension. Subsequently, ELISA screening and sequencing analysis were performed using the antigen protein huCD3E-AA1-27-huFc, resulting in the acquisition of modified molecules 63-scFv (SEQ ID NO: 11), 78-scFv (SEQ ID NO: 12), 79-scFv (SEQ ID NO: 13), 16-scFv (SEQ ID NO: 75), 23-scFv (SEQ ID NO: 76), 34-scFv (SEQ ID NO: 77), 43-scFv (SEQ ID NO: 78), 67-scFv (SEQ ID NO: 79), and 73-scFv (SEQ ID NO: 80).

[0178] Example 3: Construction, expression, and purification of candidate antibodies 3.1 Plasmid Construction The coding sequences for antibodies were constructed by ligating the C-terminus of the constant region Fc (SEQ ID NO: 25) of human IgG1 to the scFv sequences of the modified molecules obtained through screening. These scFv antibody sequences were inserted into the eukaryotic expression vector pcDNA3.4-TOPO (Invitrogen), transformed into E. coli DH5α, and cultured overnight at 37°C. Subsequently, the plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids for eukaryotic expression. Antibody molecules linked to the constant region Fc of human IgG1 were named as P1-3-scFv-LH, P2-3-scFv-LH, P3-1-scFv-LH, P1-1-scFv-LH, P2-1-scFv-LH, P2-2-scFv-LH, P3-4-scFv-LH, 63-scFv-LH, 78-scFv-LH, 79-scFv-LH, 16-scFv-LH, 23-scFv-LH, 34-scFv-LH, 43-scFv-LH, 67-scFv-LH, and 73-scFv-LH. Here, "LH" indicates that the variable domains are arranged in the order VL-VH.

[0179] 3.2 Expression and Purification of Candidate Antibodies Candidate antibodies were expressed using the ExpiCHO transient expression system (Thermo Fisher, A29133). Specifically, on the day of transfection, the cell density was approximately 7 × 10⁶. 6 ~1 × 10 7 Confirm that the cells / mL and viability >98%, and then use fresh ExpiCHO medium preheated to 37°C to reach a final concentration of 6 × 10⁶. 6 The concentration was adjusted to 1 / mL. Next, OptiPRO was pre-cooled to 4°C. TM Dilute the plasmid constructed in Example 3.1 in SFM (add 1 μg of plasmid to 1 mL of medium), and then use OptiPRO TM ExpiFectamine in SFM TM Dilute CHO and mix the two in equal volumes, then stir gently to form ExpiFectamine TMA CHO / plasmid DNA mixture was prepared. After incubation at room temperature for 1-5 minutes, it was slowly added to the prepared cell suspension and cultured in a shaking incubator at 37°C and 8% CO2 while gently mixing.

[0180] ExpiCHO is available 18-22 hours after transfection. TM Enhancer and ExpiCHO TM Feed was added to the culture medium, and cultivation was continued under conditions of 32°C and 5% CO2. On day 5 after transfection, the same amount of ExpiCHO was added. TM The feed was added again and mixed slowly to homogenize the cell suspension. On day 7 post-transfection, the culture supernatant expressing the target protein was centrifuged at 15,000 g for 10 minutes, and the resulting supernatant was purified using MabSelect SuRe LX (GE, 17547403). The target protein was eluted with 100 mM sodium acetate buffer (pH 3.0), neutralized with 1 M Tris-HCl, and then dialyzed into PBS buffer using an ultrafiltration concentration cartridge (Millipore, UFC901096) to finally obtain the purified antibody.

[0181] Example 4 Evaluation of the physicochemical properties of candidate antibodies 4.1 SDS-PAGE analysis of candidate antibodies Sample preparation under non-reducing conditions: 1 μg of the candidate antibody and quality control product IPI (i.e., ipilimumab) obtained in Example 3 was added to 5× SDS loading buffer and 40 mM iodoacetamide, and heated in a dry bath at 75°C for 10 minutes. After cooling to room temperature, the mixture was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was collected.

[0182] Sample preparation under reducing conditions: 2 μg of candidate antibody and quality control product IPI were mixed with 5×SDS loading buffer and 5 mM DTT, and heated in a dry bath at 100°C for 10 minutes. After cooling to room temperature, the mixture was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was collected.

[0183] The obtained supernatant was added to a Bis-Tris 4-15% gradient gel (manufactured by Jinsrui Biotechnology Co., Ltd.), and gel electrophoresis was performed. Protein bands were then visualized by Coomassie brilliant blue staining. The gel background was made clear with a destaining solution, and images were taken with an EPSON V550 color scanner. The purity of the non-reduced and reduced bands was then calculated using peak area normalization with ImageJ software.

[0184] The results are shown in Table 1. The bands of the candidate antibodies were located at approximately 104 kD under non-reducing conditions and approximately 60 kD under reducing conditions, which was consistent with the predicted molecular weight. The antibody molecules P1-3-scFv-LH, P2-3-scFv-LH, P3-1-scFv-LH, 63-scFv-LH, 78-scFv-LH, and 79-scFv-LH were confirmed to have antibody purity on a reducing gel, and all had a purity of 93% or higher.

[0185] 4.2 Evaluation of candidate antibody monomer purity by SEC-HPLC Materials and Methods: 1. Liquid phase: 150 mmol / L phosphate buffer (pH 7.4) 2. Sample preparation: Candidate antibody and quality control product IPI were diluted to 0.5 mg / mL in the liquid phase.

[0186] An Agilent HPLC 1100 system and an XBridge BEH SEC column (3.5 μm, 7.8 mm ID × 30 cm, Waters) were used, with a flow rate of 0.8 mL / min, an injection volume of 20 μL, and detection wavelengths of 280 nm and 214 nm. 150 mmol / L phosphate buffer (blank), IPI quality control solution, and each sample solution were sequentially injected, and the proportions of high molecular weight polymers, antibody monomers, and low molecular weight components were calculated using area normalization.

[0187] The results are shown in Table 1. The antibody molecules P1-3-scFv-LH, P2-3-scFv-LH, P3-1-scFv-LH, 63-scFv-LH, 78-scFv-LH, and 79-scFv-LH were detected as antibody monomers by SEC-HPLC, and the monomer purity of all of them was 97% or higher.

[0188] [Table 1]

[0189] Example 5 Modification of anti-CD3 antibody and evaluation of its physicochemical properties Furthermore, combination mutations were introduced into the molecules with good physicochemical properties obtained in Example 4, which had undergone humanization and affinity maturation: P1-3-scFv, P2-3-scFv, P3-1-scFv, 63-scFv, 78-scFv, and 79-scFv. As a result, the following modified molecules were obtained: P1-3-3-scFv (SEQ ID NO: 14), P2-3-3-scFv (SEQ ID NO: 15), P2-3-5-scFv (SEQ ID NO: 16), 63-P2-3-scFv (SEQ ID NO: 17), 63-P3-1-scFv (SEQ ID NO: 18), 78-P1-3-scFv (SEQ ID NO: 19), 78-P2-3-scFv (SEQ ID NO: 20), 78-P3-1-scFv (SEQ ID NO: 21), 79-P1-3-scFv (SEQ ID NO: 22), 79-P2-3-scFv (SEQ ID NO: 23), and 79-P3-1-scFv (SEQ ID NO: 24). Antibodies were constructed, expressed, and purified for each of the obtained molecules. The specific methods followed those described in Example 3.

[0190] This yielded the following antibody molecules: P1-3-3-scFv-LH, P2-3-3-scFv-LH, P2-3-5-scFv-LH, 63-P2-3-scFv-LH, 63-P3-1-scFv-LH, 78-P1-3-scF v-LH, 78-P2-3-scFv-LH, 78-P3-1-scFv-LH, 79-P1-3-scFv-LH, 79-P2-3-scFv-LH, and 79-P3-1-scFv-LH.

[0191] These antibody molecules were analyzed by SDS-PAGE and monomer purity was evaluated by SEC-HPLC. The detailed procedure is described in Example 4, and the results are shown in Table 2. Furthermore, all of the modified molecules showed a purity of 95% or higher by SDS-PAGE, and monomer purity by SEC-HPLC was also confirmed to be 95% or higher for all except 79-P3-1-scFv-LH.

[0192] [Table 2]

[0193] Example 6: Affinity measurement of candidate antibodies 6.1 Evaluation of Affinity Activity of Modified Antibodies Based on FACS Method Logarithmic growth phase huCD3D-CD3E-CHO-K1 cells or Jurkat cells (human T lymphoblastic leukemia cells) were collected, centrifuged at 300 g, and the supernatant was removed. The cells were resuspended in FACS buffer (PBS containing 1% BSA), and the cells were counted to determine the concentration of the suspension at 1 × 10⁻⁶. 6 The concentration was adjusted to 1 / mL. Subsequently, 100 μL of cell suspension was added to each well, dispensed into a 96-well round-bottom plate, and centrifuged at 300 g to remove the supernatant.

[0194] Each well contains candidate antibodies (63-scFv-LH, 78-scFv-LH, 79-scFv-LH, P1-3-scFv-LH, P2-3-scFv-LH, P3-1-scFv-LH, P1-3-3-scFv-LH, P2-3-3-scFv-LH, P2-3-5-scFv-LH, 63-P2-3-scFv-LH, 63-P3-1-scFv-LH, 78-P1-3-scFv-LH, 78-P2-3-scFv-LH, 78-P3-1-scFv-LH, 79-P1-3-scFv-LH, 79-P2-3-scFv-LH, 79-P3-1-scFv-LH) and a control antibody at different concentrations. Diluted H2C-scFv-LH was added, the cells were resuspended, and incubated at 4°C for 60 minutes.

[0195] After incubation, the cell suspension was washed three times, resuspended with PE-labeled anti-human IgG-Fc antibody (Abcam, 98596), and incubated at 4°C for 30 minutes. The cell mixture after incubation was washed three more times, resuspended in 200 μL of FACS buffer, and measured and analyzed using a flow cytometer (Beckman, CytoFLEX AOO-1-1102). PRISM was used for data analysis. TM (GraphPad Software, San Diego, CA) 50 The value was calculated.

[0196] The results of the FACS binding measurement are shown in Figures 2A to 2D. From Figures 2A and 2B, P1-3-scFv-LH showed the upper limit and EC values ​​for binding to huCD3D-CD3E-CHO-K1 cells. 50 Both were inferior to the control antibody H2C-scFv-LH, and EC 50 The affinity calculated from this data was approximately 3.65 times lower than that of the control antibody, indicating low affinity. P2-3-scFv-LH, 63-scFv-LH, 78-scFv-LH, and 79-scFv-LH exhibited EC levels nearly equivalent to the control antibody H2C-scFv-LH against huCD3D-CD3E-CHO-K1 cells. 50The results showed that 63-scFv-LH and 78-scFv-LH had slightly lower upper limits (Figure 2A). P3-1-scFv-LH is more EC than H2C-scFv-LH 50 The coefficient was small and showed higher affinity (Figure 2B). From Figures 2C and 2D, P2-3-3-scFv-LH, P2-3-5-scFv-LH, and 63-P3-1-scFv-LH have lower EC values ​​than H2C-scFv-LH for Jurkat cells. 50 This demonstrated a higher affinity than the control antibody. 63-P2-3-scFv-LH showed binding to a similar degree to the control antibody. On the other hand, P1-3-3-scFv-LH bound to Jurkat cells more effectively than H2C-scFv-LH. 50 The MFI was remarkably high, with approximately a twofold decrease in MFI at concentrations of 1.25 μg / mL, 0.3125 μg / mL, 0.0781 μg / mL, and 0.0195 μg / mL, confirming low affinity. 78-P1-3-scFv-LH showed very weak binding to Jurkat cells, exhibiting approximately 1000-fold decrease in affinity compared to the control antibody, indicating extremely low affinity (Figure 2C). 78-P3-1-scFv-LH had a lower EC than H2C-scFv-LH. 50 The results showed that the upper limit was lower than that of the control antibody. 78-P2-3-scFv-LH and 79-P2-3-scFv-LH had lower upper limits than the control antibody, and EC 50 They were almost equivalent. 79-P1-3-scFv-LH was effective against Jurkat cells. 50 It was inferior to the control antibody in both the upper limit and EC 50 The calculated affinity decreased by approximately 3.65 times, indicating low affinity (Figure 2D).

[0197] 6.2 Evaluation of cross-reactivity of modified antibodies against monkey antigens using FACS method CynoCD3E-HEK293 cells in the logarithmic growth phase were harvested and centrifuged at 300 g to remove the supernatant. The cells were resuspended in FACS buffer (PBS containing 1% BSA), the cell count was measured, and the concentration of the suspension was adjusted to 1 × 10⁻⁶. 6 The concentration was adjusted to 1 / mL.

[0198] Subsequently, 100 μL of cell suspension was added to each well, dispensed into a 96-well round-bottom plate, and centrifuged at 300 g to remove the supernatant. Dilutions of candidate antibodies (63-P2-3-scFv-LH, 63-P3-1-scFv-LH, 78-P1-3-scFv-LH, 78-P2-3-scFv-LH, 78-P3-1-scFv-LH, 79-P1-3-scFv-LH, 79-P2-3-scFv-LH) and the control antibody H2C-scFv-LH were added to each well. The initial concentration of 78-P1-3-scFv-LH was 50 μg / mL, and a 3-fold serial dilution was performed. For the other molecules, the initial concentration was 5 μg / mL, and a 3-fold serial dilution was performed similarly. The cells were resuspended and incubated at 4°C for 60 minutes. After incubation, the cell mixture was washed three times, and PE-labeled anti-human IgG-Fc flow cytometry antibody (Invitrogen, 12-4998-82) was added and the cells were resuspended and incubated at 4°C for 30 minutes. After incubation, the cells were washed three more times and resuspended in 200 μL of FACS buffer. The cells were then measured and analyzed using a flow cytometer (Beckman, CytoFLEX AOO-1-1102). PRISM was used for data analysis. TM (GraphPad Software, San Diego, CA) 50 The value was calculated.

[0199] The results of the FACS measurements are shown in Figure 3. From Figure 3, it was confirmed that the candidate antibodies 63-P2-3-scFv-LH, 63-P3-1-scFv-LH, 78-P1-3-scFv-LH, 78-P2-3-scFv-LH, 78-P3-1-scFv-LH, 79-P1-3-scFv-LH, and 79-P2-3-scFv-LH all bound to CynoCD3E-HEK293 cells and exhibited cross-reactivity to monkey CD3. Among these, 63-P3-1-scFv-LH and 78-P3-1-scFv-LH showed slightly higher binding activity to monkey CD3-overexpressing cells CynoCD3E-HEK293 than the control antibody H2C-scFv-LH. On the other hand, 63-P2-3-scFv-LH, 78-P2-3-scFv-LH, and 79-P2-3-scFv-LH showed binding activity equivalent to the control antibody, while 78-P1-3-scFv-LH and 79-P1-3-scFv-LH showed significantly lower binding activity compared to the control antibody H2C-scFv-LH.

[0200] 6.3 Affinity evaluation of candidate antibodies by BLI method In this example, the affinity of candidate antibodies and control antibodies to the huCD3E AA1-27-huFc antigen protein was measured using a Gator instrument.

[0201] Protein biotinylation: The antigen protein huCD3E AA1-27-huFc was diluted to a final concentration of 1 mg / mL, pre-mixed with biotin, and reacted at 4°C for 1 hour. Subsequently, the mixture was filtered using an ultrafiltration tube to remove unreacted free biotin, yielding biotinylated huCD3E AA1-27-huFc (hereinafter, huCD3E-AA1-27-huFc-Biotin).

[0202] Preparation of Q Buffer: 2 g of BSA (IgG-free, Jackson ImmunoResearch Lab) and 2 mL of 10% Tween 20 (Thermo) were added to 1000 mL of 1×PBS (Thermo), mixed, and the pH was adjusted to 7.40. After filtration, the solution was aliquoted and stored.

[0203] Preparation of regeneration buffer: 0.38 g of glycine and 4.38 g of sodium chloride were added to 500 mL of pure water and mixed. The pH was adjusted to 2.0 to prepare the sensor regeneration buffer. After filtration, the solution was dispensed and stored.

[0204] Measurement: huCD3E-AA1-27-huFc-Biotin was diluted to 75 nM in Q Buffer, and candidate antibodies or control antibodies were serially diluted 2-fold in Q Buffer to create a final concentration series of 40, 20, 10, 5, 2.5, 1.25, 0.62, and 0 nM.

[0205] Measurements were performed under light-shielding conditions. The sensors (SA Probes, ProbeLife) were pre-moistened with Q Buffer, and the test was started 10 minutes later on a sample plate (Greiner, 655209).

[0206] After confirming that it was working correctly, the setup was performed according to the configuration program. First, antigen binding was performed for 600 seconds, followed by equilibration in Q Buffer for 30 seconds. Then, the sensor bound with the antigen huCD3E-AA1-27-huFc-Biotin was transferred to antibody dilutions of various concentrations and bound for 120 seconds. Subsequently, the sensor was returned to Q Buffer, and dissociation was measured for 180 seconds. Finally, KD, Ka, and Kd were calculated using binding and dissociation data at different antigen and antibody concentrations.

[0207] The results are shown in Figures 4A to 4Q and Table 3. P1-3-scFv-LH, P1-3-3-scFv-LH, 78-P1-3-scFv, and 79-P1-3-scFv clearly have lower affinity for the antigen protein than the maternal antibody, and the KD value of 78-P1-3-scFv-LH in particular is 10. -10 from 10 -8 The order rose, and a significant decrease in affinity was observed. On the other hand, the modified antibody 78-P2-3-scFv-LH showed higher affinity for the antigen protein than the parent antibody.

[0208] [Table 3]

[0209] 6.4 Affinity evaluation of candidate antibodies based on the SPR method In this example, the affinity between the candidate antibody and the huCD3E AA1-27-huFc antigen protein was measured using a Biacore instrument. Protein coupling: The huCD3E-AA1-27-huFc protein obtained in Example 1.2 was diluted to 5.8 μg / mL in sodium acetate (NaAc) buffer at pH 4.5. The flow rate was set to 10 μL / min, and the tip was activated with a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (activation time: default value 420 s). Based on the set coupling amount, the antigen protein huCD3E AA1-27-huFc was immobilized at approximately 75 RU levels, and unreacted active groups were blocked with ethanolamine. Sample measurement conditions: PBS buffer (pH 7.4) containing 0.05% Tween-20 was used as the running buffer. The control antibody was measured using the same buffer solution, and the antibody concentration series was set to 4 nM and 20 nM. The flow rate during analysis was 30 μL / min, the binding time was 180 s, and the dissociation time was 300 s. After dissociation was complete, a 20-second regeneration treatment was performed using 10 mM Gly-HCl (pH 2.0) to completely remove the antibody bound to the antigen.

[0210] Parameter analysis: The experiment was conducted in multi-cycle mode, and the obtained response signals were recorded with analysis time on the x-axis and response value on the y-axis. After subtracting double references from the data, a 1:1 Langmuir coupling model was fitted using BIAcore T200 analysis software to calculate affinity parameters such as the coupling constant (Ka), dissociation constant (Kd), and equilibrium dissociation constant (KD).

[0211] Results: The results are shown in Figures 5A to 5H and Table 4. The candidate antibodies 63-P2-3-scFv-LH, 63-P3-1-scFv-LH, 78-P2-3-scFv-LH, 78-P3-1-scFv-LH, and 79-P2-3-scFv-LH showed affinity for the antigen protein almost equivalent to that of the parent antibody. On the other hand, the modified antibodies 78-P1-3-scFv-LH and 79-P1-3-scFv-LH showed significantly lower affinity compared to the parent antibody. Specifically, the Koff values ​​for 78-P1-3-scFv-LH and 79-P1-3-scFv-LH are 1.77 × 10⁻⁶ each. -2 and 8.23 ​​× 10 -3 The values ​​decreased, and the equilibrium dissociation constants (KD) of these antibodies with the antigen were 8.71 × 10⁻⁶ each. -9 and 1.81 × 10 -9 That was the case.

[0212] [Table 4]

[0213] Example 7: Evaluation of T-cell activation ability of candidate antibodies based on reporter gene method In this example, the luciferase reporter gene system was used to evaluate the ability of antibodies before and after modification to activate the NF-κB signaling pathway. Anti-CD3 antibody binds to CD3 to activate downstream NF-κB signal transduction and induce the expression of luciferase. By stimulating with stepwise changes in antibody concentration, an antibody concentration-dependent fluorescence reading curve can be obtained, and the T cell activation ability of the antibody can be evaluated.

[0214] The specific method is shown below. Test method: 100 μL of serially diluted candidate antibodies and control antibodies were added to each well of a 96-well white bottomless cell culture plate and incubated overnight at 4°C. The next day, NF-κB-Jurkat cells in the logarithmic growth phase were collected and centrifuged at 300 g to remove the supernatant. The cells were resuspended in FACS buffer (PBS containing 1% BSA), the cell count was measured, and the concentration was adjusted to 1×10 6 cells / mL. After washing the plate three times with PBS, 100 μL of the cell suspension was added to each well and cultured in a 37°C incubator for 6 hours. Then, 50 μL of Bright-Lite (Vazyme, product number: DD1204-03) was added to each well and incubated for 10 minutes under light-shielded conditions to measure the fluorescence signal. Data analysis was performed using PRISM TM (GraphPad Software, San Diego, CA).

[0215] Results: The results of the activation ability test are shown in Figures 图6A~图6F. The results are as follows. P2-3-scFv-LH showed a higher activation upper limit than the control antibody H2C-scFv-LH and had a stronger T cell activation ability. P1-3-scFv-LH showed a higher EC 50 value and a lower upper limit, and the EC 50The affinity calculated from the results decreased by approximately 7 times, and T cell activation ability decreased (Figure 6A). P3-1-scFv-LH showed a higher activation limit than the control antibody and had high T cell activation ability (Figure 6B). 63-scFv-LH and 78-scFv-LH showed lower activation ability than 79-scFv-LH (Figure 6C). P1-3-3-scFv-LH and P2-3-5-scFv-LH were found in EC 50 These antibodies showed higher and lower T cell activation activity than the control antibody. In particular, P1-3-3-scFv-LH showed a 7.69-fold decrease in activity compared to H2C-scFv-LH, and P2-3-5-scFv-LH showed a 6.86-fold decrease in activity (Figure 6D). 63-P2-3-scFv-LH showed a lower activation limit than the control antibody, and 63-P3-1-scFv-LH showed EC 50 The value was approximately twice as high, indicating reduced activation ability. 78-P2-3-scFv-LH had a low activation limit and high EC 50 It showed low T cell activation ability. 78-P1-3-scFv-LH showed extremely weak T cell activation ability (Figure 6E). 79-P2-3-scFv-LH had upper limits and EC levels similar to the control antibody. 50 The results showed that the activation ability was equivalent. 78-P3-1-scFv-LH showed EC compared to the control antibody. 50 The activation activity was 2.62 times higher, indicating low activation activity. 79-P1-3-scFv-LH showed lower activation activity than the control antibody, and its fluorescence intensity at a maximum concentration of 1000 μg / mL was approximately 1 / 3.7 of that of the control antibody (Figure 6F).

[0216] Discussion: The research results suggest that antibodies with low T cell activation ability can prevent T cell overactivation and reduce the risk of cytokine storms. In this example, the 78-P2-3-scFv-LH antibody molecule suppressed T cell activation ability while maintaining high affinity. Therefore, CD3 bispecific antibodies containing this antibody sequence are expected to increase binding affinity to CD3 on the T cell surface, bring tumor cells and T cells closer together, promote interaction between the TCR and tumor surface pMHC, induce tumor cell killing through the secretion of perforating agents and granzymes, while avoiding excessive T cell activation and reducing the risk of abnormal cytokine release.

[0217] Example 8 Evaluation of cytokine secretion capacity of candidate antibodies In this example, the ability of modified antibodies to induce cytokine IFN-γ secretion was measured using the ELISA method. Anti-CD3 antibodies activate T cells by binding to CD3 on T cells, inducing the secretion of cytokines such as IL-2, IL-6, IL-10, IFN-γ, and TNF-α. By stimulating T cells with an anti-CD3 antibody concentration gradient, a concentration-dependent amount of cytokine secretion can be obtained, thereby allowing evaluation of the T cell activating ability of the anti-CD3 antibody.

[0218] Methods: First, candidate antibodies at an initial concentration of 8 μg / mL were serially diluted and added to 96-well plates (Corning, 3370), and incubated overnight at 4°C. The following day, the plate was washed three times with pre-cooled PBS, and peripheral blood mononuclear cells (PBMCs) were added in a 1 × 10⁶ solution. 6 The cells / mL were adjusted. 100 μL of PBMC suspension and 100 μL of a solution containing 1 μg / mL of CD28 antibody (Biolegend, 302934) were added to each well. The plates were placed in a cell culture incubator at 37°C and 5% CO2, incubated for 48 hours, then centrifuged and the supernatant was collected. The IFN-γ concentration was measured by ELISA. The obtained data were processed using PRISM. TM The analysis was performed using GraphPad Software (San Diego, CA).

[0219] Results: The results are shown in Figures 7A to 7E. The results revealed the following: 63-scFv-LH showed higher IFN-γ secretion than the control antibody H2C-scFv-LH at concentrations of 8 μg / mL and 1.28 μg / mL. 78-scFv-LH also induced higher IFN-γ secretion than the control antibody at concentrations of 8 μg / mL, 1.28 μg / mL, and 0.64 μg / mL (Figure 7A). 79-scFv-LH showed higher IFN-γ secretion than the control antibody at all concentrations of 8, 3.20, 1.28, 0.64, and 0.32 μg / mL, demonstrating significantly higher secretion-inducing ability. On the other hand, P1-3-scFv-LH showed lower secretion than the control antibody at concentrations of 8, 3.20, 1.28, and 0.64 μg / mL, indicating clearly reduced IFN-γ secretion-inducing ability (Figure 7B). P2-3-scFv-LH and P3-1-scFv-LH showed higher IFN-γ secretion levels than the control antibody at concentrations of 8, 3.20, 1.28, and 0.64 μg / mL, demonstrating significantly higher inducing ability than the control antibody (Figure 7C). 78-P1-3-scFv-LH showed significantly lower IFN-γ secretion levels than the control antibody at concentrations of 8 μg / mL and 3.20 μg / mL, and hardly induced secretion at low concentrations. Therefore, the IFN-γ secretion-inducing ability of 78-P1-3-scFv-LH was significantly reduced, while 78-P2-3-scFv-LH showed comparable inducing ability to the control antibody (Figure 7D). 79-P1-3-scFv-LH exhibited significantly lower IFN-γ secretion induction ability than the control antibody, and secretion levels at concentrations of 8, 3.20, 0.64, and 0.32 μg / mL were all lower than those of H2C-scFv-LH (Figure 7E).

[0220] Discussion: Existing studies have shown that low-affinity CD3 antibodies can reduce the risk of abnormal cytokine release and mitigate the toxicity of CD3 bispecific antibodies by promoting the distribution of bispecific antibodies to tumor tissue and suppressing their distribution to secondary lymphoid tissue. In this example, P1-3-scFv-LH, 79-P1-3-scFv-LH, and 78-P1-3-scFv-LH significantly reduced IFN-γ secretion, suggesting that bispecific antibodies containing these sequences may have higher safety.

[0221] While representative embodiments of the present invention have been described above, those skilled in the art should understand that these disclosures are merely examples and that various changes, modifications, and substitutions are possible within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments illustrated herein.

[0222] [Table 4] JPEG2026511221000007.jpg250170JPEG2026511221000008.jpg250170JPEG2026511221000009.jpg251170 JPEG2026511221000010.jpg254170JPEG2026511221000011.jpg254170JPEG2026511221000012.jpg249170 JPEG2026511221000013.jpg249170JPEG2026511221000014.jpg254170JPEG2026511221000015.jpg254170 JPEG2026511221000016.jpg254170JPEG2026511221000017.jpg249170JPEG2026511221000018.jpg217170

Claims

1. An antibody or antigen-binding fragment that specifically binds to CD3 and comprises any of the following: (a) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:28 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:29; (b) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:1 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:31; (c) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:32 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:33; (d) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:1 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:34; (e) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:28 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:36; (f) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:39 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:40; or (g) Three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO:28 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO:

43.

2. An antibody or antigen-binding fragment that specifically binds to CD3, comprising a heavy chain variable region and a light chain variable region, (a) The heavy chain variable region includes HCDR1, indicated by GFTFSKYAMN (SEQ ID NO: 51) based on the AbM number; HCDR2, indicated by RIRSKYNNYATY (SEQ ID NO: 46); and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47), and the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48) based on the AbM number; LCDR2, indicated by GTKFLAP (SEQ ID NO: 49); and LCDR3, indicated by ALWYSNRWV (SEQ ID NO: 50). (b) The heavy chain variable region includes HCDR1, indicated by GFTFNKYAMN (SEQ ID NO: 45) based on the AbM number; HCDR2, indicated by RIRSKYNNYATY (SEQ ID NO: 46); and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47); and the light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48) based on the AbM number; LCDR2, indicated by GTKFLAP (SEQ ID NO: 49); and LCDR3, indicated by ALWVDNRWV (SEQ ID NO: 55); (c) The heavy chain variable region includes HCDR1 represented by GFTFPKYAMN (SEQ ID NO: 52) based on the AbM number, HCDR2 represented by RIRSKYNNYETY (SEQ ID NO: 53), and HCDR3 represented by HGNFGNSYISYWAY (SEQ ID NO: 47), and the light chain variable region includes LCDR1 represented by GSSTGAVTSGYYPN (SEQ ID NO: 48) based on the AbM number, LCDR2 represented by GTHFLAP (SEQ ID NO: 54), and LCDR3 represented by ALWYSNRWV (SEQ ID NO: 50). (d) The heavy chain variable region includes HCDR1, indicated by GFTFNKYAMN (SEQ ID NO: 45) based on the AbM number; HCDR2, indicated by RIRSKYNNYATY (SEQ ID NO: 46); and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47). The light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48) based on the AbM number; LCDR2, indicated by GTKFLAP (SEQ ID NO: 49); and LCDR3, indicated by ALWQENRWV (SEQ ID NO: 56); (e) The heavy chain variable region includes HCDR1, indicated by GFTFSKYAMN (SEQ ID NO: 51) based on the AbM number; HCDR2, indicated by RIRSKYNNYATY (SEQ ID NO: 46); and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47). The light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48) based on the AbM number; LCDR2, indicated by GTKFLAP (SEQ ID NO: 49); and LCDR3, indicated by ALWVDNRWV (SEQ ID NO: 55); (f) The heavy chain variable region includes HCDR1, indicated by GFTFSKYAMN (SEQ ID NO: 51) based on the AbM number; HCDR2, indicated by RIRSKYNNYETY (SEQ ID NO: 53); and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47). The light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48) based on the AbM number; LCDR2, indicated by GTHFLAP (SEQ ID NO: 54); and LCDR3, indicated by ALWYSNRWV (SEQ ID NO: 50); or (g) The heavy chain variable region includes HCDR1, indicated by GFTFSKYAMN (SEQ ID NO: 51) based on the AbM number; HCDR2, indicated by RIRSKYNNYATY (SEQ ID NO: 46); and HCDR3, indicated by HGNFGNSYISYWAY (SEQ ID NO: 47). The light chain variable region includes LCDR1, indicated by GSSTGAVTSGYYPN (SEQ ID NO: 48) based on the AbM number; LCDR2, indicated by GTKFLAP (SEQ ID NO: 49); and LCDR3, indicated by ALWQENRWV (SEQ ID NO: 56).

3. An antibody or antigen-binding fragment that specifically binds to CD3 as described in claim 2, comprising any of the following: (a) Heavy chain variable region shown in SEQ ID NO:26 and light chain variable region shown in SEQ ID NO:27; (b) Heavy chain variable region shown in SEQ ID NO:28 and light chain variable region shown in SEQ ID NO:29; (c) Heavy chain variable region shown in SEQ ID NO:30 and light chain variable region shown in SEQ ID NO:2; (d) Heavy chain variable region shown in SEQ ID NO:1 and light chain variable region shown in SEQ ID NO:31; (e) Heavy chain variable region shown in SEQ ID NO:32 and light chain variable region shown in SEQ ID NO:33; (f) Heavy chain variable region shown in SEQ ID NO:1 and light chain variable region shown in SEQ ID NO:34; (g) Heavy chain variable region shown in SEQ ID NO:30 and light chain variable region shown in SEQ ID NO:27; (h) Heavy chain variable region shown in SEQ ID NO:35 and light chain variable region shown in SEQ ID NO:29; (i) Heavy chain variable region shown in SEQ ID NO:30 and light chain variable region shown in SEQ ID NO:29; (j) Heavy chain variable region shown in SEQ ID NO:28 and light chain variable region shown in SEQ ID NO:36; (k) Heavy chain variable region shown in SEQ ID NO:30 and light chain variable region shown in SEQ ID NO:31; (l) Heavy chain variable region shown in SEQ ID NO:37 and light chain variable region shown in SEQ ID NO:38; (m) Heavy chain variable region shown in SEQ ID NO:39 and light chain variable region shown in SEQ ID NO:40; (n) Heavy chain variable region shown in SEQ ID NO:41 and light chain variable region shown in SEQ ID NO:33; (o) Heavy chain variable region shown in SEQ ID NO:26 and light chain variable region shown in SEQ ID NO:42; (p) Heavy chain variable region shown in SEQ ID NO:28 and light chain variable region shown in SEQ ID NO:43; or (q) The heavy chain variable region shown in SEQ ID NO:30 and the light chain variable region shown in SEQ ID NO:

44.

4. An antibody or antigen-binding fragment that specifically binds to CD3 as described in any one of claims 1 to 3, which is an IgG1, IgG2, IgG3, or IgG4 antibody; Optionally, an IgG1 or IgG4 antibody; Any IgG1 antibody is acceptable.

5. An antibody or antigen-binding fragment that specifically binds to CD3 according to any one of claims 1 to 4, The antigen-binding fragments are Fab, Fab', and F(ab'). 2 Fv, single-chain Fv (scFv), single-chain Fab, or diabody.

6. An antibody or antigen-binding fragment that specifically binds to CD3 according to any one of claims 1 to 5, having one or more of the following properties: (i) In measurements by SEC-HPLC, the monomer purity exceeds 90%, preferably exceeding 92%, 94%, 96%, or 97%; (ii) In measurements by reduced or non-reduced SDS-PAGE, the purity exceeds 90%, preferably exceeding 91%, 92%, or 93%; (iii) It specifically binds to CD3 expressed on the cell surface.

7. An isolated nucleic acid encoding an anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1 to 6.

8. A vector comprising the nucleic acid described in claim 7, preferably an expression vector.

9. A host cell comprising the nucleic acid described in claim 7 or the vector described in claim 8, Preferably, the host cell is a prokaryotic cell or a eukaryotic cell. More preferably, selected from E. coli cells, yeast cells, mammalian cells, or other cells suitable for the production of antibodies or their antigen-binding fragments, Most preferably, the host cells are 293 cells or CHO cells.

10. A method for producing an anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1 to 6, The process includes culturing the host cells described in claim 9 under conditions suitable for expressing the nucleic acid encoding the anti-CD3 antibody or its antigen-binding fragment described in any one of claims 1 to 6, A method further comprising the optional step of recovering the anti-CD3 antibody or its antigen-binding fragment from a culture of the host cells.

11. A pharmaceutical composition comprising an anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1 to 6, and optionally a pharmaceutically acceptable carrier.

12. Uses of the anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1 to 6, or the pharmaceutical composition according to claim 11, Applications used in the manufacture of pharmaceuticals for the prevention or treatment of cancer, infectious diseases, or autoimmune diseases in test subjects.

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