Binding molecule targeting CD3 and cea

Abstract

Provided is a bispecific antibody targeting a CD3 molecule and a tumor-associated antigen (TAA). The tumor-associated antigen is CEA. The bispecific antibody includes a first binding domain targeting the CD3 molecule and a second binding domain targeting the CEA, wherein the first binding domain only binds to the epsilon (ε) subunit in the delta (δ) - epsilon (ε) heterodimer of the CD3 molecule, and does not bind to the epsilon (ε) subunit in the gamma (γ) - epsilon (ε) heterodimer of the CD3 molecule. The provided CD3 × CEA bispecific antibody has a strong activation effect on immune cells and triggers less cytokine release activity, and can also effectively recruit T cells at a tumor cell site, thereby achieving a good tumor cell killing effect.

Description

Binding molecules targeting CD3 and CEA

[0001] Cross-reference to related applications

[0002] This patent application claims the priority benefit of Chinese patent application No. CN202410828045.1, filed on June 25, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the field of biological medicine, and specifically, the present application relates to a binding molecule targeting human CD3 and CEA, and the application of the binding molecule. BACKGROUND

[0004] CD3 plays a key role in T cell activation and signal transduction. CD3 molecules are mainly distributed on the surface of mature T lymphocytes and are important differentiation antigens on the membrane of T cells. The subunits of CD3 molecules combine with the subunits of T cell receptors (TCR) to form heterodimers, which together constitute the TCR-CD3 complex. When TCR binds to antigen, the CD3 target is activated, promoting downstream signal transduction and triggering the immune response of T cells, so that immune cells attack infected areas, tumors and other abnormal cells. Therefore, CD3 is a key regulatory point of T cell immune response, and intervention on the CD3 target can regulate the degree of T cell activation and the intensity of immune response, which has important significance in the treatment of immune diseases and tumor immunotherapy.

[0005] CD3 protein has important clinical applications in the medical field. For example, CAR-T cell therapy in immunotherapy uses the knowledge of CD3 zeta signal to design artificial T cells that can more effectively recognize and attack tumor cells, and this treatment has achieved remarkable results in the treatment of some leukemia and lymphoma.

[0006] OKT3 is the first approved therapeutic CD3 antibody. OKT3 was first approved by the US FDA in 1985 for the treatment of acute rejection after organ transplantation, which is a mouse monoclonal antibody that can specifically react with the TCR-CD3 complex on the surface of T cells by binding to a glycoprotein CD3 epsilon chain, thereby activating circulating T cells. It is reported that OKT3 cannot bind to CD3-epsilon chain single transfected cells. That is, the recognition of OKT3 to CD3 epsilon chain is based on the conformational epitope on two or more CD3 subunits. This interaction leads to transient activation of T cells, release of cytokines, and inhibition of T cell proliferation and differentiation.

[0007] However, the use of anti-CD3 antibodies such as OKT3 also has certain problems. For example, the use of OKT3 is limited by the toxic dose response syndrome. This syndrome is believed to be associated with OKT3-mediated T cell activation and cytokine release. OKT3 is a potent mitogen that can promote T cell proliferation and cytokine secretion, triggering a series of side effects, including fever, chills, nausea, vomiting, and headache, summarized as "flu-like", "cytokine release" or "first dose" syndrome. A small number of patients experience more serious side effects such as cardiorespiratory distress, epilepsy, encephalopathy, meningitis, renal dysfunction, and graft thrombosis.

[0008] The long-term activation of primary T cells by anti-CD3 antibodies such as OKT3 can also lead to T cell anergy. T cell anergy is a tolerance mechanism in which lymphocytes are functionally inactivated after encountering antigens, but survive for a long time in a hypo-responsive state. T cell anergy is associated with persistent high-intensity activation of antigens in vitro or stimulation in an environment lacking costimulation or high co-inhibition.

[0009] Bispecific antibodies (BsAb), simply "dual antibodies", are a class of bifunctional antibody molecules that can specifically bind to two different antigens at the same time. Such molecules can bridge target cells (expressing one of the antigens, such as tumor cells expressing tumor-associated antigens) and functional molecules or their cells (such as T cells expressing T cell receptors), stimulate and guide the body's immune response, so as to better control and kill target cells, especially in tumor immunotherapy, which has broad application prospects.

[0010] Bispecific antibodies targeting CD3 as one of the target antigens are a class of dual antibodies that have been studied and applied more deeply. Bispecific antibodies with CD3 and tumor antigen binding activity can recruit T cells to tumors by binding TCR-CD3 complex, thereby bridging T cells and tumors and promoting T cell activation, and then killing tumors. There are many bispecific antibodies of different structural forms in the research, clinical trial or approved marketing stages. In addition to targeting CD3 as one of the target antigens, these bispecific antibodies also target CD19, CD20, CD123, CD33, CD38, B cell maturation antigen (BCMA) and other tumor-associated antigens.

[0011] However, the use of bispecific antibodies also has certain problems, the most important of which is that it can lead to an increase in the circulating levels of inflammatory cytokines, including interferon-γ and interleukin-6, thereby leading to cytotoxicity. This symptom of an increase in the levels of cytokines in the whole body of a patient is also referred to as a cytokine release storm (CRS). Cytokine release storm is a systemic inflammatory response, which is associated with infectious and non-infectious diseases, and can be induced by infection, drugs, and the like. CRS mainly affects all important organs of the human body, and in severe cases can lead to multiple organ failure. CRS stimulates macrophages for a long time, which leads to macrophage activation syndrome, which in turn can lead to high fever, elevated ferritin levels, and hypertriglyceridemia. In CRS, a large number of immune cells (B cells, T cells and / or natural killer cells, macrophages, dendritic cells, and monocytes) are activated and then release more inflammatory cytokines. It has been reported that cytokine release usually begins within a few minutes to a few hours after infusion of antibodies such as CD28 superagonists and anti-CD3 (OKT3).

[0012] The degree of activation of immune cells determines the severity of CRS. In the use of bispecific antibodies targeting CD3 as one of the target antigens, the activation of T cells depends on the role played by the activating CD3 antibody portion.

[0013] In summary, there is still a need in the art for a new antibody targeting CD3, which should reduce the anergy of T cells while reducing the cytokine release syndrome caused by activation; and in the case of the preparation of a bispecific antibody using the new antibody, the bispecific antibody should not induce a high level of killing of tumor cells by T cells, while at the same time causing a smaller amount of cytokines to be produced by immune cells, thereby reducing the inflammatory response in vivo when used. SUMMARY

[0014] To solve the above technical problems, the purpose of the present application is to provide an antibody targeting a human CD3 molecule, which has essentially the same, or even stronger, activating effect on immune cells such as T cells (e.g., causing strong T cell proliferation, mediating significant tumor cell killing by T cells) compared to existing anti-CD3 antibodies, but at the same time causes a smaller amount of cytokine, particularly inflammatory cytokine, release. Further, the purpose of the present application is to construct a bispecific antibody by including a CD3-binding moiety of the antibody targeting a human CD3 molecule and an antibody or a moiety thereof that binds to a tumor-associated antigen, so that the resulting bispecific antibody retains the strong activating effect of the antibody on immune cells and the activity of causing less cytokine release, while being able to effectively recruit T cells at the site of tumor cells and achieve good tumor cell killing.

[0015] The present application provides the following technical solutions.

[0016] In one aspect, the present application provides a bispecific antibody targeting CD3 molecule and tumor associated antigen (TAA), the tumor associated antigen being CEA, and the bispecific antibody comprising a first binding domain targeting CD3 molecule and a second binding domain targeting CEA, wherein the first binding domain only binds to epsilon (ε) subunit in delta (δ)-epsilon (ε) heterodimer of the CD3 molecule, but not to epsilon (ε) subunit in gamma (γ)-epsilon (ε) heterodimer of the CD3.

[0017] Depending on the context, the term "targeting" is used interchangeably with "binding" in the present application.

[0018] In the context of the present application, the CD3 molecule refers to CD3 molecule of mammal; preferably, the CD3 molecule is CD3 molecule of primate, preferably human. Preferably, the CD3 molecule refers to CD3 molecule on the surface of T cell.

[0019] In the context of the present application, the term "CEA" is the abbreviation of carcinoembryonic antigen. CEA is originally extracted from colon cancer and embryonic tissue, can be expressed in a variety of malignant tumors, and has been widely used in clinic for diagnosis, treatment monitoring and prognosis evaluation of tumor.

[0020] Accordingly, the bispecific antibody provided by the present application can be referred to as "CD3 x CEA bispecific antibody" for short.

[0021] In terms of structure, according to the specific embodiments of the present application, the CD3 x CEA bispecific antibody can take the form of homodimer, i.e. having two identical heavy chains and two identical light chains, the heavy chain comprising domains connected in the following order: anti-CD3 VH-CH1-FC-anti-CEA scfv; the light chain comprising domains connected in the following order: anti-CD3 VL-CL. The CD3 x CEA bispecific antibody can also take the form of heterodimer, i.e. having two different heavy chains and two different light chains, one of which comprises domains connected in the following order: anti-CD3 VH-CH1-FC, and the other comprises domains connected in the following order: anti-CEA VH-CH1-FC; one of which comprises domains connected in the following order: anti-CD3 VL-CL, and the other comprises domains connected in the following order: anti-CEA VL-CL. Bispecific antibodies constructed in other structural types are also encompassed in the present application, as long as they comprise a first binding domain targeting CD3 molecule and a second binding domain targeting CEA.

[0022] The CD3 x CEA bispecific antibody provided by the present application can also be obtained by KiH technology, for example, using the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 as the heavy chain constant region of the two heavy chains of the bispecific antibody.

[0023] In the CD3 x CEA bispecific antibody provided by the present application, the first binding domain comprises a heavy chain and a light chain, which specifically bind to CD3 molecules and comprise a combination of CDRs (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3) selected from the following:

[0024] (i) CDRH1 comprising the amino acid sequence (GYTFSRY) shown in SEQ ID NO: 9, CDRH2 comprising the amino acid sequence (LPGSGS) shown in SEQ ID NO: 10, CDRH3 comprising the amino acid sequence (CYRYDAYGMDY) shown in SEQ ID NO: 11; and, CDRL1 comprising the amino acid sequence (RASQSISGYLH) shown in SEQ ID NO: 13, CDRL2 comprising the amino acid sequence (YASQSIS) shown in SEQ ID NO: 14, CDRL3 comprising the amino acid sequence (QNGHSFPLT) shown in SEQ ID NO: 15;

[0025] (ii) CDRH1 comprising the amino acid sequence (GYTFSRYW) shown in SEQ ID NO: 19, CDRH2 comprising the amino acid sequence (ILPGSGST) shown in SEQ ID NO: 20, CDRH3 comprising the amino acid sequence (ARCYRYDAYGMDY) shown in SEQ ID NO: 21; and, CDRL1 comprising the amino acid sequence (QSISGY) shown in SEQ ID NO: 22, CDRL2 comprising the amino acid sequence (YAS) shown in SEQ ID NO: 23, CDRL3 comprising the amino acid sequence (QNGHSFPLT) shown in SEQ ID NO: 15;

[0026] (iii) a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 24 (RYWIE), a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 25 (EILPGSGSTNYNEKFKG), a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 11 (CYRYDAYGMDY); and, a CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 13 (RASQSISGYLH), a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (YASQSIS), a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 15 (QNGHSFPLT); and

[0027] (iv) a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 9 (GYTFSRY), a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 10 (LPGSGS), a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 11 (CYRYDAYGMDY); and, a CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 26 (SGYLHWY), a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 27 (LIKYASQSI), a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 28 (QNGHSFPL).

[0028] Preferably, said first binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity with said amino acid sequence; and / or, a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity with said amino acid sequence.

[0029] In the context of the present application, "at least 75% identity" is any percentage number of identity between 75% and 100%, for example 75%, 80%, 85%, 90%, and even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% identity.

[0030] In the context of the present application, both the "heavy chain variable region" and the "light chain variable region" can comprise the above-mentioned CDR combinations as well as spacer framework regions, the arrangement of the individual domains being: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further optionally, the "at least 75% identity" leading to a maximum of 25% difference in the amino acid sequence can be present in any of the framework regions in the heavy chain variable region or the light chain variable region, or in any of the domains or sequences outside the heavy chain variable region and the light chain variable region. The difference can be caused by deletion, addition or substitution of amino acids at any position.

[0031] In the CD3 x CEA bispecific antibody provided by the present application, the second binding domain comprises a heavy chain and a light chain, which specifically bind to a CEA molecule and comprise a CDR combination (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3) selected from the group consisting of:

[0032] CDRH1 comprising the amino acid sequence (GFNIKDTY) shown in SEQ ID NO: 35, CDRH2 comprising the amino acid sequence (IDPANGNS) shown in SEQ ID NO: 36, CDRH3 comprising the amino acid sequence (APFGYYVSDYAMAY) shown in SEQ ID NO: 37; and, CDRL1 comprising the amino acid sequence (ESVDIFGVGF) shown in SEQ ID NO: 38, CDRL2 comprising the amino acid sequence (RAS) shown in SEQ ID NO: 39, CDRL3 comprising the amino acid sequence (QQTNEDPYT) shown in SEQ ID NO: 40.

[0033] Further, the second binding domain specifically binds to CEA and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity to said amino acid sequence; and, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 75% identity to said amino acid sequence.

[0034] In another aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the CD3 x CEA bispecific antibody provided by the present application.

[0035] The nucleic acid molecule of the present application can be cloned into a vector, which in turn is used to transform or transfect a host cell. Therefore, in a further aspect, the present application also provides a vector comprising the nucleic acid molecule of the present application. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc. The vector or nucleic acid molecule of the present application can be used to transform or transfect a host cell. Therefore, in a further aspect, the present application provides a host cell comprising or transformed or transfected with the nucleic acid molecule and / or vector of the present application. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or an insect, fungal, plant or animal cell.

[0036] The CD3 x CEA bispecific antibody provided by the present application can be obtained using any method known in the art.

[0037] The CD3 x CEA bispecific antibody, nucleic acid molecule, vector and / or host cell provided by the present application can be comprised in a composition, more particularly in a pharmaceutical composition, such as a pharmaceutical preparation, for various purposes according to actual needs. Therefore, in a further aspect, the present application also provides a composition, such as a pharmaceutical composition, comprising the antibody or fragment thereof, nucleic acid molecule, vector and / or host cell of the present application, and optionally a pharmaceutically acceptable excipient.

[0038] Experiments have shown that the CD3 x CEA bispecific antibody provided by the present application can effectively activate T cells via its first binding domain targeting CD3 molecules, and the inflammatory cytokines produced by T cells in response to the first binding domain are relatively less in terms of releasing inflammatory cytokines associated with cytokine storm; at the same time, the CD3 x CEA bispecific antibody can effectively recruit T cells at CEA-expressing tumor cells, achieving good tumor cell killing effect. By recruiting and triggering T cells to kill CEA-expressing tumor cells, the CD3 x CEA bispecific antibody provided by the present application can be used for immunotherapy, especially for the immunotherapy of tumors.

[0039] Therefore, in a further aspect, the present application also provides the use of the CD3 x CEA bispecific antibody, the nucleic acid molecule, the vector, the host cell or the composition in the preparation of a medicament for treating graft versus host disease, or for treating a tumor. The tumor can be a solid tumor, such as gastric cancer, rectal cancer, breast cancer, kidney cancer, ovarian cancer, etc., or a hematological tumor, such as lymphoma, leukemia, myeloma, etc.

[0040] Alternatively, the present application also provides a method for treating graft versus host disease or tumor, comprising administering the CD3 x CEA bispecific antibody, the nucleic acid molecule, the vector, the host cell or the composition to a subject in need thereof. Wherein, the tumor is a solid tumor or a hematological tumor, the solid tumor is for example gastric cancer, rectal cancer, breast cancer, kidney cancer, ovarian cancer, etc., the hematological tumor is for example lymphoma, leukemia, myeloma, etc. The subject is a mammal, preferably a primate, more preferably a human.

[0041] Compared with the prior art, the present application firstly provides a novel antibody targeting human CD3 molecule. Taking 4B1 provided in the embodiments of the present application as an example, experiments show that the antibody provided by the present application has very low level of inflammatory related cytokines produced by activated human T cells, even less than one third of the most widely used anti-CD3 antibody OKT3, but at the same time, the tumor killing effect of activated human T cells is basically consistent with the level of OKT3. Therefore, the antibody targeting human CD3 molecule provided by the present application has greater application advantages in reducing side effects such as cytokine storm in the process of immunotherapy for tumor patients.

[0042] Based on the novel antibody, the present application provides a prepared CD3 x CEA bispecific antibody. Taking the bispecific antibody constructed by 4B1 as an example, experiments show that different structural forms of CD3 x CEA bispecific antibodies are constructed by using the structure part of the monoclonal antibody 4B1 which binds to CD3, the CD3 x CEA bispecific antibodies retain the strong activation effect of the antibody 4B1 on immune cells and the activity of inducing less cytokine release, at the same time, can effectively recruit T cells at the tumor cell site, and achieve good tumor cell killing effect. BRIEF DESCRIPTION OF DRAWINGS

[0043] Hereinafter, the embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0044] Figure 1: Binding kinetics curve of monoclonal antibody and natural TCR-CD3 protein complex.

[0045] Figure 2: Activity detection of monoclonal antibody in activating T cells to release cytokines.

[0046] Figure 3: Activity detection of monoclonal antibody in causing T cell proliferation.

[0047] Figure 4: Schematic diagram of monoclonal bispecific antibody structure.

[0048] Figure 5: Activity detection of bispecific antibody in mediating PBMCs to kill tumor cells.

[0049] Figure 6: Detection of the activity of bispecific antibody in mediating PBMCs to secrete cytokines.

[0050] Figure 7: Detection of the activity of bispecific antibody in mediating PBMCs to kill tumor cells.

[0051] Figure 8: Pre-incubation of bispecific antibody with T cells.

[0052] Figure 9: Detection of the activity of bispecific antibody in mediating T cells to kill tumor cells and secrete cytokines after pre-incubation of bispecific antibody with T cells.

[0053] Figure 10: Establishment of mouse model and administration scheme for detection of the activity of monoclonal antibody and bispecific antibody in vivo.

[0054] Figure 11: Tumor progression curve for detection of the activity in vivo.

[0055] Figure 12: In vivo tumor imaging for detection of the activity in vivo.

[0056] Figure 13: Detection of the activity of bispecific antibody in mediating cytokine secretion in mice in vivo.

[0057] Figure 14: Effect of monoclonal antibody and bispecific antibody on the survival of mice.

[0058] Figure 15: Electron microscopy analysis of the binding of Fab region of monoclonal antibody to TCR-CD3 protein complex.

[0059] Figure 16: Detection of the efficiency of binding of monoclonal antibody to human T cells.

[0060] Best mode for carrying out the invention

[0061] The present application is described below with reference to specific examples. Those skilled in the art will understand that these examples are only used to illustrate the present application, and do not limit the scope of the present application in any way.

[0062] In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the raw materials, reagents and materials used are commercially available unless otherwise specified. Among them:

[0063] When constructing a chimeric antibody, the constant region sequence used is as follows:

[0064] Human IgG1 CL (SEQ ID NO: 1):

[0065] Human IgG1 CH1 (SEQ ID NO: 2):

[0066] Human IgG1 FC (SEQ ID NO: 3):

[0067] For the construction of bispecific antibodies, human IgGl CL and the following Knob, Hole containing constant region sequences were used:

[0068] Heavy chain constant region (CH1 and FC-Knob) (SEQ ID NO: 4):

[0069] Heavy chain constant region (CH1 and FC-Hole) (SEQ ID NO: 5):

[0070] The method for the production of chimeric monoclonal antibodies and bispecific antibodies is as follows:

[0071] Both chimeric monoclonal antibodies and bispecific antibodies were expressed in HEK-293F cells. Heavy chain plasmid and light chain plasmid were co-transfected in culture medium in proportion. Transfected HEK-293F cells were cultured in 5% carbon dioxide (CO2) incubator at 37°C for 3 days, and oscillated. The supernatant was centrifuged at 10000 rpm for 5 min to obtain clear expression supernatant, and the antibody molecules were purified by protein A. The subsequent molecular sieve evaluation showed that the antibody protein was of high purity and lacked aggregation (<1%).

[0072] The positive control antibodies (OKT3 and UCHT1) used in the examples were all prepared by using well-known variable region sequences and the constant region sequences described above.

[0073] The present application provides the sequence of antibody 4B1 (the signal peptide is in bold and underlined): 4B1:

[0074] > Heavy chain (SEQ ID NO: 6)

[0075] > Light chain (SEQ ID NO: 7)

[0076] The variable region and constant region of the heavy chain and light chain of antibody 4B1 and the full-length sequence are as follows:

[0077] Using different CDR definition schemes, the CDR regions of antibody 4B1 were further obtained, as follows:

[0078] The anti-CEA (T48.6), anti-CD19 (B43), and anti-HER2 (HUA21) antibodies were employed in the embodiments of the present application. The variable region sequences of the light and heavy chains of the three antibodies are as follows, in which the underlined bold portions are the CDR regions (partitioned in the IMGT manner):

[0079] Anti-CEA antibody T48.6:

[0080] > Heavy chain variable region (VH: SEQ ID NO: 29; CDRH1 / CDRH2 / CDRH3: SEQ ID NO: 35 / SEQ ID NO: 36 / SEQ ID NO: 37)

[0081] > Light chain variable region (VL: SEQ ID NO: 30; CDRL1 / CDRL2 / CDRL3: SEQ ID NO: 38 / SEQ ID NO: 39 / SEQ ID NO: 40)

[0082] Anti-CD19 antibody B43:

[0083] > Heavy chain variable region (VH: SEQ ID NO: 31; CDRH1 / CDRH2 / CDRH3: SEQ ID NO: 41 / SEQ ID NO: 42 / SEQ ID NO: 43)

[0084] > Light chain variable region (VL: SEQ ID NO: 32; CDRL1 / CDRL2 / CDRL3: SEQ ID NO: 44 / SEQ ID NO: 45 / SEQ ID NO: 46)

[0085] Anti-HER2 antibody HUA21:

[0086] > Heavy chain variable region (VH: SEQ ID NO: 33; CDRH1 / CDRH2 / CDRH3: SEQ ID NO: 47 / SEQ ID NO: 48 / SEQ ID NO: 49)

[0087] > Light chain variable region (VL: SEQ ID NO: 34; CDRL1 / CDRL2 / CDRL3: SEQ ID NO: 50 / SEQ ID NO: 51 / SEQ ID NO: 52)

[0088] Preparation and screening of monoclonal antibodies

[0089] Two Bal b / c mice were immunized with human full-length TCR-CD3 complex (PDB database sequence number: 6JXR (Structure of human T cell receptor-CD3 complex)) and serum titers were determined by indirect ELISA.

[0090] The human CD3 antigen was coated on a solid carrier, and a sample containing mouse serum to be tested was added to bind to the solid-phase antigen (Ag-Ab), and then an enzyme-labeled anti-CD3 antibody (secondary antibody) was added to bind to the Ag-Ab complex to form a complex. Substrate was added, and the enzyme on the complex catalyzed the substrate to develop color. The degree of color development of the enzyme and the substrate was used to determine the titer of the antibody to be tested. There was a washing step between each step. If the sample does not contain the corresponding antibody, the enzyme-labeled antibody is washed away, the substrate does not develop color and shows a negative reaction. If the OD of the test well is greater than 0.1 and greater than 2.1 times the OD of the negative control well (P / N>2.1, where P is the OD value of the test serum at a certain dilution, and N is the OD value of the negative serum at the corresponding dilution), it is determined to be positive. The highest dilution of serum that is determined to be positive is the serum antibody titer. Monoclonal antibodies with titers greater than 1:100K are selected for further functional experiments.

[0091] Using the feature that the surface BCR of memory B cells (MBC) specifically binds to antigens and the surface markers of B cells, antigen-specific MBCs were sorted from prepared PBMCs by flow cytometry. The sorted antigen-specific MBCs were obtained by extracting total RNA, RT-PCR amplification, vector construction, high-throughput expression to obtain cell supernatant containing monoclonal antibodies, and then detected by indirect ELISA. A certain number of positive clone cells were selected, the variable region sequences of the antibody light and heavy chains in the positive clone cells were measured, a commercially available human IgG1 constant region vector was used to construct a heavy-light chain expression vector, expression and purification were performed, and finally monoclonal antibodies were obtained.

[0092] Several murine monoclonal antibodies were obtained, and were named 4B1, 4B12, 4G3, 4D10 and 4F7, respectively.

[0093] Example 2 Affinity determination of monoclonal antibodies

[0094] The binding affinity of the monoclonal antibodies provided by the application and the positive control antibody to the natural TCR-CD3 protein complex was determined by bio-layer interferometry, and the equilibrium dissociation constant (KD) value was calculated.

[0095] BioLayer Interferometry assays were performed on an Octet RED96 system (ForteBio) at 30°C with 25 mM HEPES (pH 7.5), 150 mM NaCl and 0.06% gypsogenin TCR-CD3 as buffer. TCR-CD3 proteins were biotinylated with NHS-LC-LC biotin and then loaded onto streptavidin biosensors (ForteBio) which were equilibrated in running buffer for 10 minutes. After 2 minutes of incubation, the biosensors were re-acted with different concentrations of monoclonal antibodies for 2 minutes and then dissociated for 3 minutes. Data were analyzed using global fitting algorithm in Octet data analysis software v.9.0 (ForteBio).

[0096] Results are shown in Figure 1.

[0097] Three independent experiments showed that for the target protein, 4B1, 4F7 exhibited similar high affinity (pM level) as UCHT1, while OKT3 exhibited lower affinity (0.6 nM). The rest of the antibodies 4G3 (1.6 nM), 4B12 (144 nM) and 4D10 (1800 nM) exhibited different degrees of affinity.

[0098] Example 3 Detection of the activity of monoclonal antibodies in activating T cells to release cytokines

[0099] Human primary T cells were purified from PBMC of healthy donors using human CD3 negative selection kit. Different concentrations of monoclonal antibodies were pre-coated on 96-well plates overnight at 4°C, and then 1e5 human primary T cells were added to each well of the plates.

[0100] CD69: At 24h, the cultured T cells were taken and the ratio of CD69 positive cells was detected by flow cytometry.

[0101] Culture supernatant cytokines: At 48h, the culture supernatant was taken and detected by LEGEND-plex Human Thelper 1 (Th1) Cytokine Kit (containing IL-6, IFN-g and TNF-a), and the specific steps were referred to the kit instructions.

[0102] Intracellular cytokines: At 48h, the T cells were taken, surface stained, fixed with 1% paraformaldehyde (4 degrees for 30 minutes), broken membrane with 0.2% tween 20 (37°C for 20 minutes), and intracellularly stained with perforin and GZMB, and the percentage of T cell intracellular perforin and GZMB expression was detected by flow cytometry.

[0103] Results are shown in Figure 2.

[0104] The experimental results show that among the five monoclonal antibodies, 4B1 and UCHT1 highly activate T cells after co-culture for 24 hours (2A in FIG. 2), and the expression of CD69 is 9% higher than that of OKT3, followed by 4B12, 4F7, 4G3 and 4D10. After continuing to culture to 48 hours (2B in FIG. 2), compared with other monoclonal antibodies screened, 4B1 activated T cells produce more significant levels of cytotoxic cytokines GZMB and Perforin, which are equal to OKT3.

[0105] However, for the inflammatory cytokines related to the cytokine storm produced by T cell response to monoclonal antibody activation, 4B1 mediates the production of less inflammation-related factors than OKT3; and UCHT1 produces very strong cytokine levels after activation, even higher than OKT3.

[0106] According to the above results, it can be preliminarily inferred that compared with OKT3, monoclonal antibody 4B1 can produce lower levels of cytokines on the basis of causing high levels of T cell killing.

[0107] Example 4 Detection of the activity of monoclonal antibodies in causing T cell proliferation

[0108] The Leu234 and Leu235 mutations to alanine and the Pro329 mutation to glycine were introduced into 4B1 and OKT3, respectively, to prepare their effectorless human IgG1 (Fc silence) variants, removing the FC-mediated complement effect.

[0109] Human primary T cells were purified from PBMC of healthy donors using human CD3 negative selection kit. Different concentrations of the above monoclonal antibody variants were coated in advance on 96-well plates, and after overnight incubation at 4°C, 1e5 human primary T cells were added to each well of the plate, and 3 μg / mL anti-huCD28 was added. After 96 hours of antibody incubation, T cells were taken, and the percentage of depleted PD-1 on CD4+ and CD8+ T cells was detected by flow cytometry.

[0110] The results are shown in FIG. 3.

[0111] The experimental results show that 4B1 causes stronger T cell proliferation; and after 4 days of culture, the expression level of the cell surface exhaustion indicator molecule PD-1 caused by 4B1 is lower than that of OKT3 stimulation, whether for CD4+ or CD8+ T cells.

[0112] Example 5 Construction of bispecific antibodies (BsAb)

[0113] The monoclonal antibody 4B1 and the control antibody OKT3 provided by the application are used to construct two structural forms of bispecific antibodies respectively. The structure of the bispecific antibody is shown in Figure 4, wherein the bispecific antibody with the structure shown on the left side of Figure 4 is named CD3 / TAA (i.e. "IgG-scFv format"), and the bispecific antibody with the structure shown on the right side of Figure 4 is named CD3xTAA (i.e. "knobs-into-holes format"), and TAA is respectively carcinoembryonic antigen CEA, HER2 and CD19.

[0114] Firstly, the light and heavy chain variable region sequences of anti-CEA (T48.6), anti-CD19 (B43) and anti-HER2 (HUA21) antibodies are obtained from the Protein Data Bank of the United States.

[0115] For the antibody of CD3 / TAA format, the heavy chain C-terminus of OKT3 and 4B1 monoclonal antibody variants (i.e. Fc silence) is connected to the scfv form of anti-TAA through a (GGGGS)1 linker, and the light and heavy chain variable regions of the scfv are connected using a (GGGGS)4 long linker to construct a heavy chain of "anti-CD3 VH-CH1-FC-anti-TAA scfv". The light chain of OKT3 and 4B1 monoclonal antibodies is obtained. As described above, the heavy chain plasmid and the light chain plasmid are co-transfected into cells at a ratio of 1:1 to obtain bispecific antibodies of CD3 / TAA format, which are respectively named OKT3 / TAA and 4B1 / TAA.

[0116] For the antibody of CD3xTAA format, the heavy chain variable region of OKT3 and 4B1 monoclonal antibodies is respectively connected to the heavy chain constant region (CH1 and FC containing hole mutation) to construct a heavy chain, and the heavy chain variable region of the above anti-TAA is connected to the heavy chain constant region (CH1 and FC containing knob mutation) to construct a heavy chain. The light chain of OKT3 and 4B1 monoclonal antibodies is obtained. Similarly, the light chain of human IgG1 of anti-TAA is constructed. As described above, the two heavy chain plasmids and the light chain plasmid are co-transfected into cells at a ratio of 1:1:1 to obtain bispecific antibodies of CD3xTAA format, which are respectively named OKT3xTAA and 4B1xTAA.

[0117] Example 6 Detection of the activity of bispecific antibodies in mediating PBMCs to kill tumor cells and secrete cytokines

[0118] (1) Targeting CEA

[0119] The human gastric cancer cell line MKN45 expressing CEA antigen is purchased from the National Cell Line Resource Library (Beijing, China) and is cultured in 1640 medium (Gibco) added with 1% L-glutamine, 1% penicillin / streptomycin and 10% fetal bovine serum.

[0120] Human MKN45 cells were mixed with in vitro isolated human CD3+ T cells at 10:1 E:T ratio. Cells were incubated with limited dilution of OKT3 BsAb, 4B1 BsAb or control BsAb that cannot bind to specific tumor cell line. After 48 hours incubation at 37℃, lactate dehydrogenase (LDH) release assay was performed using LDH Cytotoxicity Detection Kit (Biovision) according to the manufacturer's instruction.

[0121] Results are shown in Figure 5 and Figure 6.

[0122] The results showed that both BsAbs against carcinoembryonic antigen (CEA) had high activity in killing human MKN45 gastric cancer cell line (high expression of CEA antigen) by endogenous T cells in PBMCs prepared from healthy donors. Figure 5 shows the representative dose-response curves of 9 healthy donors, and the summary of EC50 values and killing degree, which showed that the average EC50 of CD3_4B1 / CEA caused by antibody killing tumor was higher than that of CD3_OKT3 / CEA, but there was no difference in the average killing degree between the two groups after 48 hours of culture. Figure 6 shows the results of cytokine levels, i.e. interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in the supernatant of culture solution, which showed that the cytokine release stimulated by CD3_4B1 / CEA was significantly lower than that of CD3_OKT3 / CEA group.

[0123] (2) Targeting CEA, HER2 or CD19

[0124] Further in vitro detection of the killing activity of the bispecific antibodies constructed in the present application on carcinoembryonic antigen expressing cells, HER2 expressing tumor cells (SKOV3 cells, as an example of solid malignant tumor), and CD19 expressing tumor cells (RAJI cells, as an example of hematological malignant tumor).

[0125] Human tumor cell lines expressing specific antigens were mixed with in vitro isolated human CD3+ T cells at 10:1 E:T ratio. 96-well plates contained 3e4 tumor cells and 3e5 human primary T cells per well. Cells were incubated with gradient concentrations of OKT3 BsAb and 4B1 BsAb. After 24 (RAJI) or 48 (SKOV3) hours incubation at 37℃, lactate dehydrogenase (LDH) release assay was performed using LDH Cytotoxicity Detection Kit according to the manufacturer's instruction.

[0126] Results are shown in Figure 7.

[0127] The results of the three tumor killing models showed that the average EC50 of antibody killing tumor caused by CD3_4B1 / TAA was higher than that of CD3_OKT3 / TAA, but the average killing percentage of the two was not different.

[0128] Example 7 Detection of the activity of bispecific antibodies mediating T cell killing tumor cells and secreting cytokines after pre-incubation with T cells

[0129] Because BsAb mainly acts on T cells, in order to exclude the influence of other cells in PBMCs, a treatment method of pre-incubation of bispecific antibodies with T cells was established, as shown in Figure 8.

[0130] As shown in the figure, after pre-incubation of T cells with BsAb for 25 min (flow identification of all antibodies bound to T cells) and washing steps, the target cells, i.e. tumor cells, were co-cultured, and then the killing percentage of tumor cells and Th1 cytokines (IL-2, IL-6, IL-10, IFN-γ and TNF-α) in the supernatant were detected; other conditions refer to the detection method when PBMCs were used in Example 6.

[0131] The results are shown in Figure 9.

[0132] The experimental results showed that compared with PBMCs-BsAb+tumor cells, IL-6, IL-10 and TNF-α produced in the case of pre-incubation of T cells were significantly reduced; and after pre-incubation, the ratio of the maximum cytokine release of 4B1 / OKT3 was significantly lower than that of the PBMCs-BsAb+tumor cell group, and the killing percentage of tumor cells of the two groups was basically flat. It is suggested that when 4B1 antibody is used to construct bispecific antibodies for killing tumors in patients, the side effects caused by cytokine storm in patients can be reduced, and at the same time, the effect of bispecific antibodies on the killing ability of tumors will not be affected.

[0133] Example 8 Detection of the in vivo anti-tumor, cytokine release stimulating activity of bispecific antibodies and detection of the effect on animal survival

[0134] In order to confirm that the in vitro killing and cytokine release stimulating characteristics of bispecific antibodies containing CD3_4B1 are consistent with the in vivo therapeutic effect, RAJI lymphoblastic lymphoma xenograft mouse models were used to compare the effector functions of CD3_4B1xCD19 and CD3_OKT3xCD19.

[0135] The mouse model was established and the dosing regimen is shown in Figure 10. As shown in Figure 10, 5e5 RAJI lymphoblastoid lymphocytes stably expressing luciferase (Lucifrase, LUC) were injected into the tail vein of the mouse on day 0, and the tumor progression of the mouse was observed. After confirming that the tumor progression was as expected, the pre-incubated T cells and PBMCs were injected into the tail vein on day 7. 5e6 T cells were pre-incubated with different concentration gradients of bispecific antibodies for 30 minutes, and the excess bispecific antibodies were removed by PBS washing twice. The pre-incubated T cells were mixed with 1e7 PBMCs and injected into the mouse through the tail vein, and then the bispecific antibodies constructed by the application and 4B1 or OKT3 monoclonal antibodies were injected into the tail vein of the mouse at a dose of 30, 10, 2, and 0.2 μg per mouse on days 10, 14, 18, and 22, respectively.

[0136] (1) Tumor progression

[0137] The tumor burden of the mouse was evaluated using bioluminescence imaging before each antibody administration and on days 7-22. Each mouse was intraperitoneally injected with 10 mg / ml d-luc luminescent substrate, and after 10 minutes of anesthesia, the light intensity change was detected by bioluminescence imaging technology (BLI) to monitor the disease development in real time.

[0138] The results are shown in Figures 11 and 12.

[0139] The experimental results show that the CD3_4B1xCD19 molecule can produce strong tumor cell killing effect at various dose levels, and the 10 μg dose shows almost complete tumor clearance, and the 30 μg dose is similar to the 10 μg dose, but shows a higher level of inflammatory factors, which indicates that the 10 μg dose has the best therapeutic effect on the mouse under the premise of minimum cytokines. Moreover, the body chemiluminescence photos show that CD3_4B1xCD19 effectively controls the growth of RAJI tumor cells, and the mouse maintains a very low tumor level within 20 days after treatment. It is worth noting that the tumor burden of CD3_4B1xCD19 at a dose of 10 μg is more obvious than that of CD3_OKT3xCD19 molecule in terms of tumor clearance effect.

[0140] (2) Cytokine secretion

[0141] It is known from the study of cytokine storm concurrent with immunotherapy with BsAb in clinical patients that the cytokine level usually reaches a peak after the first immunotherapy, causing irreversible damage to the whole body of the patient, including the cardiovascular and nervous systems.

[0142] To investigate the cytokine levels caused by the two BsAbs in mice, the peripheral blood serum of mice was taken at different time points (2, 6, and 24 hours) within 24 hours after the first BsAb treatment: tumor progression on the 7th day. After centrifugation of about 100 ul of mouse serum at 3000 rpm at room temperature for 10 minutes, the supernatant was taken. The secretion of human Th1-type cytokines was detected by LEGEND-plex Human T helper 1 (Th1) Cytokine Kit (containing IL-10, IL-2, IL-6, IFN-γ, and TNF-α), and flow cytometry analysis was performed according to the kit instructions.

[0143] The results are shown in Figure 13.

[0144] The experimental results show that the five cytokines have similar metabolic peak degrees, which reach the peak of secretion at 2 hours, gradually decrease at 6 hours, and basically return to the baseline within 24 hours. Consistent with the previous in vitro results, the cytokines produced by CD3_4B1 x CD19 activated immune system are significantly lower than those produced by CD3_OKT3 x CD19. This indicates that the bispecific antibody based on CD3_4B1 can effectively reduce the cytokine storm that bursts in a short time, achieving the purpose of optimizing treatment.

[0145] (3) Animal survival

[0146] It is well known that RAJI lymphoma has strong proliferation and invasion ability in vivo, which can greatly threaten the survival of NKG mice in a short period of time. Therefore, the effect of treatment of the two immunotherapeutic bispecific antibodies on the survival of tumor-bearing mice was detected.

[0147] From the 7th to the 35th day of the above experiment, the survival of mice was recorded by daily observation, and the Kaplan-Meier mouse survival curve was drawn (PRISM 8.0 software).

[0148] The results are shown in Figure 14.

[0149] From the results of the mouse survival curve, it can be seen that the mice in the control group and the vehicle group basically all died within 25 days of tumor inoculation. After treatment with CD3_4B1 x CD19 and CD3_OKT3 x CD19 formulations, the mice basically all survived within 35 days.

[0150] Example 9 Epitope characterization of monoclonal antibody binding to CD3

[0151] As shown in the above examples, the anti-CD3 antibody 4B1 and bispecific antibodies constructed using 4B1 provided by the present application can cause stronger T cell proliferation and can mediate stronger tumor cell killing activity of T cells, but at the same time cause T cells to produce less inflammatory cytokines related to cytokine storm, relative to the control antibody OKT3 and bispecific antibodies constructed using OKT3. The following experiments were performed to further study the mechanism of action of 4B1 and its functional domains.

[0152] Recombinant vectors for expressing light chains and heavy chains of anti-CD3 antibodies OKT3, UCHT1, and 4B1 were constructed using pcDNA3.4, and then HEK293 free style cells were co-transfected, recombinant antibodies were expressed, supernatants were collected, and anti-CD3 antibodies OKT3, UCHT1, and 4B1 were purified. Then, Fab was obtained by papain digestion. The TCR-CD3 complex was incubated with Fab of the three antibodies at a 1:1.5 molar ratio at 4°C for 3 h, and then gel column filtration was performed to obtain 4B1-TCR-CD3, OKT3-TCR-CD3, and UCHT1-TCR-CD3 protein complexes.

[0153] The 4B1-TCR-CD3 protein complex was concentrated for cryo-EM analysis and image reconstruction, and a cryo-EM structure image with a global resolution of 3.2 A was obtained, as shown in Fig. 15A. As shown in Fig. 15A, the Fab molecules of 4B1 form a 1:1 stoichiometric complex with the TCR-CD3 complex, and the Fab molecules of 4B1 bind to the CD3δ-ε heterodimer. At the same time, the cryo-EM structure images of OKT3-TCR-CD3 and UCHT1-TCR-CD3 complexes were determined at resolutions of 3.5 A and 3.4 A, respectively, as shown in Figs. 15B and 15C. As shown in the figures, the Fab molecules of OKT3 and UCHT1 both form a 2:1 stoichiometric complex with the TCR-CD3 complex, compared with the 1:1 stoichiometry between 4B1 Fab and TCR-CD3.

[0154] ​​​Analysis of the binding epitopes of OKT3, UCHT1, 4B1 on CD3 shows that the Fab molecules of 4B1, OKT3 and UCHT1 recognize the overlapping epitopes on the CD3e subunit of CD3d-e heterodimer, but their interactions with CD3e are significantly different: there is an angle deviation of about 45° of 4B1 Fab relative to OKT3 Fab and UCHT1 Fab, so as to interact with CD3e of CD3d-e heterodimer, see 15D in Figure 15; if 4B1 Fab is docked with the CD3e subunit in CD3g-e heterodimer, there will be serious spatial conflict with the TCR b subunit, see 15E in Figure 15. These observations confirm the 1:1 stoichiometry between the Fab molecules of 4B1 and the TCR-CD3 complex.

[0155] Further, the binding efficiency of 4B1, OKT3 and UCHT1 antibodies to human T cells was detected by flow cytometry.

[0156] Human CD3+T cells were collected, washed with PBS, and 5x10 5 The CD3+T cells were incubated with different concentrations of antibodies at 4°C for 30 minutes; anti-human IgG antibody was used as isotype control. After incubation, the cells were washed with PBS, anti-human IgG-FITC antibody was added and incubated at 4°C for 30 minutes in the dark, and then washed again. The FITC fluorescence intensity on the cell surface was acquired, and analyzed by FlowJo software.

[0157] The results show that both antibodies exhibit effective binding to the TCR-CD3 complex. However, the mean fluorescence intensity (MFI) analysis shows that the amount of antibody bound on a single CD3+T cell by OKT3 at saturating concentration is twice the amount bound by 4B1; see Figure 16. In combination with the above electron microscopy structure analysis, it is further confirmed that OKT3 binds to the CD3e subunit of CD3d-e heterodimer and the CD3e subunit of CD3g-e heterodimer, while the anti-CD3 antibody 4B1 of the present application only binds to the CD3e subunit of CD3d-e heterodimer.

[0158] The above description of specific embodiments of the present application does not limit the present application, and those skilled in the art can make various changes or modifications to the present application without departing from the spirit of the present application, and all such changes or modifications shall fall within the scope of the appended claims of the present application.

Claims

1. A bispecific antibody targeting CD3 molecules and tumor-associated antigens (TAAs), characterized in that, The tumor-associated antigen is CEA, and the bispecific antibody comprises a first binding domain targeting the CD3 molecule and a second binding domain targeting CEA, wherein the first binding domain binds only to the epsilon(ε) subunit of the delta(δ)-epsilon(ε) heterodimer of the CD3 molecule, and does not bind to the epsilon(ε) subunit of the gamma(γ)-epsilon(ε) heterodimer of the CD3.

2. The bispecific antibody according to claim 1, characterized in that, The first binding domain comprises a heavy chain and a light chain, specifically binds to the CD3 molecule, and contains a combination of CDRs selected from the following (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3): (i) CDRH1 containing the amino acid sequence shown in SEQ ID NO:9, CDRH2 containing the amino acid sequence shown in SEQ ID NO:10, and CDRH3 containing the amino acid sequence shown in SEQ ID NO:11; and CDRL1 containing the amino acid sequence shown in SEQ ID NO:13, CDRL2 containing the amino acid sequence shown in SEQ ID NO:14, and CDRL3 containing the amino acid sequence shown in SEQ ID NO:15; (ii) CDRH1 comprising the amino acid sequence shown in SEQ ID NO:19, CDRH2 comprising the amino acid sequence shown in SEQ ID NO:20, and CDRH3 comprising the amino acid sequence shown in SEQ ID NO:21; and CDRL1 comprising the amino acid sequence shown in SEQ ID NO:22, CDRL2 comprising the amino acid sequence shown in SEQ ID NO:23, and CDRL3 comprising the amino acid sequence shown in SEQ ID NO:15; (iii) CDRH1 comprising the amino acid sequence shown in SEQ ID NO:24, CDRH2 comprising the amino acid sequence shown in SEQ ID NO:25, and CDRH3 comprising the amino acid sequence shown in SEQ ID NO:11; and CDRL1 comprising the amino acid sequence shown in SEQ ID NO:13, CDRL2 comprising the amino acid sequence shown in SEQ ID NO:14, and CDRL3 comprising the amino acid sequence shown in SEQ ID NO:15; and (iv) CDRH1 comprising the amino acid sequence shown in SEQ ID NO:9, CDRH2 comprising the amino acid sequence shown in SEQ ID NO:10, CDRH3 comprising the amino acid sequence shown in SEQ ID NO:11; and CDRL1 comprising the amino acid sequence shown in SEQ ID NO:26, CDRL2 comprising the amino acid sequence shown in SEQ ID NO:27, and CDRL3 comprising the amino acid sequence shown in SEQ ID NO:

28.

3. The bispecific antibody according to claim 1 or 2, characterized in that, The first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with the amino acid sequence; and / or, the light chain variable region comprises an amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 75% identity with the amino acid sequence.

4. The bispecific antibody according to any one of claims 1 to 3, characterized in that, The second binding domain comprises a heavy chain and a light chain, specifically binds to the CEA molecule, and contains a combination of CDRs selected from the following (CDRH1, CDRH2, CDRH3; CDRL1, CDRL2, CDRL3): CDRH1 comprising the amino acid sequence shown in SEQ ID NO:35, CDRH2 comprising the amino acid sequence shown in SEQ ID NO:36, CDRH3 comprising the amino acid sequence shown in SEQ ID NO:37; and CDRL1 comprising the amino acid sequence shown in SEQ ID NO:38, CDRL2 comprising the amino acid sequence shown in SEQ ID NO:39, and CDRL3 comprising the amino acid sequence shown in SEQ ID NO:

40.

5. The bispecific antibody according to any one of claims 1 to 4, characterized in that, The second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having at least 75% identity with the amino acid sequence; and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 75% identity with the amino acid sequence.

6. A nucleic acid molecule comprising a nucleotide sequence encoding a bispecific antibody according to any one of claims 1 to 5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising the nucleic acid molecule of claim 6 and / or the vector of claim 7.

9. A composition comprising the bispecific antibody of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, and / or the host cell of claim 8; Preferably, the composition is a pharmaceutical composition, which further comprises optional pharmaceutically acceptable excipients.

10. Use of the bispecific antibody of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, and / or the composition of claim 9 in the preparation of a medicament, wherein the medicament is used to treat graft-versus-host disease or to treat tumors.

11. A method for treating graft-versus-host disease or tumor, the method comprising administering to a subject in need the bispecific antibody of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, and / or the composition of claim 9.

12. The use according to claim 10 or the method according to claim 11, characterized in that, The tumor is a solid tumor or a hematologic tumor; Preferably, the solid tumor is gastric cancer, rectal cancer, breast cancer, kidney cancer, or ovarian cancer; or, the hematologic malignancy is lymphoma, leukemia, or myeloma. Preferably, the subject is a mammal, and more preferably a human.

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