CN110172100B

CN110172100B - Anti-human CD3E antibodies and uses thereof

Info

Publication number: CN110172100B
Application number: CN201910372193.6A
Authority: CN
Inventors: 刘志刚; 刘玉兰; 郝小勃; 郭晶晶
Original assignee: Beijing Wisdomab Biotechnology Co ltd
Current assignee: Beijing Wisdomab Biotechnology Co ltd
Priority date: 2019-05-06
Filing date: 2019-05-06
Publication date: 2021-03-19
Anticipated expiration: 2039-05-06
Also published as: CN110172100A

Abstract

The present application discloses antibodies or antigen-binding portions thereof that bind to human CD3E, polynucleotides encoding the antibodies or antigen-binding portions thereof, vectors comprising the polynucleotides, host cells comprising the polynucleotides or vectors, methods of making and purifying the antibodies, and uses of the antibodies or antigen-binding portions thereof.

Description

Anti-human CD3E antibodies and uses thereof

Technical Field

The present application relates generally to the fields of genetic engineering and antibody medicine; in particular to the field of anti-human CD3E antibodies and application thereof. The present application develops a novel anti-human CD3E antibody and provides for the use of the antibody in the prevention or treatment of tumors.

Background

The CD3 molecule is a human immune T cell surface marker antigen and is composed of 4 subunits, namely CD3 epsilon (CD3E), CD3 delta (CD3D), CD3 gamma (CD3G) and CD3 zeta (CD3Z), which are transmembrane proteins. The 6 polypeptide chains, consisting of 4 subunits, surround the T Cell Receptor (TCR) by charge adsorption, forming a TCR-CD3 complex containing 8 polypeptide chains. This complex has the functions of T cell activation signaling and stabilization of TCR structure. The cytoplasmic domain of the CD3 molecule has a common sequence called immunoreceptor tyrosine-based activation motif (ITAM), which allows tyrosine protein kinase p561ck in T cells to phosphorylate tyrosine residues of ITAM in CD3 molecules and then recruit tyrosine protein kinases (e.g., ZAP-70) containing SH2(Scr homology 2) domain due to TCR specific recognition and binding of antigenic peptides presented by MHC (major histocompatibility complex) molecules. ITAM phosphorylation and ZAP-70 binding are among the important biochemical reactions of T cells to activate early signaling processes. Thus, the CD3 molecule functions to conduct the activation signal generated by the recognition of antigen by TCR.

The anti-CD 3E antibody binds to the CD3E subunit of the TCR receptor complex on the surface of T cells and provides a first signal for T cell activation (similar to binding of the MHC-peptide complex on an antigen presenting cell to the TCR) which facilitates T cell activation. And the bispecific antibody aiming at the CD3E and the tumor cell surface antigen (TAA) can realize the enrichment of T cells at the periphery of tumor cells and improve the killing efficiency of the T cells to the tumor cells, thereby having important significance for tumor treatment.

Summary of The Invention

In a first aspect, the application provides an antibody that binds human CD3E, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 amino acid sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 amino acid sequences, wherein

The amino acid sequence of the HCDR1 is GFKFSGY, the amino acid sequence of the HCDR2 is WFDGSR, the amino acid sequence of the HCDR3 is QMGYWHFGL, the amino acid sequence of the LCDR1 is RASQGINNSLT, the amino acid sequence of the LCDR2 is GASNRET, and the amino acid sequence of the LCDR3 is QQWLKLPPT; or

The amino acid sequence of the HCDR1 is GFTFSNA, the amino acid sequence of the HCDR2 is KDKSNNYA, the amino acid sequence of the HCDR3 is VHYGVRFFYTMDV, the amino acid sequence of the LCDR1 is RPSQSLVHNNGNTYLS, the amino acid sequence of the LCDR2 is KVSNRFS, and the amino acid sequence of the LCDR3 is GQGTQYPFT; or

The HCDR1 amino acid sequence is GFTFSNA, the HCDR2 amino acid sequence is KSKSKSDNYA, the HCDR3 amino acid sequence is VHYGAYYGVDA, the LCDR1 amino acid sequence is RSSQSLVHSNRNTYLN, the LCDR2 amino acid sequence is KVSNRFS, and the LCDR3 amino acid sequence is GQGTHAPYA;

wherein the HCDR and LCDR amino acid sequences are defined according to Chothia.

In some embodiments of the first aspect, the heavy chain variable region of the antibody has the amino acid sequence set forth in SEQ ID NO 19, 21, or 23.

In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the antibody is as set forth in SEQ ID NOs 20, 22, or 24.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO. 19, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO. 20; or

The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 21, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 22; or

The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 23, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 24.

In a second aspect, the application provides an antibody that binds human CD3E, wherein the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity to any one of SEQ ID NOs 19, 21 or 23 and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity to any one of SEQ ID NOs 20, 22 or 24.

In some embodiments of the first and second aspects, the antibody is a monoclonal antibody.

In some embodiments of the first and second aspects, the antibody is a whole antibody, a Fab fragment, a F (ab')₂Fragment or single chain Fv fragment (scFv).

In some embodiments of the first and second aspects, the antibody is a fully human antibody.

In some embodiments of the first and second aspects, the antibody further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, and an IgG4 subtype.

In some embodiments of the first and second aspects, the antibody further comprises a light chain constant region selected from the kappa subtype or the lambda subtype.

In some embodiments of the first and second aspects, the antibody is capable of mediating T cell activation.

In a third aspect, the present application provides a nucleic acid molecule encoding the antibody or antigen-binding portion thereof of the first or second aspect.

In a fourth aspect, the present application provides a bispecific antibody comprising a first arm directed to human CD3E and a second arm directed to a tumor cell surface antigen (TAA), wherein the first arm directed to human CD3E comprises:

the amino acid sequence is HCDR1 of GFKFSGY, the amino acid sequence is HCDR2 of WFDGSR, the amino acid sequence is HCDR3 of QMGYWHFGL, the amino acid sequence is LCDR1 of RASQGINNSLT, the amino acid sequence is LCDR2 of GASNRET, and the amino acid sequence is LCDR3 of QQWLKLPPT; or

The amino acid sequence is HCDR1 of GFTFSNA, the amino acid sequence is HCDR2 of KDKSNNYA, the amino acid sequence is HCDR3 of VHYGVRFFYTMDV, the amino acid sequence is LCDR1 of RPSQSLVHNNGNTYLS, the amino acid sequence is LCDR2 of KVSNRFS, and the amino acid sequence is LCDR3 of GQGTQYPFT; or

The amino acid sequence is HCDR1 of GFTFSNA, the amino acid sequence is HCDR2 of KSKSKSDNYA, the amino acid sequence is HCDR3 of VHYGAYYGVDA, the amino acid sequence is LCDR1 of RSSQSLVHSNRNTYLN, the amino acid sequence is LCDR2 of KVSNRFS, and the amino acid sequence is LCDR3 of GQGTHAPYA;

In some embodiments of the fourth aspect, the first arm directed to human CD3E comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NOs 19, 21, or 23.

In some embodiments of the fourth aspect, the first arm to human CD3E comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NOs 20, 22, or 24.

In some embodiments of the fourth aspect, the first arm for human CD3E comprises:

a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 19 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 20; or

A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 21 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 22; or

Heavy chain variable region with amino acid sequence shown as SEQ ID NO. 23, and light chain variable region with amino acid sequence shown as SEQ ID NO. 24.

In a fifth aspect, the present application provides a bispecific antibody comprising a first arm directed to human CD3E and a second arm directed to a tumor cell surface antigen (TAA), wherein the first arm directed to human CD3E comprises: a heavy chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs 19, 21 or 23, and a light chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs 20, 22 or 24.

In some embodiments of the fourth and fifth aspects, the first arm for human CD3E comprises a Fab fragment for human CD3E, or a single chain Fv fragment (scFv).

In some embodiments of the fourth and fifth aspects, the first arm directed to human CD3E further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, and an IgG4 subtype.

In some embodiments of the fourth and fifth aspects, the first arm against human CD3E further comprises a light chain constant region selected from the kappa subtype or the lambda subtype.

In some embodiments of the fourth and fifth aspects, the tumor cell surface antigen (TAA) comprises HER2, BCMA, CD123, GPC3, FAP, VEGFR, EGFR, PD-L1, CD19, CD125, HGFR, FGFR2, IGF-1R, SALL4, or LASEP 1. In some embodiments of the fourth and fifth aspects, the bispecific antibody is capable of effecting an enrichment of T cells in the vicinity of tumor cells.

In some embodiments of the fourth and fifth aspects, the bispecific antibody increases the killing efficiency of T cells against tumor cells.

In a sixth aspect, the present application provides a pharmaceutical composition comprising an antibody of the first or second aspect, or a bispecific antibody of the fourth or fifth aspect, and a pharmaceutically acceptable excipient, diluent or carrier.

In some embodiments, the pharmaceutical composition is for preventing or treating a tumor.

In some embodiments, the tumor is a malignant tumor.

In a seventh aspect, the present application provides the use of an antibody of the first or second aspect, or a bispecific antibody of the fourth or fifth aspect, in the manufacture of a medicament for the prevention or treatment of a tumour.

In some embodiments, the tumor is a malignant tumor.

In an eighth aspect, the application provides a method of preventing or treating a tumor comprising administering to an individual in need thereof an antibody of the first or second aspect, or a bispecific antibody of the fourth or fifth aspect, or a pharmaceutical composition of the seventh aspect.

In some embodiments, the tumor is a malignant tumor.

Drawings

FIG. 1 shows ELISA assays for the binding ability of anti-human CD3E monoclonal antibody (phage scFv) to different CD 3E.

FIG. 2 shows ELISA analysis of the binding ability of various anti-human CD3E monoclonal antibodies to CD3E recombinant protein.

FIG. 3 shows flow cytometry analysis of the binding capacity of different anti-human CD3E monoclonal antibodies to PBMCs of different species.

FIG. 4 shows the results of the measurement of the biological activity of T cells activated by different anti-CD 3E monoclonal antibodies.

Description of the amino acid sequence

SEQ ID NO:1 shows the amino acid sequence of the extracellular region of recombinant human (homo sapiens) CD3E (huCD 3E-ECD).

SEQ ID NO:2 shows the amino acid sequence of the extracellular region of recombinant human (homo sapiens) CD3D (huCD 3D-ECD).

SEQ ID NO. 3 shows the amino acid sequence of the extracellular region of recombinant cynomolgus monkey (Macaca fascicularis) CD3E (mfCD 3E-ECD).

SEQ ID NO. 4 shows the amino acid sequence of the extracellular region of recombinant cynomolgus monkey (Macaca fascicularis) CD3D (mfCD 3D-ECD).

SEQ ID NO:5 shows the amino acid sequence of the extracellular domain of mouse (mus musculus) CD3E (mCD 3E-ECD).

SEQ ID NO 6 shows the amino acid sequence of the extracellular domain of mouse (mus musculus) CD3D (mCD 3D-ECD).

SEQ ID NO 7 shows the amino acid sequence of His tag (His).

SEQ ID NO 8 shows the amino acid sequence of the Fc fragment (mFc) of the mouse (mus musculus) antibody IgG2 a.

SEQ ID NO 9 shows the amino acid sequence of an Fc fragment mutant (FcK) of human IgG1 antibody.

SEQ ID NO 10 shows the amino acid sequence of an Fc fragment mutant (FcH) of human IgG1 antibody.

SEQ ID NO:11 shows the amino acid sequence of the constant region of the human (homo sapiens) IgG1 subtype heavy chain.

SEQ ID NO:12 shows the amino acid sequence of the constant region of the human (homo sapiens) IgG2 subtype heavy chain.

SEQ ID NO 13 shows the amino acid sequence of the constant region of the human (homo sapiens) IgG4 subtype heavy chain.

SEQ ID NO:14 shows the amino acid sequence of the constant region of the kappa subtype human (homo sapiens) light chain.

SEQ ID NO 15 shows the amino acid sequence of the human (homo sapiens) subtype lambda light chain constant region.

SEQ ID NO 16 shows the amino acid sequence of the anti-human CD3E single-chain antibody S1C3, and SEQ ID NO 19 and 20 show the amino acid sequences of the VH and VL sequences, respectively.

SEQ ID NO 17 shows the amino acid sequence of the anti-human CD3E single-chain antibody S1F6, and SEQ ID NO 21 and 22 show the amino acid sequences of the VH and VL sequences, respectively.

SEQ ID NO 18 shows the amino acid sequence of the anti-human CD3E single-chain antibody S4C1, and SEQ ID NO 23 and 24 show the amino acid sequences of their VH and VL sequences, respectively.

Detailed Description

The inventors of the present application obtained a novel anti-human CD3E antibody by antibody engineering techniques. In various aspects of the present application, there are provided novel anti-human CD3E antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, methods of making and purifying the antibodies, and medical and biological applications of the antibodies or antigen-binding fragments thereof. According to the amino acid sequence of the variable region of the antibody provided by the application, a full-length antibody molecule can be constructed to be used as a medicine for preventing or treating tumors.

The practice of the present application employs, unless otherwise indicated, conventional molecular biology, microbiology, cell biology, biochemistry, and immunology techniques.

Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.

Definition of

The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like. As used herein, "antibody" includes not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (e.g., Fab ', F (ab')₂Fv), variants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of immunoglobulin molecules comprising an antigen recognition site of a desired specificity, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies.

Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second and third constant regions (CH1, CH2 and CH 3). Each light chain contains a light chain variable region (VL) and a constant region (CL). Full-length antibodies can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), but the antibodies need not belong to any particular class. Depending on the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The heavy chain constant domains corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

The term "antigen-binding fragment or antigen-binding portion" as used herein refers to a portion or region of an intact antibody molecule that is responsible for binding an antigen. The antigen-binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementarity determining regions CDR1, CDR2, and CDR 3.

It is well known to those skilled in the art that the complementarity determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions of the variable region that have the greatest impact on the affinity and specificity of an antibody. There are two common definitions of CDR amino acid sequences for VH or VL, namely the Chothia definition and the Kabat definition. (see, e.g., Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); A1-Lazikani et al, J.mol.biol.273: 927-. For a given antibody variable region amino acid sequence, according to Chothia definition or Kabat definition to determine VH and VL amino acid sequence in CDR amino acid sequence. In embodiments of the present application, Chothia is used to define CDR amino acid sequences.

For a given antibody variable region amino acid sequence, the CDR amino acid sequence within the variable region amino acid sequence can be analyzed in a variety of ways, such as can be determined using the online software Abysis (http:// www.abysis.org /).

Examples of antigen-binding fragments include, but are not limited to: (1) a Fab fragment, which can be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) f (ab')₂A fragment, which may be a bivalent fragment having two Fab 'fragments linked by a disulfide bridge of the hinge region (i.e., a dimer of Fab'); (3) (ii) an Fv fragment having VL and VH domains of a single arm of an antibody; (4) single chain fv (scfv), which may be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) (scFv)₂It may comprise two VH domains connected by a peptide linker and two VL domains, the two VL domains being combined with the two VH domains via a disulphide bridge.

The term "bispecific antibody" as used herein is an antibody having the ability to bind two different antigens, which may consist of two Fc fragments and two antigen binding moieties fused thereto, respectively.

The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, e.g. binding of an antibody to an epitope of an antigen.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of naturally occurring mutations in a small number of individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding amino acid sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy and/or light chain is identical or homologous to corresponding amino acid sequences in antibodies derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The term "tumor" as used herein refers to a neoplasm or solid lesion formed by abnormal cell growth. Tumors may be benign, premalignant or malignant.

The term "malignancy" as used herein refers to or describes a physiological condition of a mammal that is typically characterized by unregulated cell growth. Exemplary malignancies include: carcinoma, solid tumor, melanoma sarcoma, hematologic tumor, germ cell tumor, and blastoma. More specific examples of malignancies include: kidney cancer, lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, liver cancer (hepatoma), stomach cancer including gastrointestinal cancer, prostate cancer, pancreatic cancer, peritoneal cancer, hepatocellular cancer, glioblastoma, ovarian cancer, liver cancer (liver cancer), urinary tract cancer, hepatoma, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, squamous cell carcinoma (e.g., squamous cell carcinoma), vulval cancer, thyroid cancer, anal cancer, penile cancer, melanoma, multiple myeloma and B-cell lymphoma, brain and head and neck cancer, and associated metastases.

The term "hematological tumor" as used herein refers to a tumor caused by uncontrolled growth and proliferation of abnormal cells, the site of origin of which in most cases is bone marrow, which is where the blood cells originate. Exemplary hematological tumors include various leukemias, multiple myeloma, and malignant lymphoma. More specific examples of hematological tumors include: acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), Hairy Cell Leukemia (HCL), T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, juvenile myelomonocytic leukemia, B-cell prolymphocytic leukemia, Burkitt's leukemia and adult T-cell leukemia, non-Hodgkin's lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, primary macroglobulinemia ((ALL) ((II)

macrogolbulilinimia), splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, intranodal marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, burkitt lymphoma, B-cell chronic lymphocytic lymphoma, classical hodgkin lymphoma, hodgkin lymphoma predominantly nodal, adult T-cell lymphoma, extranodal rhino-type NK/T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blast cell NK-cell lymphoma, mycosis fungoides, sence syndrome, primary skin CD30 positive T-cell lymphoproliferative disorder, primary skin anaplastic large cell lymphoma, lymphoma-like papulosis, lymphomatoid papulosis, lymphomatosis, Angioimmunoblastic T-cell lymphoma, non-finger peripheral T-cell lymphoma, and anaplastic large cell lymphoma.

The term "solid tumor" as used herein refers to a tangible mass that can be palpated by clinical examination such as X-ray film, CT scan, B-ultrasound or palpation. Clinically diagnosed solid tumors are both malignant and benign. Malignant solid tumors include: hodgkin lymphoma in children: the lymphocyte is main type, nodule sclerosis type, mixed cell type, lymphocyte depletion type; non-hodgkin's lymphoma in children: lymphoblastic lymphoma, small non-dividing cell lymphoma (burkitt/non-burkitt lymphoma), diffuse large B cell lymphoma, anaplastic large cell lymphoma, and the like; kidney tumor in children: wilms' tumor, clear cell carcinoma of kidney, striated myosarcoma of kidney, clear cell sarcoma of kidney, renal primitive extraembryonic phylloma, etc.; neuroblastoma in children: neuroblastoma, nodal neuroma; pediatric extracranial germ cell tumors: mature teratoma, immature teratoma, endoblastoma (yolk sac tumor), seminoma, dysgerminoma, chorioepithelioma, embryo cancer, etc.; osteosarcoma and chondrosarcoma; rhabdomyosarcoma of children: embryonic, acinar, polymorphous, etc.; soft tissue sarcoma in children: fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant schwannoma, acinar soft tissue sarcoma, epithelioid sarcoma, hyaline cell sarcoma, malignant melanoma, synovial sarcoma, fibroproliferative small round cell tumor, etc.; ewing's family sarcoma: ewing's sarcoma, primitive neuroectodermal leaf tumors; liver tumor of children: hepatoblastoma (embryonal, fetal, undifferentiated), hepatocellular carcinoma; retinoblastoma; other tumors: posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, thymoma, pneumocoblastoma, pancreatoblastoma, islet cell tumor of pancreas, ileocecal carcinoid, mesothelioma, etc. Benign solid tumors include: lymphangioma, hemangioma, thyroid cyst, and the like.

In some embodiments, the heavy chain variable region of the antibody has the amino acid sequence set forth in SEQ ID NO 19, 21, or 23.

In some embodiments, the amino acid sequence of the variable region of the light chain of the antibody is set forth in SEQ ID NOs 20, 22, or 24.

In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO. 19, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO. 20; or

In some embodiments of the first and second aspects, the heavy chain constant region is of the IgG1 subtype.

In some embodiments of the first and second aspects, the light chain constant region is of the kappa subtype.

In some embodiments, the nucleic acid molecule is operably linked to a regulatory amino acid sequence that can be recognized by a host cell transformed with the vector.

Since bispecific antibodies have two different antigen binding portions for two different antigens, the antigen binding portions may be in the form of either single chain antibodies (scfv) or Fab fragments.

Bispecific antibodies are also described herein as having two "arms," e.g., bounded by a middle, the bispecific antibody can be split into two arms. The arms of a bispecific antibody may consist of an Fc fragment and an antigen binding portion (Fab fragment or single chain antibody). For arms composed of an Fc fragment and a Fab fragment, which are structurally similar to conventional antibodies, containing the entire heavy and light chains, the structure of such arms can be represented as Fc + Fab, and also as heavy chain (the variable region of the heavy chain in Fc + Fab and the CH1 fragment) + light chain (the light chain portion in Fab). When both arms contain an antigen binding moiety in the form of a Fab fragment, the structure of the bispecific antibody thus formed is close to that of a natural antibody.

In some specific embodiments of the fourth and fifth aspects, the heavy chain constant region is of the IgG1 subtype.

In some specific embodiments of the fourth and fifth aspects, the light chain constant region is of the kappa subtype.

In some embodiments of the fourth and fifth aspects, the tumor cell surface antigen (TAA) comprises HER2, BCMA, CD123, GPC3, FAP, VEGFR, EGFR, PD-L1, CD19, CD125, HGFR, FGFR2, IGF-1R, SALL4, or LASEP 1.

In some embodiments of the fourth and fifth aspects, the bispecific antibody is capable of effecting an enrichment of T cells in the vicinity of tumor cells.

In some embodiments, the tumor is a malignant tumor.

In some embodiments, the pharmaceutical composition may further comprise one or more of the following: lubricants, such as talc, magnesium stearate and mineral oil; a wetting agent; an emulsifier; a suspending agent; preservatives, such as benzoic acid, sorbic acid and calcium propionate; sweeteners and/or flavoring agents, and the like.

In some embodiments, the pharmaceutical compositions herein can be formulated in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules.

In some embodiments, the pharmaceutical compositions of the present application may be delivered using any physiologically acceptable mode of administration, including, but not limited to: oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.

In some embodiments, pharmaceutical compositions for therapeutic use may be formulated for storage in lyophilized formulations or aqueous solutions by mixing the agent with the desired purity, optionally with pharmaceutically acceptable carriers, excipients, and the like.

In some embodiments, the tumor is a malignant tumor.

In other aspects, the present application also provides isolated nucleic acid molecules encoding the antibodies of the invention or the light or heavy chains thereof as well as vectors comprising the nucleic acid molecules, host cells comprising the vectors, and methods of producing the antibodies. In some embodiments, the nucleic acid molecule is operably linked to a regulatory amino acid sequence that can be recognized by a host cell transformed with the vector. In some embodiments, the method of producing an antibody comprises culturing a host cell to facilitate expression of the nucleic acid. In some embodiments, the method of producing an antibody further comprises recovering the antibody from the host cell culture medium.

In addition, antibodies described herein that specifically bind to human CD3E can also be used to detect the presence of CD3E in a biological sample. Antibody-based detection methods are well known in the art and include, for example, ELISA, immunoblotting, radioimmunoassay, immunofluorescence, immunoprecipitation, and other related techniques.

It should be understood that the above detailed description is only for the purpose of making the content of the present application more clearly understood by those skilled in the art, and is not intended to be limiting in any way. Various modifications and changes to the described embodiments will be apparent to those skilled in the art.

Examples

The following examples are for the purpose of illustration only and are not intended to limit the scope of the present application.

Example 1: preparation of recombinant proteins

A number of different recombinant proteins were used in the preparation of anti-CD 3E monoclonal antibodies, including recombinant human CD3E extracellular domain (huCD3E-ECD, SEQ ID NO:1), human CD3D extracellular domain (huCD3D-ECD, SEQ ID NO:2), cynomolgus monkey CD3E extracellular domain (mfCD3E-ECD, SEQ ID NO:3), cynomolgus monkey CD3D extracellular domain (mfCD3D-ECD, SEQ ID NO:4), mouse CD3E extracellular domain (mCD3E-ECD, SEQ ID NO:5), mouse CD3D extracellular domain (mCD3D-ECD, SEQ ID NO: 6). These recombinant proteins have a number of post-translational modifications (e.g., glycosylation or disulfide bonding) and thus, the use of mammalian cell expression systems would be more advantageous in maintaining the structure and function of the recombinant protein. In addition, for the convenience of purification, a His tag (SEQ ID NO:7) or Fc fragment (mFc, SEQ ID NO:8) of mouse antibody IgG2a was added to the C-terminus of the recombinant protein of the non-antibody class. In addition, on the cell surface, CD3E was able to form heterodimers with CD3D or CD3G, so to prepare CD3E antigen with native conformation, we used Fc fragment mutants of human IgG1 antibody based on the KIH (Knob-into-Hole) technique (FcK, SEQ ID NO:9 or FcH, SEQ ID NO:10), fused CD3E to the N-terminus of Fc (FcK) containing Knob mutation, fused CD3D to the N-terminus of Fc (FcH) containing Hole mutation, prepared CD3E and CD3D heterodimers. In preparing a recombinant antibody, the antibody heavy chain constant region may be of human IgG1 subtype (SEQ ID NO:11), IgG2 subtype (SEQ ID NO:12) or IgG4 subtype (SEQ ID NO:13), and the light chain constant region may be of human kappa subtype (SEQ ID NO:14) or lambda subtype (SEQ ID NO: 15).

Genes (including His tag, mFc or Fc coding gene) of the above-mentioned various recombinant proteins were designed and synthesized based on the amino acid sequences of various recombinant proteins of interest in the Uniprot database. The synthesized recombinant protein genes are cloned to a proper eukaryotic expression vector (such as pcDNA3.1 of Invitrogen company) by utilizing the conventional molecular biology technology, and then the prepared recombinant protein expression plasmid is transfected into HEK293 cells (such as HEK293F of Invitrogen company) by utilizing liposomes (such as 293fectin of Invitrogen company) or other transfection reagents (such as PEI and the like), and the cells are cultured for 3-5 days under the serum-free suspension culture condition. The culture supernatant is then harvested by centrifugation or the like.

The recombinant protein expressed by His tag fusion is purified in one step by using a metal chelating affinity column (e.g., HisTrap FF from GE). The recombinant protein and recombinant antibody expressed by the mFc fusion are purified in one step by a ProteinA/G affinity chromatography column (e.g., Mabselect SURE from GE, Inc.). The recombinant protein storage buffer is then replaced with PBS buffer (pH7.0) or other suitable buffer using a desalting column (e.g., Hitrap desalting, Inc., etc.). If necessary, the antibody sample may be sterilized by filtration, and then stored at-20 ℃.

Example 2: screening of anti-human CD3E monoclonal antibody by phage display antibody library technology

Recombinant human CD3ECD3D heterodimer (huCD3ECD3D) prepared in example 1 is used as antigen, phage presenting a human single-chain antibody library is screened by using a solid phase screening strategy (the experimental technical process can be seen in example 1 in Chinese patent application No. 201510097117.0), and finally 3 humanized single-chain antibodies S1C3(SEQ ID NO:16, the amino acid sequences of VH and VL are respectively SEQ ID NO:19 and 20), S1F6(SEQ ID NO:17, the amino acid sequences of VH and VL are respectively SEQ ID NO:21 and 22), and S4C1(SEQ ID NO:18, the amino acid sequences of VH and VL are respectively SEQ ID NO:23 and 24) which specifically bind to human CD3E are obtained. Homology analysis of the amino acid sequences of the light and heavy chain variable regions of 3 humanized single-chain antibody molecules was performed by using an NCBI website online tool IGBLAST, and the results showed that VH of S1C3 is from IGHV3-33, and VL is from IGKV 1-33; VH of S1F6 comes from IGHV3-15, VL comes from IGKV 2-29; VH of S4C1 comes from IGHV3-15, VL comes from IGKV 2-30.

The prepared recombinant human CD3ECD3D heterodimer (huCD3ECD3D), cynomolgus monkey CD3ECD3D heterodimer (mfCD3ECD3D), mouse CD3ECD3D heterodimer (mCD3ECD3D), human CD3E (huCD3E-his), human CD3D (huCD3D-his), cynomolgus monkey CD3E (mfCD3E-his) and mouse CD3E (mCD3E-his) were coated with 96-well ELISA plates (1. mu.g/ml, 100. mu.l/well) respectively and overnight at 4 ℃. After blocking with blocking solution PBS-0.1% Tween 20-3% milk at 37 ℃ for 1 hour, phage supernatants (phage scFv) of recombinant anti-CD 3E monoclonal antibodies S1C3, S1F6 and S4C1 were added, 100. mu.l/well, and bound at 37 ℃ for 1 hour. The ELISA plates were washed with PBST buffer, and HRP anti-M13 antibody (Sinobiological, 11973-MM05T-H) was added to bind for 1 hour at 37 ℃. Washing ELISA plate with PBST buffer, adding OPD substrate developing solution, and applying 1M H after 5-10 min₂SO₄Terminate the color development toThe optical density values were measured with a microplate reader at a double wavelength of 492nm/630 nm. The ELISA assay results (figure 1) showed that S1C3 recognized huCD3ECD3D heterodimer, did not recognize human CD3E subunit (huCD3E-his), and crossed with no species of cynomolgus monkey CD3E and mouse CD 3E; S1F6 recognizes human CD3E subunit (huCD3E-his), has clear binding signals with huCD3E-his and huCD3ECD3D, has species crossing with cynomolgus monkey CD3E, and does not recognize mouse CD 3E; S4C1 recognizes human CD3E subunit (huCD3E-his), has clear binding signals with both huCD3E-his and huCD3ECD3D, has species cross with cynomolgus monkey CD3E, and does not recognize mouse CD 3E.

Example 3: analysis of anti-human CD3E monoclonal antibody binding to CD3E recombinant protein

Genes encoding light chains and heavy chains of single-chain antibodies S1C3, S1F6 and S4C1, respectively, were cloned into eukaryotic expression vectors using conventional molecular biology methods to prepare recombinant human antibodies in the form of human IgG 1-kappa.

The prepared recombinant human CD3E (huCD3E-his), human CD3D (huCD3D-his), cynomolgus monkey CD3E (mfCD3E-his) and mouse CD3E (mCD3E-his) were coated on 96-well ELISA plates (1. mu.g/ml, 100. mu.l/well), respectively, and overnight at 4 ℃. After blocking with blocking solution PBS-0.1% Tween 20-3% milk at 37 ℃ for 1 hour, each of the recombinant anti-CD 3E monoclonal antibodies S1C3, S1F6 and S4C1 (1.0. mu.g/100. mu.l/well) was added and bound at 37 ℃ for 1 hour. The ELISA plates were washed with PBST buffer, and HRP mouse anti-human IgG (bios, bsm-0297M-HRP) was added and bound for 1 hour at 37 ℃. Washing ELISA plate with PBST buffer, adding OPD substrate developing solution, and applying 1M H after 5-10 min₂SO₄The color development was stopped and the optical density value was determined at a double wavelength of 492nm/630nm using a microplate reader. The ELISA assay (figure 2) showed that S1C3 was unable to recognize CD3E subunit alone, S1F6 and S4C1 recognized both human CD3E and cynomolgus monkey CD3E, with no species crossover to mouse CD 3E.

Example 4: anti-human CD3E monoclonal antibody binding PBMC assay

Anti-human CD3E monoclonal antibodies S1C3, S1F6 and S4C1 (all at 10. mu.g/ml) were mixed with human PBMC, rhesus PBMC and mouse PBMC, respectively, in V-type 96-well plates (cells diluted to 10 x 10)⁶Ml, mixing the antibody and cells in equal volumes of 100. mu.l/well each), incubating at 4 ℃ for 60 min;washing with PBS buffer for 2 times; incubated with goat anti-human IgG antibody-FITC (1:400 dilution, 100. mu.l/well) at 4 ℃ for 30min, washed 2 times with PBS buffer and detected by flow cytometry. The results (figure 3 and table 1) show that S1C3 can bind to human PBMC, but not rhesus and mouse PBMC; S1F6 and S4C1 recognize both human and rhesus PBMC, but do not bind mouse PBMC.

TABLE 1 binding of anti-CD 3E monoclonal antibodies to human, rhesus and mouse PBMCs

MFI(FITC)	Control (no antibody added)	S1C3	S1F6	S4C1
					Human PBMC	11860	58155	18401	16730
Rhesus monkey PBMC	3470	3430	20431	18564
					Mouse PBMC	3818	3426	2550	1454

Combining the experimental results in example 3 and example 4, the following conclusions can be drawn: S1C3 does not recognize the human CD3E single subunit, but can normally bind to the CD3ECD3D heterodimer on the surface of human T cells; S1C3 did not bind to cynomolgus monkey CD3E at molecular level in example 3, and S1C3 did not bind to rhesus monkey cell surface CD3E in example 4, so there was no species crossover of S1C3 with both cynomolgus and rhesus monkeys; there was also no species crossover of S1C3 with mouse CD 3E; S1F6 and S4C1 recognized the human CD3E single subunit, were species crossed with cynomolgus and rhesus CD3E, and did not recognize mouse CD 3E.

Example 5: affinity assay for anti-human CD3E monoclonal antibody binding to CD3E

The affinity of each anti-CD 3E monoclonal antibody (S1C3, S1F6, and S4C1) was determined using Biacore X100 plus. The amino conjugate kit, the human antibody capture kit, the CM5 chip, and related reagents and consumables such as 10 XHBS-EP at pH7.4 were purchased from GE healthcare. According to the instruction in the kit, an antibody of an anti-human Fc fragment is coupled to the surface of a CM5 chip by using an amino coupling method, then antibody protein is diluted to a proper concentration (S1C 3: 1 mug/ml, S1F 6: 3 mug/ml, S4C 1: 1 mug/ml), and injection is carried out for 30S at 10 mug/min, so that about 100RU of the antibody is captured by the anti-human Fc antibody. A series of concentration gradients (100nM, 33.3nM, 11.1nM, 3.7nM and 1.23nM) were applied to huCD3ECD3D or mfCD3ECD3D, 30. mu.l/min was injected for 120s, dissociated for 600s, the chip surface was regenerated with 10mM Glycine-HCl pH1.7, and the affinity of each monoclonal antibody was determined at 25 ℃. Biacore data were analyzed using Biacore X100 evaluation software version 2.0.1, and the fitting results are shown in table 2 and table 3, respectively.

TABLE 2 affinity constants for anti-CD 3E monoclonal antibody binding to huCD3ECD3D

Class of antibody	Kon	Koff	KD
				S1C3	8.143E+4	3.563E-3	4.375E-8
S1F6	7.396E+4	1.503E-3	2.032E-8
				S4C1	1.333E+5	1.558E-3	1.169E-8

TABLE 3 affinity constants for anti-CD 3E monoclonal antibody binding to mfCD3ECD3D

Class of antibody	Kon	Koff	KD
				S1C3	Is not bonded	-	-
S1F6	2.601E+4	7.353E-4	2.827E-8
				S4C1	2.497E+4	1.539E-3	6.163E-8

Example 6: analysis of anti-human CD3E monoclonal antibody mediated T cell activation

The bioactivity analysis of the anti-CD 3E monoclonal antibody activated T cells was performed based on the Jurkat Dual cell line. The coating concentration of the anti-CD 28 monoclonal antibody is 4 mug/ml, the initial concentration of the anti-CD 3E monoclonal antibody is 30 mug/ml, 2-time gradient dilution is carried out, 10 concentration gradients are obtained, the anti-CD 28 monoclonal antibody and the anti-CD 3E monoclonal antibody with different concentrations are mixed in equal volumes, the mixture is paved in a 96-well plate, 3 multiple wells are arranged in each concentration, 50 mug/well is arranged, and the mixture is placed in a clean workbench to be dried in the air overnight air. The following day Jurkat Dual cells were counted and resuspended to 1 x10 cells with media without selection pressure⁶Adding 100 mu l of each cell/ml into a 96-well plate, continuously culturing for 20h, centrifuging at 1500rpm for 5min, adding 50 mu l of supernatant into a new 96-well plate, adding 50 mu l of Quanti luc detection solution, reading at full wavelength by using a microplate reader, and obtaining detection results shown in figure 4, wherein the Jurkat Dual cells can be activated by S1C3, S1F6 and S4C 1. EC (EC)₅₀The values (table 4) show that S1C3, S1F6, and S4C1 have no significant difference in activating activity on Jurkat Dual cells.

TABLE 4 anti-CD 3E monoclonal antibodiesEC activating Jurkat Dual cells₅₀Value of

	S1C3	S1F6	S4C1
				EC₅₀(M)	3.679*10^-9	2.724*10^-9	3.432*10^-9

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Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

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<213> Artificial Sequence (Artificial Sequence)

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Gln Ile Lys Asp Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ala Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Tyr Val His Tyr Gly Val Arg Phe Phe Tyr Thr Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr

130 135 140

Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile

145 150 155 160

Ser Cys Arg Pro Ser Gln Ser Leu Val His Asn Asn Gly Asn Thr Tyr

165 170 175

Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

180 185 190

Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

210 215 220

Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly Thr Gln Tyr Pro Phe

225 230 235 240

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

245 250

<210> 18

<211> 249

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Lys Ser Lys Ser Asp Asn Tyr Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser

130 135 140

Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys

145 150 155 160

Arg Ser Ser Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu Asn

165 170 175

Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys

180 185 190

Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly

195 200 205

Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp

210 215 220

Val Gly Val Tyr Tyr Cys Gly Gln Gly Thr His Ala Pro Tyr Ala Phe

225 230 235 240

Gly Gln Gly Thr Lys Leu Glu Ile Lys

245

<210> 19

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Phe Asp Gly Ser Arg Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Met Gly Tyr Trp His Phe Gly Leu Trp Gly Arg Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 20

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Asn Ser

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Leu Lys Leu Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 21

<211> 124

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Gln Ile Lys Asp Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ala Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Tyr Val His Tyr Gly Val Arg Phe Phe Tyr Thr Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 22

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Pro Ser Gln Ser Leu Val His Asn

20 25 30

Asn Gly Asn Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly

85 90 95

Thr Gln Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 23

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Lys Ser Lys Ser Asp Asn Tyr Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 24

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly

85 90 95

Thr His Ala Pro Tyr Ala Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Claims

1. An antibody that binds human CD3E, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 amino acid sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 amino acid sequences, wherein

2. The antibody of claim 1, wherein the heavy chain variable region of the antibody has the amino acid sequence set forth in SEQ ID NO 19, 21 or 23.

3. The antibody of claim 1, wherein the variable region of the light chain of said antibody has the amino acid sequence shown in SEQ ID NO 20, 22 or 24.

4. The antibody of claim 1, wherein

The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 19, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 20; or

5. The antibody of any one of claims 1-4, wherein the antibody is a whole antibody, a Fab fragment, F (ab')₂Fragment or single chain Fv fragment (scFv).

6. The antibody of any one of claims 1-4, wherein the antibody is a fully human antibody.

7. The antibody of any one of claims 1-4, wherein the antibody is a monoclonal antibody.

8. The antibody of any one of claims 1-4, wherein the antibody further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype.

9. The antibody of claim 8, wherein the heavy chain constant region is of the IgG1 subtype.

10. The antibody of any one of claims 1-4, wherein the antibody further comprises a light chain constant region selected from the kappa subtype or the lambda subtype.

11. The antibody of claim 10, wherein the light chain constant region is of the kappa subtype.

12. The antibody of any one of claims 1-4, wherein the antibody is capable of mediating T cell activation.

13. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1-12.

14. A bispecific antibody comprising a first arm directed to human CD3E and a second arm directed to a tumor cell surface antigen (TAA), wherein the first arm directed to human CD3E comprises:

15. The bispecific antibody of claim 14, wherein the first arm directed to human CD3E comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NOs 19, 21, or 23.

16. The bispecific antibody of claim 14, wherein the first arm directed to human CD3E comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NOs 20, 22 or 24.

17. The bispecific antibody of claim 14, wherein the first arm directed to human CD3E comprises:

18. The bispecific antibody of any one of claims 14-17, wherein the first arm directed to human CD3E comprises a Fab fragment or a single chain Fv fragment (scFv) directed to human CD 3E.

19. The bispecific antibody of any one of claims 14-17, wherein the first arm directed to human CD3E further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype.

20. The bispecific antibody of claim 19, wherein the heavy chain constant region is of the IgG1 subtype.

21. The bispecific antibody of any one of claims 14-17, wherein the first arm directed to human CD3E further comprises a light chain constant region selected from the kappa subtype or the lambda subtype.

22. The bispecific antibody of claim 21, wherein the light chain constant region is of the kappa subtype.

23. The bispecific antibody of any one of claims 14-17, wherein

The bispecific antibody can realize the enrichment of T cells in the periphery of tumor cells, and/or

The bispecific antibody can improve the killing efficiency of T cells to tumor cells.

24. A pharmaceutical composition comprising the antibody of any one of claims 1-12 or the bispecific antibody of any one of claims 14-23, and a pharmaceutically acceptable excipient, diluent or carrier.

25. Use of the antibody of any one of claims 1-12 or the bispecific antibody of any one of claims 14-23 in the manufacture of a medicament for the prevention or treatment of a tumor.

26. The use of claim 25, wherein the tumor is a malignant tumor.