Therapeutic agents comprising multispecific antibodies and their use in tumor therapy

Abstract

The present invention provides a therapeutic agent comprising a labeled polypeptide introduced into tumor cells and a multispecific antibody capable of binding to and identifying the labeled polypeptide, the multispecific antibody, a kit comprising the therapeutic agent, and uses of the multispecific antibody of the therapeutic agent in preparation of drugs for treating tumors and / or cancers.

Description

Therapeutic agents comprising multispecific antibodies and their use in tumor treatment Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a therapeutic agent comprising a multispecific antibody and its application in tumor treatment. Background Art

[0002] According to data released by the World Health Organization (WHO), cancer has become one of the most serious diseases that threaten human health, with a continuously increasing number of cases and a high mortality rate. Current traditional cancer treatments still have many unmet clinical needs. Oncolytic virotherapy offers a new type of tumor immunotherapy. Oncolytic viruses selectively replicate in tumor cells, destroying them by direct lysis, stimulating the host's anti-tumor immune response to kill tumor cells without affecting the growth of normal cells, offering hope for a wide range of malignant tumor patients (Shi T et al., Front Immunol 2020;11:683). Compared with other tumor immunotherapies, oncolytic virotherapy offers multiple advantages, including high tumor killing efficiency, good targeting, low drug resistance, and low cost (Heo et al., 2012).

[0003] Like other anti-tumor therapies, oncolytic virus therapy also has some limitations, such as virus delivery to the target, penetration into the tumor mass, and the induction of a strong antiviral immune response, which is limited by physical barriers and host immunity. Therefore, oncolytic virus therapy often needs to be combined with chemotherapy or immunotherapy to enhance the therapeutic effect. Globally, most oncolytic virus drugs that are already on the market or under research are also undergoing combination therapy trials, such as in combination with immune checkpoints or CAR-T cell therapy.

[0004] Bispecific antibodies (BsAbs) are antibodies that can simultaneously and specifically bind to two antigens or antigenic epitopes. Since Nisonoff and his collaborators first proposed the concept of BsAbs in 1960, they have rapidly developed with the gradual maturity of genetically engineered antibody technology. Because BsAbs can target multiple antigens or antigenic epitopes, they offer advantages over monoclonal antibodies and are increasingly demonstrating higher efficacy than monoclonal antibody combination therapies. For example, specific immune effector cells can be redirected to adjacent tumor cells to enhance tumor killing; binding specificity can be increased through the interaction of two different cell surface antigens; and compared to single antibody combinations, development costs and clinical trials can be reduced. Bispecific antibodies have become a research hotspot in the field of antibody engineering and have broad application prospects in areas such as tumor treatment and autoimmune diseases. Furthermore, with technological advancements, antibodies targeting more antigens or more antigenic epitopes, such as trispecific antibodies or multifunctional fusion antibodies, have emerged.

[0005] Despite the emergence of a variety of therapies, there are still unmet clinical needs and an urgent need to develop new immunotherapies.

[0006] Summary of the Invention

[0007] The present invention provides a therapeutic agent comprising a multispecific antibody and its use in tumor treatment. By using the first active ingredient in the therapeutic agent, tumor cells or cancer cells are made to carry a labeled polypeptide, and the multispecific antibody is used to recognize the labeled polypeptide marked on the tumor cells or cancer cells. The other antigen-binding portion of the multispecific antibody can recruit immune cells such as T cells, NK cells, etc. to achieve the killing of tumor cells or cancer cells. The therapeutic agent provided by the present invention can significantly improve the effect of tumor treatment. Furthermore, by administering an oncolytic virus, tumor cells or cancer cells can be made to carry a labeled polypeptide, and the tumor killing effect can be achieved before the administration of the multispecific antibody, and the purpose of tumor treatment can be significantly improved after the administration of the multispecific antibody. To this end, the present invention also provides a new immunotherapy, comprising administering a therapeutically effective amount of an oncolytic virus and a multispecific antibody to a subject, respectively, and achieving the purpose of tumor treatment by combining oncolytic virus therapy and multispecific antibody therapy. The immunotherapy provided by the present invention will provide a new and creative therapy for the treatment of tumors.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] A first aspect of the present invention provides a therapeutic agent comprising:

[0010] (a) a first composition comprising a first active ingredient, the first active ingredient comprising or containing a nucleic acid having a marker polypeptide encoding sequence for introduction into tumor cells and / or cancer cells; the marker polypeptide having an operably linked extracellular antigenic determinant, a spacer portion, and a transmembrane portion; the amino acid sequence of the extracellular antigenic determinant containing the amino acid sequence of one or more antigenic epitope polypeptides; and the amino acid sequence of a mammalian cell membrane protein or secretory protein not containing the amino acid sequence of the antigenic epitope polypeptide in a natural state; and

[0011] (b) a second composition comprising a second active ingredient, wherein the second active ingredient comprises a multispecific antibody, wherein the multispecific antibody comprises at least a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion can specifically recognize and bind to the extracellular antigenic determinant region of the marker polypeptide;

[0012] The second antigen binding portion is capable of specifically recognizing and binding to an immune cell antigen or a first cytokine or its receptor;

[0013] The multispecific antibody may also optionally include a third antigen binding portion, which specifically recognizes and binds to a tumor antigen or an immune checkpoint or a second cytokine or its receptor.

[0014] The second aspect of the present invention provides a multispecific antibody comprising at least a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion comprises one or more domains capable of specifically recognizing and binding to the amino acid sequence of Strep Tag I or Strep Tag II, and the second antigen-binding portion comprises one or more domains capable of specifically recognizing and binding to an immune cell antigen or a first cytokine or its receptor; the multispecific antibody may also optionally comprise a third antigen-binding portion comprising one or more domains capable of specifically recognizing and binding to a tumor antigen or an immune checkpoint or a second cytokine or its receptor. The first antigen-binding portion can specifically recognize and bind to Strep Tag I or Strep Tag II, which have been confirmed to be absent or almost absent in mammals. By making tumor cells in mammals, especially humans, carry corresponding tags, the first antigen-binding portion can specifically target these tags, thereby enabling the multispecific antibody to specifically target tumor cells. The second antigen-binding portion is then used to recruit immune cells or cytokines or their receptors to specifically kill tumor cells, thereby achieving the effect of tumor-specific targeting and killing. The multispecific antibody may further comprise a third antigen-binding portion capable of specifically targeting a tumor antigen or an immune checkpoint or a cytokine or its receptor as needed to further enhance the effect of tumor targeting and killing.

[0015] The third aspect of the present invention provides a polynucleotide encoding the multispecific antibody according to the second aspect of the present invention.

[0016] The fourth aspect of the present invention provides a construct comprising the polynucleotide according to the third aspect of the present invention.

[0017] The fifth aspect of the present invention provides a host cell, wherein the host cell comprises the construct according to the fourth aspect of the present invention.

[0018] The sixth aspect of the present invention provides a method for producing the multispecific antibody of the second aspect, comprising: culturing the host cell of the fifth aspect, and collecting the multispecific antibody from the culture.

[0019] The seventh aspect of the present invention provides a use of a therapeutic agent or a multispecific antibody in the preparation of a medicament for treating tumors and / or cancer, wherein the therapeutic agent is the therapeutic agent mentioned in the first aspect of the present invention, and the multispecific antibody is the multispecific antibody described in the second aspect of the present invention.

[0020] The eighth aspect of the present invention provides a medicine kit for a synergistic combination drug for treating tumors and / or cancer, comprising: a first container, the first container containing a first composition of the therapeutic agent according to the first aspect; a second container, the second container containing a second composition of the therapeutic agent according to the first aspect or the multispecific antibody according to the second aspect, wherein the first container and the second container are independent; and instructions stating the timing and method of administration.

[0021] The ninth aspect of the present invention provides a method for treating tumors and / or cancer, comprising: administering to a subject a first composition of the therapeutic agent according to any one of the first aspects; and administering to the subject a second composition of the therapeutic agent according to the first aspect, or administering to the subject a multispecific antibody according to the second aspect.

[0022] The tenth aspect of the present invention provides a novel immunotherapy, comprising: administering to a subject a therapeutically effective amount of an oncolytic virus, the genome of the oncolytic virus containing a coding sequence for a marker polypeptide, the marker polypeptide having an operably linked extracellular antigenic determinant, a spacer portion, and a transmembrane portion, the amino acid sequence of the extracellular antigenic determinant containing the amino acid sequence of one or more antigenic epitope polypeptides, and in a natural state, the amino acid sequence of the cell membrane protein or secretory protein of the subject does not contain the amino acid sequence of the antigenic epitope polypeptide; and

[0023] administering to the subject a therapeutically effective amount of a multispecific antibody, wherein the multispecific antibody comprises at least a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion is capable of specifically recognizing and binding to the extracellular antigenic determinant region of the marker polypeptide, and the second antigen-binding portion is capable of specifically recognizing and binding to an immune cell antigen or a first cytokine or a receptor thereof;

[0024] The multispecific antibody may also optionally include a third antigen binding portion, which is capable of specifically recognizing and binding to a tumor antigen or an immune checkpoint or a second cytokine or its receptor.

[0025] The present invention has achieved the following beneficial technical effects:

[0026] The present invention provides a therapeutic agent, which contains a first composition and a second composition, wherein the first composition contains a first active ingredient, which marks the surface of tumors and / or cancer cells by an exogenous labeled polypeptide, and is recognized by the multispecific antibody in the second composition and the labeled polypeptide. By combining the two, the strategy can effectively kill tumor cells. Moreover, when an oncolytic virus is used as a vector to mediate the expression of an exogenous labeled polypeptide in tumor cells, based on the existing oncolytic killing effect of the oncolytic virus, the multispecific antibody targeting the labeled polypeptide can effectively eliminate residual tumor cells infected by the oncolytic virus. At the same time, because the oncolytic virus destroys the microenvironment of the tumor, the killing ability of the multispecific antibody for the tumor will be further improved, thereby significantly improving the effectiveness of tumor treatment, especially the treatment effect of solid tumors. Specifically, the present invention utilizes a marker polypeptide amino acid sequence having an extracellular antigenic determinant, a spacer portion, and a transmembrane portion, and a nucleic acid encoding the marker polypeptide, so that after the nucleic acid is transfected into tumor cells and / or cancer cells, or after the nucleic acid is inserted into the viral genome so that the resulting recombinant virus infects tumor cells and / or cancer cells, the tumor cells and / or cancer cells can express the marker polypeptide, and the marker polypeptide will ultimately be modified on the surface of the tumor cells and / or cancer cells. Because the amino acid sequence of the extracellular antigenic determinant of the marker polypeptide contains the amino acid sequence of one or more antigenic epitope polypeptides, it can effectively solve the problem of heterogeneity of solid tumor antigen expression and tumor escape from immune surveillance. At the same time, it is used in combination with a multispecific antibody that can recognize the extracellular antigenic determinant, and the multispecific antibody is used to recognize and bind to immune cells, thereby achieving immune cell-tumor cell bridging and specifically killing tumor cells. Moreover, compared to the killing of immune cells modified by CAR, the killing of immune cells mediated by multispecific antibodies can greatly reduce drug costs and toxic side effects. In particular, CAR-T cells have a long survival time in the body. The continued presence of CAR-T cells targeting tagged peptides in the body will hinder the subsequent multiple administration of nucleic acids or recombinant viruses encoding the tagged peptides. Multispecific antibodies, on the other hand, have a certain half-life and will not persist in the body. The drug concentration of multispecific antibodies in the body can be maintained more flexibly by adjusting the dosing cycle and frequency.

[0027] Furthermore, when an oncolytic virus is used as a carrier, the oncolytic virus can be specifically replicated in tumor cells to introduce the marker polypeptide encoding nucleic acid into tumor cells and / or cancer cells, so that the oncolytic virus can significantly enhance the expression of the above-mentioned antigen epitope peptide on the surface of tumor cells while killing tumor cells and / or cancer cells, thereby showing a synergistic therapeutic effect in combination with the immune cells recruited by the multispecific antibody, and more importantly, avoiding the off-target (on-target off-tumor) effect on normal tissue. The oncolytic virus destroys the microenvironment of the tumor, so that the killing ability of the multispecific antibody for tumor cells is further improved, thereby improving the effectiveness of tumor (especially solid tumor) treatment. In addition, multispecific antibodies can also effectively remove tumor cells that cannot complete the replication cycle and produce a sufficient number of daughter viruses after being infected by the oncolytic virus and undergo lysis; thereby achieving further synergy. In addition, the antigens released by the tumor cells lysed by the oncolytic virus can further activate the body's own anti-tumor immunity, thereby achieving a better tumor killing effect than using oncolytic virus or multispecific antibody alone, achieving a synergistic therapeutic effect.

[0028] In a further invention, the present invention improves the amino acid sequence and / or protein structure of a multispecific antibody targeting a marker polypeptide, including humanizing the first and second antigen-binding moieties and the heavy and light chain constant domains, thereby reducing the immunogenicity of the therapeutic agent of the present invention. Furthermore, by modifying the first and second antigen-binding moieties of the multispecific antibody, the affinity of at least these two moieties for the target antigen is improved, allowing the multispecific antibody to not only effectively exert an anti-tumor effect but also avoid safety issues such as immune storms caused by excessive affinity for immune cell antigens. For example, by humanizing the first antigen-binding portion of a targeted marker polypeptide, while ensuring affinity with the marker polypeptide target antigen, the potential immunogenicity can be effectively reduced, thereby reducing the risk of production in the human body, and maintaining a very high affinity level with the marker polypeptide target antigen, thereby ensuring the tumor-killing ability of the multispecific antibody; and, by humanizing the second antigen-binding portion of a targeted immune cell antigen, the multispecific antibody maintains a relatively moderate affinity activity for the target antigen on the immune cell when recruiting immune cells, thereby achieving tumor killing while avoiding safety issues. The design and screening of the present invention enable the multispecific antibody to exhibit particularly suitable affinity as a whole. The modified multispecific antibody has excellent anti-tumor effect and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is a schematic diagram of utilizing a therapeutic agent to exert a therapeutic effect according to an embodiment of the present invention.

[0030] FIG2 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention, wherein a is a schematic structural diagram of one bispecific antibody, and b is a schematic structural diagram of another bispecific antibody.

[0031] FIG3 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention, wherein the gray oval in the first antigen-binding portion represents VL, and the black oval represents VH.

[0032] FIG4 is a schematic structural diagram of a bispecific antibody provided according to an embodiment of the present invention, wherein the gray oval in the first antigen-binding portion represents VL, and the black oval represents VH.

[0033] Fig. 5 is the result of the binding activity of bispecific antibodies and T cells detected by FACS according to embodiment 1 of the present invention. A to D in Fig. 5 are all flow histograms, wherein A in Fig. 5 is the flow detection atlas (blank control) without adding antibody staining, B in Fig. 5 is the flow detection atlas (negative control) with addition of isotype control antibody staining, C in Fig. 5 is the flow detection atlas (positive control) with addition of anti-CD3 monoclonal antibody staining, and D in Fig. 5 is the flow detection atlas (double anti) with addition of double anti-staining. In the figure, the horizontal coordinate is the fluorescence intensity reading shown by the flow cytometer, and the vertical coordinate represents the relative cell number.

[0034] Figure 6 is the result of detecting the binding activity of bispecific antibodies to wild-type HCT116 cell lines using FACS according to Example 1 of the present invention. Figure 6 A is a blank control, Figure 6 B is the result of 0.1nM double antibody staining, Figure 6 C is the result of 1nM double antibody staining, Figure 6 D is the result of 10nM double antibody staining, and Figure 6 E is the result of 100nM double antibody staining. The horizontal axis in the figure is the fluorescence intensity reading displayed by the flow cytometer, and the vertical axis represents the relative cell number.

[0035] Figure 7 shows the results of FACS detection of the binding activity of bispecific antibodies to HCT116-TT3 cells according to Example 1 of the present invention. Figure 7 A is a blank control, Figure 7 B is the result of 0.1 nM bispecific antibody staining, Figure 7 C is the result of 1 nM bispecific antibody staining, Figure 7 D is the result of 10 nM bispecific antibody staining, and Figure 7 E is the result of 100 nM bispecific antibody staining. The horizontal axis in the figure is the fluorescence intensity reading displayed by the flow cytometer, and the vertical axis represents the relative cell number.

[0036] Figure 8 is a graph showing the affinity results of the bispecific antibody with the TT3 end and the CD3 end according to Example 1 of the present invention, wherein A, B, and C are the affinity results of different bispecific antibodies with the CD3 end, and D, E, and F are the affinity results of different bispecific antibodies with the TT3 end.

[0037] FIG9 shows the binding specificity results of bispecific antibodies to wild-type tumor cell lines HCT116 (shown in FIG9A ) and MC38 (shown in FIG9B ) using FACS according to Example 1 of the present invention. The concentrations of the bispecific antibodies were 10 nM (white) and 100 nM (black slash). The horizontal axis represents different monoclonal antibodies or bispecific antibodies, and the vertical axis represents the antibody binding percentage.

[0038] Figure 10 is a graph showing the results of in vitro combined killing of bispecific antibodies and pre-activated and expanded T cells using RTCA real-time killing instrumentation according to Example 2 of the present invention. The horizontal axis in the figure is time in hours, with the tumor cell line plating as the starting time; the vertical axis is the cell growth index; the effector cell: target cell (E:T) ratio is 2:1; each point shown in the figure is the mean of two replicate wells.

[0039] Figure 10A shows the results of the combined killing of wild-type HCT116 cell lines by anti-CD3 monoclonal antibodies and T cells in vitro. The black circles represent the HCT116 group (HCT116), the dark gray triangles represent the HCT116+T cell group (HCT116+T), the gray squares represent the HCT116+T cells+10nM anti-CD3 monoclonal antibody group (HCT116+T+10nM CD3 Ab), the light gray inverted triangles represent the HCT116+T cells+1nM anti-CD3 monoclonal antibody group (HCT116+T+1nM CD3 Ab), and the gray-white diamonds represent the HCT116+T cells+0.1nM anti-CD3 monoclonal antibody group (HCT116+T+0.1nM CD3 Ab).

[0040] Figure 10B shows the results of the combined killing of wild-type HCT116 cell lines by bispecific antibodies and T cells in vitro. The black circle represents the HCT116 group (HCT116), the gray square represents the HCT116+T cell+10nM bispecific antibody group (HCT116+T+10nM BK-A-101), the light gray inverted triangle represents the HCT116+T cell+1nM bispecific antibody group (HCT116+T+1nM BK-A-101), and the gray-white diamond represents the HCT116+T cell+0.1nM bispecific antibody group (HCT116+0.1nM+BK-A-101).

[0041] Figure 10C shows the results of the combined killing of HCT116-TT3 cell line by anti-CD3 monoclonal antibody and T cells in vitro. The black circles represent the HCT116-TT3 group (HCT116-TT3), the dark gray triangles represent the HCT116-TT3+T cell group (HCT116-TT3+T), the gray squares represent the HCT116-TT3+T cells+10nM anti-CD3 monoclonal antibody group (HCT116-TT3+T+10nM CD3 Ab), the light gray inverted triangles represent the HCT116-TT3+T cells+1nM anti-CD3 monoclonal antibody group (HCT116-TT3+T+1nM CD3 Ab), and the gray-white diamonds represent the HCT116-TT3+T cells+0.1nM anti-CD3 monoclonal antibody group (HCT116-TT3+T+0.1nM CD3 Ab).

[0042] Figure 10D shows the results of the combined killing of HCT116-TT3 cell lines by bispecific antibodies and T cells in vitro. The black circles represent the HCT116-TT3 group (HCT116-TT3), the gray squares represent the HCT116-TT3+T cells+10nM bispecific antibody group (HCT116-TT3+T+10nM BK-A-101), the light gray inverted triangles represent the HCT116-TT3+T cells+1nM bispecific antibody group (HCT116-TT3+T+1nM BK-A-101), and the gray-white diamonds represent the HCT116-TT3+T cells+0.1nM bispecific antibody group (HCT116-TT3+T+0.1nM BK-A-101).

[0043] Figure 11 is a graph showing the results of in vitro combined killing of bispecific antibodies and T cells detected by RTCA according to Example 2 of the present invention. The horizontal axis in the figure is time, in hours, with the tumor cell line plating as the starting time; the vertical axis is the cell growth index; effector cells: target cells (E:T) = 5:1. The dual antibody concentration in Figure A is 0.1nM, and the dual antibody concentration in Figure B is 1nM. Labels 1-9 represent HCT116-TT3+T cells+dual antibody BK-D-01~BK-D-09 groups, label 10 represents HCT116-TT3+T cells+BK-A-101 group, label 11 represents only HCT116-TT3 cells, without the addition of dual antibodies and T cell treatment, and label 12 represents HCT116-TT3 cells with only T cell treatment added and no dual antibodies added.

[0044] Figure 12 is a graph showing the results of in vitro combined killing of bispecific antibodies and T cells detected by RTCA according to Example 2 of the present invention. The horizontal axis in the figure is time, measured in hours, with the tumor cell line plating as the starting time; the vertical axis is the cell growth index; effector cells: target cells (E:T) = 5:1. The dual antibody concentration in Figure A is 100nM, and the tumor cell line is the HCT116 cell line; the dual antibody concentration in Figure B is 10nM, and the tumor cell line is the SKHEP1 cell line. Among them, labels 1-6 represent wild-type tumor cell lines + T cells + dual antibody BK-D-01, 04, 05, 06, 07, and 08 groups, respectively, label 7 represents wild-type tumor cell lines + T cells + TT3 monoclonal antibody-1 group, label 8 represents wild-type tumor cell lines + T cells + CD3 monoclonal antibody SP34 group, label 9 represents only wild-type tumor cell line cells, without the addition of antibodies and T cells, and label 10 represents wild-type tumor cell line cells with only T cell treatment added and no antibodies added.

[0045] Figure 13 is a graph showing the results of in vitro combined killing of bispecific antibodies and PMBC cells by RTCA according to Example 3 of the present invention. The horizontal axis in the figure represents time in hours, with the tumor cell line plating as the starting time; the vertical axis represents the cell growth index; the effector cell:target cell (E:T) ratio is 2:1; each point shown in the figure is the mean of two replicate wells. The black circle (label 1) represents the HCT116-TT3 group (HCT116-TT3), the dark gray square (label 2) represents the HCT116-TT3+PBMC group (HCT116-TT3+PBMC), the gray triangle (label 3) represents the HCT116-TT3+PBMC+10nM dual antibody group (HCT116-TT3+PBMC+10nM BK-A-101), and the gray-white inverted triangle (label 4) represents the HCT116-TT3+PBMC+1nM dual antibody group (HCT116-TT3+PBMC+1nM BK-A-101).

[0046] Figure 14 shows the binding activity of DDvv-TT3-infected tumor cell lines to the bispecific antibody BK-A-101 using FACS analysis, according to Example 4 of the present invention. Panel A shows the results for the human pancreatic cancer cell line (PANC-1), Panel B shows the results for the human melanoma cell line (A375), Panel C shows the results for the human glioma cell line (U251), and Panel D shows the results for the human ovarian cancer cell line (SKOV3). The horizontal axis represents different MOIs of infection with DDvv-TT3, and the vertical axis represents the percentage of flow cytometry positive cells in the cell population. Circles represent the results of staining with an isotype control antibody (negative control), squares represent the results of staining with the bispecific antibody (BK-A-101), and triangles represent the results of staining with a commercial antibody (GenScript) (positive control).

[0047] Figure 15 is the result of using FACS to detect the activation of bispecific antibodies in vitro cultured T cells and PBMCs according to Example 5 of the present invention. The concentrations of the bispecific antibodies are 0.01nM (white), 0.1nM (black slash), 1nM (black dot), 10nM (black horizontal bar), 100nM (black cross line), and 250nM (black grid). Figure A shows the results of in vitro cultured T cells, and Figure B shows the results of PBMCs. The horizontal axis represents different bispecific antibodies, and the vertical axis represents the expression ratio of CD25, an early activation marker on the surface of T cells.

[0048] Figure 16 is a diagram of the ELISA assay for detecting the IFN-γ content in the supernatant after 3 days of co-culture of bispecific antibodies and PBMC according to Example 5 of the present invention. The concentrations of the bispecific antibodies are 0.1nM (black dots), 1nM (black grid), 10nM (black vertical bar), 100nM (black diagonal bar), and 250nM (black cross lines). Figure A shows the supernatant detection results of 3 days of co-culture of bispecific antibodies and PBMC, Figure B shows the supernatant detection results of 3 days of co-culture of bispecific antibodies and PBMC and SKHEP1 cell lines, and Figure C shows the supernatant detection results of 3 days of co-culture of bispecific antibodies and PBMC and SKHEP1-TT3 cell lines. In the figure, the horizontal axis represents the CD3 monoclonal antibody SP34 and different bispecific antibodies BK-A-101 and BK-D-01, 04, 05, 06, 07, and 08, and the vertical axis represents the IFN-γ content (pg / mL) in the co-culture supernatant.

[0049] Figure 17 is a diagram of the in vitro combined killing results of bispecific antibodies, T cells and recombinant oncolytic poxviruses detected by RTCA according to Example 6 of the present invention. Figures A and C are the results of real-time killing in vitro, where the abscissa is time in hours, with tumor cell line plating as the starting time, and the ordinate is the cell growth index. Effector cell: target cell (E: T) = 10: 1, the concentration of the bispecific antibody is 1 nM, and the MOI of the recombinant oncolytic poxvirus DDvv-TT3 is 1. Figures B and D are the results of the tumor inhibition rate at the node for 80 hours of real-time killing in vitro, where the abscissa is the different experimental groups and the ordinate is the tumor growth inhibition rate. Figures A and B are wild-type HCT116 cell lines, and Figures C and D are wild-type HCT116 cell lines + DDvv-TT3. In Figure A, number 1 is the HCT116 group, number 2 is the HCT116+T cell group, numbers 3-5 are HCT116+T cells+bispecific antibodies BK-D-04, 06, and 07, and number 6 is HCT116+T cells+BK-A-101; in Figure C, number 1 is the HCT116 group, number 2 is the HCT116+DDvv-TT3 group, number 3 is the HCT116+DDvv-TT3+T group, numbers 4-6 are HCT116+DDvv-TT3+T cells+bispecific antibodies BK-D-04, 06, and 07, and number 7 is the HCT116+DDvv-TT3+T cells+BK-A-101 group.

[0050] It should be noted that the same numerical labels (such as 1, 2, 3, etc.) in Figures 11, 12, 3 and 17 do not correspond to exactly the same content, and the meanings represented by the numerical labels in each figure have been listed above. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is described in detail below with reference to specific examples. At the same time, in order to facilitate understanding by those skilled in the art, some terms of the present invention are explained and illustrated. It should be noted that these explanations and illustrations are only used to facilitate understanding by those skilled in the art and should not be regarded as limiting the scope of protection of the present invention.

[0052] As used in this specification and claims, "a", "an" and "the" include plural referents unless the context clearly indicates otherwise. The words "tumor", "cancer", "tumor cell", and "cancer cell" have the meanings commonly recognized in the art.

[0053] As used herein, the term "oncolytic virus" refers to a virus that can selectively replicate in and lyse tumor cells.

[0054] The term "therapeutically effective amount" herein refers to the amount of a drug, formulation, or active ingredient that can exhibit a detectable therapeutic effect or inhibitory effect. The detectable therapeutic effect or inhibitory effect mentioned above can be detected by any detection method known in the art.

[0055] The terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the technical features being referred to. They are also not intended to indicate a sequential order. For example, the terms "first composition" and "second composition," or "first active ingredient" and "second active ingredient" are not intended to indicate a sequential order or to characterize importance; they are only used to distinguish.

[0056] As used herein, the term "MOI," or multiplicity of infection, refers to the ratio of virus to cell number and refers to the average number of viral particles per cell. MOI = pfu / cell. PFU (Plaque Forming Unit) is a measure of the number of infectious viral particles.

[0057] The present invention provides a therapeutic agent comprising:

[0058] (a) a first composition comprising a first active ingredient, the first active ingredient comprising or containing a nucleic acid having a marker polypeptide encoding sequence for introduction into tumor cells and / or cancer cells; the marker polypeptide having an operably linked extracellular antigenic determinant, a spacer portion, and a transmembrane portion; the amino acid sequence of the extracellular antigenic determinant containing the amino acid sequence of one or more antigenic epitope polypeptides; and the amino acid sequence of a mammalian cell membrane protein or secretory protein not containing the amino acid sequence of the antigenic epitope polypeptide in a natural state; and

[0059] (b) a second composition comprising a second active ingredient, wherein the second active ingredient comprises a multispecific antibody, wherein the multispecific antibody comprises at least a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion can specifically recognize and bind to the extracellular antigenic determinant region of the marker polypeptide;

[0060] The second antigen binding portion is capable of specifically recognizing and binding to an immune cell antigen or a first cytokine or its receptor;

[0061] The multispecific antibody may also optionally include a third antigen binding portion, which specifically recognizes and binds to a tumor antigen or an immune checkpoint or a second cytokine or its receptor.

[0062] For easier understanding, FIG1 shows a schematic diagram of the therapeutic effect exerted by the provided therapeutic agent. In FIG1 , an IgG-like bispecific antibody is used as an example, and TT3 is used as an example of a marker polypeptide. An antigenic epitope polypeptide (the antigenic epitope polypeptide is at least a part of the extracellular antigenic determinant region, and the spacer and transmembrane portions form the TT3 marker polypeptide) that is not contained in the amino acid sequence of an artificially synthesized mammalian cell membrane protein or secretory protein is carried to tumor cells and marked on the surface of the tumor cells. On the one hand, the bispecific antibody can specifically recognize and bind to TT3, and on the other hand, it can specifically recognize and bind to immune cells, such as T cells and NK cells shown in the figure. The bispecific antibody connects immune cells and tumor cells to redirect tumor cell lysis (redirected tumor lysis) and activates immune cells to produce cytotoxic molecules including perforin and granzymes to kill tumor cells, thereby exerting a specific killing effect. It should be noted that the IgG-like bispecific antibody shown in Figure 1 is only used as an example, and does not mean that the multispecific antibody mentioned in this article is a specific IgG-type structure.

[0063] When an oncolytic virus is used as a vector and the oncolytic virus is specifically replicated in the tumor to express the labeled polypeptide in the tumor cells or cancer cells, the oncolytic virus can kill the tumor cells or cancer cells while significantly enhancing the expression of the antigen epitope peptide on the surface of the tumor cells, thereby combining with the antibody-bridged immune cells to show a synergistic therapeutic effect.

[0064] Labeled peptide

[0065] The marker polypeptides mentioned herein are described in PCT patent application number PCT / CN2019 / 102480 and international publication number WO2020038490A, which are hereby incorporated by reference in whole or in part as needed. The marker polypeptides mentioned herein are used to modify the surface of tumor cells and / or cancer cells, and have an operably linked extracellular antigenic determinant, a spacer portion, and a transmembrane portion, and can be modified on the surface of tumor cells and / or cancer cells by expression; the amino acid sequence of the extracellular antigenic determinant contains the amino acid sequence of one or more antigenic epitope polypeptides; wherein, in the natural state, the amino acid sequence of a mammalian cell membrane protein or secretory protein does not contain the amino acid sequence of the antigenic epitope polypeptide.

[0066] The term "extracellular antigenic determinant region" as used herein refers to the portion of the polypeptide containing the antigenic epitope located outside the cell membrane when the marker polypeptide is expressed on the cell surface.

[0067] According to an embodiment of the present invention, the amino acid sequence of the antigenic epitope polypeptide is derived from the amino acid sequence of a naturally occurring protein, or is an artificially synthesized amino acid sequence that does not exist in nature. Naturally occurring proteins include mammalian cellular proteins and proteins from organisms other than mammals. Proteins from organisms other than mammals include viral proteins, bacterial proteins, fungal proteins, protozoan proteins, plant proteins, insect proteins, and proteins from animals other than mammals.

[0068] In some embodiments of the present invention, the amino acid sequence of the antigenic epitope polypeptide is derived from the amino acid sequence of the following tags: Myc tag, HA tag, Strep tag I, Strep tag II, Flag tag, HAT tag, S tag, S1 tag, protein C tag, tag-100 tag, E2 tag, TAP tag, HSV tag, KT3 tag, V5 tag, VSV-G tag, His tag or RFP tag, etc. The amino acid sequences and nucleotide sequences of these tags are all known and can be queried from public databases commonly used in the art.

[0069] In a preferred embodiment of the present invention, the amino acid sequence of the antigenic epitope polypeptide is derived from the amino acid sequence of the following tags: human Myc tag (the corresponding marker polypeptide is denoted as TT1), influenza virus HA tag (the corresponding marker polypeptide is denoted as TT2), Strep tag II (the corresponding marker polypeptide is denoted as TT3), or Strep tag I. The amino acid sequence of the Strep tag I or Strep tag II mentioned above is at least 3 amino acids in length, for example, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, or 11, 12, 13, 14 or 15 amino acids, and may contain amino acids derived from the sequence shown in NWSHPQFEK (SEQ ID NO: 59) or AWRHPQFGG (SEQ ID NO: 60), and may also be a partial sequence or a combination of partial sequences of the sequence shown in SEQ ID NO: 59 or SEQ ID NO: 60. More specifically, the amino acid sequence of Strep tag II may be, for example, WSHPQFEK (SEQ ID NO: 58) or NWSHPQFEK (SEQ ID NO: 59). The amino acid sequence of the aforementioned Strep tag I may be, for example, AWRHPQFGG (SEQ ID NO: 60). Also preferably, the amino acid sequence of the antigenic epitope polypeptide is consistent with the amino acid sequence of positions 410 to 419 of the human Myc protein; or the amino acid sequence of the antigenic epitope polypeptide is consistent with the amino acid sequence of positions 119 to 127 of the influenza virus HA protein; or the amino acid sequence of the antigenic epitope polypeptide is consistent with Strep tag II (for a detailed description of Strep tag II, see the document “Molecular Interaction Between the Strep-tag Affinity Peptide and its Cognate Target, Streptavidin, Thomas GMSchmidt, Jurgen Koepke, Ronald Frank and Arne Skerra”). In other embodiments of the present invention, the amino acid sequence of the epitope polypeptides of TT1 and TT2 may be extended by no more than 10 amino acids upstream or downstream of the above sequences. The amino acid sequence of the human Myc protein may be the amino acid sequence numbered P01106isoform 1 in UniProtKB. The amino acid sequence of the influenza virus HA protein may be the amino acid sequence numbered Q03909 in UniProtKB.

[0070] In a specific embodiment, the amino acid sequence of the extracellular antigenic determinant region of the marker polypeptide includes the amino acid sequence of one or more epitope polypeptides, wherein when the amino acid sequence of the extracellular antigenic determinant region of the marker polypeptide includes the amino acid sequence of multiple epitope polypeptides, each two adjacent epitope polypeptides are operably connected, for example, by using a linker or directly connected without using a linker. The amino acid sequence of the linker can be (for example): G (as used in marker polypeptides C1&2a, C1&2b), GGS (as used in C1&2a, C1&2b), GGGGSGGGGS (as used in TT1-TT3).

[0071] To enhance the immunogenicity of the marker polypeptide, the extracellular antigenic determinant region of the marker polypeptide preferably comprises n antigenic epitope polypeptides, where n is an integer greater than or equal to 1, for example, n = 1, 2, 3, 4, ... Preferably, n is an integer between 1 and 10; more preferably, n is an integer between 2 and 5; and even more preferably, n = 2 or 3. For example, the extracellular antigenic determinant region of the marker polypeptide may comprise three repeats of an antigenic epitope polypeptide derived from a Myc tag (see, for example, TT1), or three repeats of an antigenic epitope polypeptide derived from an HA tag (see, for example, TT2), or three repeats of an antigenic epitope polypeptide derived from a Strep tag II (see, for example, TT3), or three repeats of an antigenic epitope polypeptide derived from a Myc tag and three repeats of an antigenic epitope polypeptide derived from an HA tag (see, for example, Cl&2a), or two repeats of an antigenic epitope polypeptide derived from a Myc tag and two repeats of an antigenic epitope polypeptide derived from an HA tag (see, for example, Cl&2b).

[0072] In one embodiment of the present invention, the amino acid sequence of the extracellular antigen determining region is as shown in SEQ ID NO: 1 (corresponding to TT1), SEQ ID NO: 2 (corresponding to TT2), SEQ ID NO: 3 (corresponding to TT3), SEQ ID NO: 4 (corresponding to Cl&2a), and SEQ ID NO: 5 (corresponding to Cl&2b).

[0073] Preferably, the transmembrane portion is derived from the transmembrane region of CD8, CD3ζ, CD4 or CD28, and its full-length amino acid sequence and nucleotide sequence are all known and can be queried from public databases commonly used in the art. Also preferably, the transmembrane portion is derived from the transmembrane region of human CD8α. More preferably, the amino acid sequence of the transmembrane portion comprises the amino acid sequence shown in SEQ ID NO:7. CD8 is a transmembrane glycosylated membrane protein composed of two subunits, α and β, which work together with T cell surface receptors to bind T cells to specific antigens. CD8 specifically binds to MHC I and mediates the killing effect of cytotoxic T cells. The transmembrane region is usually a hydrophobic α helix that spans the cell membrane.

[0074] In the marker polypeptide of the present invention, the transmembrane portion and the extracellular antigenic determinant region can be connected by a spacer. The structure of this region should be flexible, allowing the extracellular antigenic determinant region to adapt to different orientations, thereby promoting recognition and binding by the corresponding multispecific antibody. The simplest form of the spacer is the hinge region of immunoglobulin IgG1, or it can be a portion of the CH2CH3 region of an immunoglobulin. Through research and experimental exploration, it has been found that the spacer is preferably derived from the hinge region of CD8α, and the transmembrane region is preferably derived from the transmembrane region of CD8α. Preferably, the amino acid sequence of the spacer is as shown in SEQ ID NO:6. Also preferably, the spacer and transmembrane portion constitute a spacer transmembrane portion, and the amino acid sequence of the spacer transmembrane portion is identical to the amino acid sequence of positions Y to 210 of CD8α, with 118≤Y≤128, where Y is an integer. The UniProtKB number for the amino acid sequence of CD8α can be P01732. That is, the amino acid sequence of the spacer transmembrane portion is preferably selected from amino acids 118 to 210 of CD8α and includes amino acids 128 to 210. For example, the amino acid sequence of the spacer transmembrane portion is as shown in any one of the following amino acid sequence groups: amino acids 118 to 210, amino acids 119 to 210, amino acids 120 to 210, amino acids 121 to 210, amino acids 122 to 210, amino acids 123 to 210, amino acids 124 to 210, amino acids 125 to 210, amino acids 126 to 210, amino acids 127 to 210, or amino acids 128 to 210 of CD8α.

[0075] The definitions of the terms "spacer portion" and "transmembrane portion" used in this article are known in the art. For details, please refer to "Introduction to Immunology, Yu Shanqian, Higher Education Press, 2008"; and "Immunobiology, 7th Edition, Kenneth Murphy, Paul Travers, Mark Walport, etc."

[0076] In the nucleic acid of the present invention, a signal peptide coding sequence is preferably included immediately before the 5' end of the marker polypeptide coding sequence. The signal peptide has the function of directing secretion of the target protein to the cell surface. The present invention has discovered that combining the extracellular antigenic determinant with a signal peptide derived from the GM-CSF α chain enables expression of the marker polypeptide on the surface of tumor cells. The GM-CSF α chain signal peptide serves as a leader sequence that targets the marker polypeptide of the present invention to the secretory pathway. Its coding sequence, along with the coding sequence for the marker polypeptide, is first translated into protein within the cell, directing the synthesized protein into the intracellular secretory pathway. Prior to cell surface expression of the marker polypeptide, the signal peptide is removed. The full-length amino acid and nucleotide sequences of the GM-CSF α chain are known and can be retrieved from publicly available databases commonly used in the art. Preferably, the amino acid sequence of the signal peptide is selected from amino acids 1 to 22 of the human GM-CSF α chain. More preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:8. The amino acid sequence of the GM-CSF α chain is derived from UniProtKB-P15509.

[0077] In a specific embodiment of the present invention, the marker polypeptide comprises the following amino acid sequences operably linked and sequentially connected in series: an extracellular antigenic determinant (which comprises one or more amino acid sequences of the antigenic epitope polypeptide), a spacer portion, and a transmembrane portion. In a more specific embodiment of the present invention, the amino acid sequence of the antigenic epitope polypeptide of the extracellular antigenic determinant is derived from the amino acid sequence of the antigenic epitope polypeptide in the following polypeptides: a Myc tag derived from the human intracellular protein Myc, an HA tag derived from the influenza virus HA protein, an artificially synthesized sequence Strep tag II that does not exist in nature, the spacer portion is derived from the human CD8α hinge region, and the transmembrane portion is derived from the human CD8α transmembrane region.

[0078] As described above, the signal peptide and the extracellular antigenic determinant, the extracellular antigenic determinant and the spacer, and the spacer and the transmembrane portion are operably connected, for example, by using a linker or directly connected without a linker. In one embodiment of the present invention, the signal peptide and the extracellular antigenic determinant are connected by a linker, for example, GAHADITS (as used in TT1-TT3), GAHAAQLTLTKGNK (as used in C1 & 2a), or GAHA (as used in C1 & 2b). The extracellular antigenic determinant and the spacer are connected by a linker, for example, -Ala-Ser-, G, while the spacer and the transmembrane portion are directly connected without a linker.

[0079] The present invention further discovered that the tagged polypeptide can express two antigenic epitope polypeptides at the same time. Specifically, the present invention designed a tagged polypeptide Cl&2a, whose antigenic determining region contains three repeats of a Myc tag and an HA tag. The present invention also designed a tagged polypeptide Cl&2b, whose antigenic determining region contains two repeats of a Myc tag and an HA tag; in order to further stabilize the expression of the tagged polypeptide on the cell membrane surface, a TIGIT spacer was added to the spacer portion of Cl&2b, wherein the amino acid sequence of the added spacer is consistent with the amino acid sequence of positions 24 to 140 of TIGIT. The amino acid sequence of TIGIT is derived from UniProtKB-Q495Al. More preferably, the amino acid sequence of the added TIGIT spacer is shown in SEQ ID NO: 9, and the amino acid sequence of the transmembrane spacer portion of Cl&2b is shown in SEQ ID NO: 10.

[0080] Preferably, the amino acid sequence of the marker polypeptide is as shown in SEQ ID NO:11 (corresponding to TT1), SEQ ID NO:12 (corresponding to TT2), SEQ ID NO:13 (corresponding to TT3), SEQ ID NO:14 (corresponding to Cl&2a) or SEQ ID NO:15 (corresponding to Cl&2b).

[0081] SEQ ID NO: 11:

[0082] The bold part shows the antigen epitope polypeptide, the grey part shows the spacer part from the CD8 hinge region, and the underline shows the transmembrane part from the CD8 transmembrane region.

[0083] SEQ ID NO: 12:

[0084] The bold part shows the antigen epitope polypeptide, the grey part shows the spacer part from the CD8 hinge region, and the underline shows the transmembrane part from the CD8 transmembrane region.

[0085] SEQ ID NO: 13:

[0086] The bold part shows the antigen epitope polypeptide, the grey part shows the spacer part from the CD8 hinge region, and the underline shows the transmembrane part from the CD8 transmembrane region.

[0087] SEQ ID NO: 14:

[0088] The bold part shows the antigen epitope polypeptide, the grey part shows the spacer part from the CD8 hinge region, and the underline shows the transmembrane part from the CD8 transmembrane region.

[0089] SEQ ID NO: 15:

[0090] The bold part shows the antigen epitope polypeptide, the dark gray part shows the part of the spacer part derived from the TIGIT spacer region, the light gray part shows the part of the spacer part derived from the CD8 hinge region, and the underline shows the transmembrane part derived from the CD8 transmembrane region.

[0091] Nucleic acid with a marker polypeptide coding sequence

[0092] When obtaining the above-mentioned marker polypeptide coding sequence, the general rules in the art can be used to obtain the coding sequence encoding these marker polypeptides. The nucleic acid having the marker polypeptide coding sequence includes DNA or RNA; the RNA includes mRNA transcribed from the DNA. The nucleic acid sequentially comprises an operably linked promoter and the marker polypeptide coding sequence. Examples of the promoter include any promoter known in the art for initiating transcription of downstream DNA sequences, such as a CMV promoter, a T7 promoter, and the like. The above-mentioned marker polypeptide coding sequence is also described in detail in the PCT patent application with international application number PCT / CN2019 / 102480 and international publication number WO2020038490A, so it will not be described in detail here.

[0093] recombinant virus

[0094] In one embodiment of the present invention, the first active ingredient is a recombinant virus, the genome of which comprises a promoter, a signal peptide coding sequence, and the marker polypeptide coding sequence in sequence and operably linked; wherein the recombinant virus comprises a selectively replicating recombinant oncolytic virus or a replication-deficient recombinant virus. The marker polypeptide coding sequence encodes the marker polypeptide.

[0095] Preferably, the recombinant oncolytic virus is derived from a genetically mutated virus with oncolytic activity and a wild-type virus with oncolytic activity; preferably, the recombinant oncolytic virus is derived from an adenovirus, poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus and Maraba virus with oncolytic activity.

[0096] Oncolytic virus therapy has a long history, and many types of viruses have been modified for oncolytic virus research and development, including adenovirus, herpes virus, poxvirus, picornavirus, paramyxovirus, reovirus, parvovirus, and rhabdovirus. To date, several oncolytic viruses (H101, T-VEC, Delytact) have been approved for the treatment of clinical tumors. Oncolytic viruses are artificially modified viruses. The pathogenic genes of these viruses have been knocked out, and they are basically unable to replicate in normal tissue cells, but they can selectively replicate in tumor cells to exert an oncolytic effect. Taking poxvirus as an example, oncolytic poxviruses are usually constructed by genetic bioengineering using homologous recombination technology to knock out the endogenous thymidine kinase (TK) gene of the poxvirus or double knockout of TK and vaccinia growth factor (VGF) genes. TK is one of the key enzymes for DNA synthesis. During the replication process, poxviruses need to rely on the action of TK to form a high-concentration nucleic acid pool to complete the smooth replication of offspring. Poxviruses often promote the proliferation of host and surrounding cells through VGF gene products to achieve their own replication and proliferation, while poxviruses lacking VGF greatly reduce the damage of the virus to normal cells. Due to the lack of TK and VGF genes, the ability of the virus to replicate in normal cells is limited, and the expression of exogenous genes inserted in oncolytic virus vectors will also be greatly restricted. However, the TK gene is highly expressed in the environment of most tumor cells, providing a favorable environment for the replication and packaging of poxviruses, so that poxviruses have the function of selectively replicating and oncolytic in tumor cells, and highly expressing exogenous genes inserted in oncolytic virus vectors. In addition to direct oncolytic effects, oncolytic viruses can also cause tumor cells to release holographic tumor antigens to activate the body's intrinsic anti-tumor immunity, while also having a certain regulatory effect on the tumor microenvironment.

[0097] The present invention inserts the marker polypeptide coding sequence into the genome of the oncolytic virus, and utilizes the oncolytic virus to specifically replicate in tumor cells to introduce the marker polypeptide encoding nucleic acid into tumor cells and / or cancer cells, so that while the oncolytic virus kills tumor cells and / or cancer cells, the exogenous antigen epitope peptide is expressed on the surface of the tumor cell and combined with the multispecific antibody to achieve a synergistic therapeutic effect. Because the oncolytic virus destroys the microenvironment of the tumor, the therapeutic effect of the multispecific antibody can be further improved, thereby further enhancing the effectiveness of solid tumor treatment. In addition, the multispecific antibody can also effectively eliminate tumor cells that cannot complete the replication cycle and produce a sufficient number of daughter viruses after being infected by the oncolytic virus by recruiting immune cells; thereby achieving further synergistic effects. In addition, the antigens released by the tumor cells lysed by the oncolytic virus can further activate the body's own anti-tumor immunity, thereby achieving a better tumor killing effect than using oncolytic virus or multispecific antibody alone, and achieving a synergistic therapeutic effect.

[0098] In one embodiment of the present invention, the nucleic acid encoding the marker polypeptide sequence can be inserted into the genome of the resulting selectively replicable recombinant oncolytic poxvirus.

[0099] Preferably, the recombinant oncolytic virus is a recombinant oncolytic poxvirus deficient in TK and VGF gene function. The TK gene can be rendered functionally defective by inserting an exogenous nucleotide sequence. The VGF gene can be rendered functionally defective by gene knockout or insertion of an exogenous nucleotide sequence, but preferably the VGF gene is knocked out.

[0100] The term "functional defect" used in the present invention when referring to the gene of the oncolytic virus means that the oncolytic virus cannot perform the function that the gene should have, that is, loss of function, which can be achieved by (for example) inserting an exogenous fragment into the gene or knocking out the gene. Also preferably, the recombinant oncolytic poxvirus is a Wyeth strain or a WR strain.

[0101] The marker polypeptide coding sequence can be inserted into the TK gene to obtain the recombinant oncolytic virus of the present invention. The marker polypeptide coding sequence can also be inserted into the VGF gene to obtain the recombinant oncolytic virus of the present invention.

[0102] In a preferred embodiment, the recombinant oncolytic poxvirus is obtained by genetically modifying the VSC20 poxvirus. The VSC20 poxvirus is a poxvirus with a VGF gene deletion. The preparation method can be found in the scientific literature: "McCart, JA, et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. Cancer Res (2001) 61: 8751–8757.". The genetic modification includes inserting an exogenous marker polypeptide coding sequence into the TK gene of the VSC20 poxvirus, thereby rendering the TK gene functionally defective.

[0103] The genome of the recombinant virus may also be integrated with other exogenous genes, such as exogenous screening genes, including puromycin gene, gpt gene and / or LacZ gene. The exogenous screening gene can be knocked out by a gene knockout system (e.g., LoxP) during purification of the gene encoding the exogenous marker polypeptide.

[0104] In some embodiments, the present invention uses the poxvirus early / late promoter p7.5 to control the exogenous screening gene, and the artificially synthesized poxvirus early promoter pSEL to control the signal peptide sequence and the exogenous marker polypeptide coding sequence, and uses in vitro intracellular recombination technology to insert the exogenous screening gene coding sequence, the signal peptide coding sequence and the marker polypeptide coding sequence into the TK gene region of the poxvirus VSC20 strain, thereby constructing an oncolytic virus, and the two promoters respectively start the expression of their respective regulatory genes in a back-to-back manner.

[0105] In another embodiment of the present invention, a recombinant oncolytic poxvirus is constructed using Crisper-Cas9 gene editing technology. Preferably, the Crisper-Cas9 gene editing method can simultaneously perform cleavage and homologous recombination at two sites in a gene, thereby achieving the purpose of knocking out a sequence and inserting a transgene in one go.

[0106] Preferably, the Crisper-Cas9 gene editing method can simultaneously cleave the target sequence and the donor sequence to improve the efficiency of homologous recombination. The design of the cleavage and homologous recombination of the Crisper-Cas9 gene editing method is also described in detail in the PCT patent application with International Publication No. WO2020038490A (Application No. PCT / CN2019 / 102480), and will not be repeated here.

[0107] Multispecific antibodies

[0108] As used herein, the term "antibody" is used in the broadest sense to refer to a protein or polypeptide that contains an antigen binding site, antigen binding portion, or antigen binding fragment, encompassing natural antibodies and artificial antibodies of various structures, including but not limited to complete antibody forms or antigen binding fragments of antibodies.

[0109] As used herein, the terms "intact antibody" or "complete antibody" are used synonymously to refer to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (also referred to as a heavy chain constant domain, abbreviated as CH). The heavy chain constant region includes the heavy chain constant domain CHI, the heavy chain constant domain C2, and the heavy chain constant domain C3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region (also referred to as a light chain constant domain, abbreviated as CL). VH and VL can be further divided into complementarity determining regions (also referred to as hypervariable regions or hypervariable regions, abbreviated as CDRs or HVRs), interspersed with conserved framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) in the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs of the heavy chain variable region are referred to as HCDR1, HCDR2 and HCDR3, respectively, starting from the amino terminus, and the CDRs of the light chain variable region are referred to as LCDR1, LCDR2 and LCDR3, respectively, starting from the amino terminus.

[0110] "Antigen-binding fragment," "antigen-binding portion," or "antigen-binding site" refers to a portion of a full-length antibody that exhibits specific binding to an antigen. As mentioned above, the hypervariable or hypervariable regions of the heavy and light chains of an intact antibody exhibit specific binding to an antigen. Examples of antigen-binding fragments or portions include, but are not limited to, Fab, Fab', F(ab')2, bispecific Fab' and Fv fragments, linear antibodies, single-chain antibodies, single-domain antibodies, and the like. Papain digestion of an intact antibody produces two identical antigen-binding fragments, called Fab fragments, each containing the heavy and light chain variable regions and the light chain constant domain and the heavy chain constant domain CH1. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxyl terminus of the heavy chain constant domain CH1, including one or more cysteines from the antibody hinge region. Pepsin digestion of an intact antibody produces a F(ab')2 fragment. F(ab')2 fragments have two antigen-binding F(ab) moieties linked by a disulfide bond, and are bivalent antibodies. Single-chain antibodies (SCAbs) are fusion proteins composed of the variable regions of the heavy and light chains of antibodies, connected by a flexible peptide of approximately 10-25 amino acids. Single-domain antibodies (SDAbs) are antibody fragments composed of the variable regions of a single monomer. Because SDAbs are typically derived from the variable regions of the heavy chains of antibodies from camelids or sharks, they are often referred to as nanobodies.

[0111] The term "multispecific antibody" (also known as a "polyantibody") refers to an antibody comprising antigen-binding moieties that specifically bind to epitopes of at least two different biomolecules, or at least two different epitopes of the same biomolecule. For example, a multispecific antibody can be a bispecific antibody (also known as a "diabody") or a trispecific antibody. "Multi" means two or more. For convenience, quantifiers are added before the antigen-binding moieties to distinguish them. For example, they can be expressed as "first antigen-binding moiety," "second antigen-binding moiety," "third antigen-binding moiety," etc., as needed. "First," "second," and "third" do not indicate the order of binding or are not used to indicate importance. The terms "first antigen-binding moiety," "second antigen-binding moiety," and "third antigen-binding moiety" will be explained and illustrated below. Unless otherwise stated, the order of antigen binding in the listed multispecific antibodies is arbitrary. Of course, when a multispecific antibody specifically binds to different biomolecules, it may bind to more than one antigenic epitope of that specific biomolecule. According to a preferred embodiment of the present invention, the multispecific antibody comprises antigen-binding moieties that bind to different epitopes of at least two different biomolecules.

[0112] When the term "and / or" is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0113] The term "comprising" or "including" means including the elements or steps mentioned, but does not exclude other elements or steps. Of course, unless otherwise specified, "comprising" or "including" also covers the situation where it consists of the elements or steps mentioned. For example, when referring to an antibody variable region comprising a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0114] The "affinity" or "binding affinity" mentioned herein is understood according to the common meaning in the art, and is used to reflect the strength and / or stability of the binding sites between an antigen and an antibody or antigen-binding fragment.

[0115] "Specific binding" or "specifically binds to", "binds to", "specifically targets" a specific antigen or epitope, or "has specificity" or "can bind to" a specific antigen or epitope means to distinguish it from non-specific interactions, and this specific binding can be measured by some methods commonly used in the art. The ability of an antibody to bind to an antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art. For example, cells carrying an antigen can be detected by flow cytometry, and the competitive binding between the test antibody and the labeled antibody can be detected by measuring the positive rate index of the cells. Since the spatial structure of the antigen on the cell surface is closer to the form existing in the body, this method can better reflect the real situation. According to a specific embodiment of the present invention, the provided bispecific antibody and the immune cell antigen (e.g., T cell antigen (e.g., CD3 antigen)) have a molecular weight of 0.5-200nM, 1-200nM, 5-200nM, 10-200nM, 15-200nM, 0.5-150nM, 1-150nM, 5-150nM, 10-150nM, 15-150nM, 0.5-120nM, 1-120nM, 5-120nM, 1 The EC50 values ​​of the antibody and the antigen are 0-120 nM, 15-120 nM, 10-100 nM, 15-100 nM, 10-80 nM, 15-80 nM, 10-60 nM, 15-60 nM, 10-50 nM, or 15-50 nM, and the EC50 values ​​of the antibody and the antigen are 0.01-20 nM, 0.05-20 nM, 0.01-16 nM, 0.05-16 nM, 0.01-15 nM, 0.05-15 nM, 0.01-10 nM, 0.05-10 nM, 0.1-10 nM, or 0.3-5 nM, respectively. In addition, the binding activity of the antibody and the antigen can also be measured by surface plasmon resonance (SPR) or bio-film interferometry (BLI).

[0116] The multispecific antibodies, or polynucleotides, mentioned herein are generally separable or recombinant. "Separable" means capable of being identified and separated and / or recovered from cells or cell cultures expressing the polypeptide or protein. Typically, the isolated polypeptide will be prepared by at least one purification step. "Isolated antibody" means substantially free of other antibodies or antigen-binding fragments thereof having different antigenic specificities. "Recombinant" means that the antibody can be produced in an exogenous host cell using genetic recombination techniques.

[0117] "Humanized" antibodies generally refer to antibodies whose antigen-binding portion is derived from a non-human species and whose structure and sequence are based on a portion of a human immunoglobulin molecule. For example, in a humanized antibody, the entire antibody, excluding the CDRs, is encoded by a polynucleotide of human origin, which retains antigen-binding activity while reducing immunogenicity.

[0118] The CDR sequences shown herein were obtained by analysis based on existing definition schemes. For example, the CDR sequences of the antibodies shown, as well as the CDR sequences of the first antigen-binding portion and the second antigen-binding portion of the multispecific antibodies, were obtained by combining the IMGT definition scheme (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38: D301-D307 (2010); Ehrenmann, F., Lefranc, M.-P. Cold Spring Harbor Protoc., 6: 737-749 (2011)). Definition schemes such as Kabat (see, for example, U. S. Pat. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)) and Chothia (see, for example, J. Mol. Biol. 196: 901-917 (1987)) can also be obtained. It is known to those skilled in the art that the differences in CDRs due to the definition method are also included in the protection scope of the present invention.

[0119] Bispecific antibodies (BsAb, also known as bispecific antibodies) are antibodies that can specifically bind to two antigens or different epitopes of the same antigen at the same time. BsAb can target multiple antigens or antigen epitopes, and has more advantages than monoclonal antibodies, and has gradually shown higher efficacy than monoclonal antibody combination therapy. For example, specific immune effector cells can be redirected to adjacent tumor cells to enhance tumor killing; and the specificity of binding can be increased through the interaction of two different cell surface antigens; at the same time, compared with the combination of single antibodies, development costs and clinical trials can be reduced. Bispecific antibodies have broad application prospects in fields such as tumor treatment and autoimmune diseases, and have developed rapidly with the gradual maturity of genetically engineered antibody technology.

[0120] The second composition includes a second active ingredient, which includes a multispecific antibody. In addition to the multispecific antibody mentioned above, the second composition may also contain a pharmaceutically acceptable carrier as needed. Some pharmaceutically acceptable salts, such as acid addition salts or base addition salts (for example, as described in Berge, S. M. et al. (1977) J. Pharma. Sci. 66: 1-19). The multispecific antibody may be a bispecific antibody, which includes a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion can specifically recognize and bind to the extracellular antigenic determinant of the marker polypeptide; the second antigen-binding portion can specifically recognize and bind to an immune cell antigen or a first cytokine or its receptor. By recruiting or bridging immune cells or cytokines or their receptors, an immunotherapeutic effect is exerted. The first antigen-binding portion and the second antigen-binding portion are fused, and the first antigen-binding portion and the second antigen-binding portion respectively target different antigens and different epitopes. The fusion of the first antigen-binding portion and the second antigen-binding portion mentioned above can be directly connected (for example, directly connected through the terminal amino acids of the two), can be indirectly connected through a linker, and other functional region sequences, such as Fc region, CH1, CL sequence, etc., can also be connected between the two. It should be noted that, as shown in the text, the terms "first", "second" and "third" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated, nor are they used to indicate the order of precedence. The terms "first antigen-binding portion", "second antigen-binding portion" and "third antigen-binding portion" respectively refer to portions that can bind to different epitopes of different target antigens or different epitopes of the same target antigen, which can be complete antibodies or parts of complete antibodies, such as heavy chain variable regions, Fab, Fab', F(ab')2, single-chain antibodies (ScFv), or single-domain antibodies (sdAb), etc. According to a preferred embodiment of the present invention, the "first antigen-binding portion", "second antigen-binding portion" and "third antigen-binding portion" specifically bind to different epitopes of different antigens, respectively.

[0121] Herein, "linker" and "linker fragment" have the same meaning. The linker can be some oligopeptides or polypeptides, and can be any amino acid sequence that can provide flexibility. Available linkers are linkers commonly used in the art, including but not limited to the following group: GS, SG, GGS, GSG, SGG, GGG, GGGS, GGGGGSGS, GGGGSGS, GGGGSGGS, GGGGSGGGGSGGGGS, GGGGSGGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGS, GGGGSGGGGSGGGGSGGGS and the like.

[0122] The provided first antigen-binding portion is capable of specifically recognizing and binding to the extracellular antigenic determinant of the marker polypeptide. According to specific embodiments, the provided first antigen-binding portion is capable of specifically recognizing and binding to the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4 or 5 mentioned above. According to specific embodiments, the provided first antigen-binding portion comprises one or more domains capable of specifically recognizing and binding to the antigenic epitope polypeptide, wherein the amino acid sequence of the antigenic epitope polypeptide comprises the amino acid sequence of Strep tag II.

[0123] Preferably, the amino acid sequence of the light chain of the antigen binding domain targeting TT1 is consistent with the amino acid sequence of positions 1 to 111 of the light chain of the anti-Myc antibody (e.g., clone 9e10) (in other embodiments of the present invention, the amino acid sequence of the light chain of the antigen binding domain targeting TT1 may be extended by no more than 10 amino acids downstream of the above sequence), and the amino acid sequence of the heavy chain is consistent with the amino acid sequence of positions 23 to 143 of the heavy chain of the anti-Myc antibody (e.g., clone 9e10) (in other embodiments of the present invention, the amino acid sequence of the heavy chain of the antigen binding domain targeting TT1 may be extended by no more than 10 amino acids upstream and downstream of the above sequence).

[0124] Preferably, the amino acid sequence of the light chain of the antigen binding domain targeting TT2 is consistent with the amino acid sequence of positions 2 to 121 of the light chain of the anti-HA antibody (e.g., clone 12ca5) (in other embodiments of the present invention, the amino acid sequence of the light chain of the antigen binding domain targeting TT2 may be extended by no more than 10 amino acids downstream of the above sequence), and the amino acid sequence of the heavy chain is consistent with the amino acid sequence of positions 2 to 114 of the heavy chain of the anti-HA antibody (e.g., clone 12ca5) (in other embodiments of the present invention, the amino acid sequence of the heavy chain of the antigen binding domain targeting TT2 may be extended by no more than 10 amino acids downstream of the above sequence).

[0125] Preferably, the amino acid sequence of the light chain of the antigen binding domain targeting TT3 is consistent with the light chain of the anti-Strep tag II antibody (see (for example) patent document EP2871189A1), and the amino acid sequence of the heavy chain is consistent with the heavy chain of the anti-Strep tag II antibody (see (for example) patent document EP2871189A1).

[0126] The amino acid sequence of the anti-Myc antibody (clone 9e10) light chain can be derived from the amino acid sequence with PDB accession number 2ORB_L (https: / / www.ncbi.nlm.nih.gov / protein / 2ORB_L), and the amino acid sequence of the anti-Myc antibody (clone 9e10) heavy chain can be derived from the amino acid sequence with GenBank accession number CAA73271.1 (https: / / www.ncbi.nlm.nih.gov / protein / CAA73271.1?report=genbank&log$=protalign&blast_rank=1&RID=P597FX1S014), and the amino acid sequence of the linker connecting the light chain and the heavy chain is shown in SEQ ID NO: 16. According to a specific embodiment, the first antigen-binding portion has the amino acid sequence shown in SEQ ID NO: 17. The amino acid sequence of the anti-HA antibody (clone 12ca5) light chain can be derived from the amino acid sequence numbered PDB: 5XCS_B (https: / / www.ncbi.nlm.nih.gov / protein / 1258501213), and the amino acid sequence of the anti-HA antibody (clone 12ca5) heavy chain can be derived from the amino acid sequence numbered PDB: 5XCS_A (https: / / www.ncbi.nlm.nih.gov / protein / 5XCS_A), and the amino acid sequence of the linker connecting the light chain and the heavy chain can be as shown in SEQ ID NO: 18. According to a specific embodiment, the first antigen-binding portion has the amino acid sequence shown in SEQ ID NO: 19.

[0127] The amino acid sequences of the light and heavy chains of the anti-Strep Tag II antibody can be derived from the amino acid sequences disclosed in patent document EP2871189A1, and the amino acid sequence of the linker connecting the light and heavy chains can be as shown in SEQ ID NO: 16. According to specific embodiments, the first antigen-binding moiety has the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOs: 20, 21, and 22, and the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOs: 23, 24, and 25. The HCDR1, HCDR2, and HCDR3 sequences shown, as well as the LCDR1, LCDR2, and LCDR3 sequences, are obtained according to the IMGT definition protocol. According to specific embodiments, the first antigen-binding moiety has the light chain variable region sequence shown in SEQ ID NO: 26 and the heavy chain variable region sequence shown in SEQ ID NO: 27. The light chain variable region sequence and the heavy chain variable region sequence are connected by a linker, which, according to specific embodiments, can be connected by the linker shown in SEQ ID NO: 16. According to specific embodiments, the first antigen-binding moiety has the sequence shown in SEQ ID NO: 28. According to specific embodiments, the first antigen-binding portion is humanized by humanizing the light chain variable region set forth in SEQ ID NO:26 and the heavy chain variable region set forth in SEQ ID NO:27, thereby reducing immunogenicity while retaining binding activity to TT3. According to specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO:29 and a light chain variable region set forth in SEQ ID NO:32. According to specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO:29 and a light chain variable region set forth in SEQ ID NO:33. According to specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO:29 and a light chain variable region set forth in SEQ ID NO:34. According to specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO:30 and a light chain variable region set forth in SEQ ID NO:32. According to specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO:30 and a light chain variable region set forth in SEQ ID NO:33. According to a specific embodiment, the first antigen binding portion has a heavy chain variable region as shown in SEQ ID NO: 30 and a light chain variable region as shown in SEQ ID NO: 34. According to a specific embodiment, the first antigen binding portion has a heavy chain variable region as shown in SEQ ID NO: 31 and a light chain variable region as shown in SEQ ID NO: 32.According to a specific embodiment, the first antigen binding portion has a heavy chain variable region as shown in SEQ ID NO: 31 and a light chain variable region as shown in SEQ ID NO: 33. According to a specific embodiment, the first antigen binding portion has a heavy chain variable region as shown in SEQ ID NO: 31 and a light chain variable region as shown in SEQ ID NO: 34.

[0128] The first antigen-binding moiety can be connected to the second antigen-binding moiety through a linker to form a multispecific antibody. Flexible oligopeptides commonly used in the art for connecting amino acid fragments can be used as linkers for the first antigen-binding moiety and the second antigen-binding moiety. According to a specific embodiment, the amino-terminal (also known as the N-terminal) amino acid of the first antigen-binding moiety can be connected to the carboxyl-terminal (also known as the C-terminal) amino acid of the second antigen-binding moiety. According to a specific embodiment, the C-terminal amino acid of the first antigen-binding moiety can be connected to the N-terminal amino acid of the second antigen-binding moiety. When the first antigen-binding moiety and the second antigen-binding moiety are connected, they can be directly connected, can be connected through a linker, and can also be connected with other functional amino acid sequences between the two. The other functional amino acid sequences mentioned can be, for example, Fc sequences, which can mediate ADCC (antibody-dependent cell-mediated cytotoxicity), ADCP (antibody-dependent cell-mediated phagocytosis) or CDC (complement-mediated cytotoxicity), etc. CH1, CH2, CL sequences, etc. can be connected between the first antigen-binding moiety and the second antigen-binding moiety as needed. According to a specific embodiment, the C-terminal amino acid of the first antigen-binding moiety is linked to the Fc sequence and connected to the second antigen-binding moiety via a linker. According to a specific embodiment, the C-terminal amino acid of the second antigen-binding moiety forms an IgG-like antibody structure with the CH1, CL sequences, and Fc region, and the C-terminal amino acid of the Fc region is linked to the N-terminal amino acid of the first antigen-binding moiety via a linker. According to a specific embodiment, the light chain variable region and heavy chain variable region sequences of the first antigen-binding moiety can be directly connected via a linker to form a single-chain antibody (scFv) format, or they can be directly connected.

[0129] In some embodiments, the second antigen binding portion can specifically recognize and bind to the first cytokine or its receptor. The first cytokine or its receptor mentioned can be the full length, fragment or variant of the cytokine. In some embodiments, the first cytokine mentioned includes but is not limited to: interleukin-1 (IL-1α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-17a, IL-1 7b, IL-17c, IL-17d, IL-17f, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, I L-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-37, EPO, CCL1, CCL2, CCL3, CC At least one of L4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, IFNα, IFNβ, IFNγ, TNFα, TGFβ, VEGF, GM-CSF, GCSF, or their related receptors. By binding to cytokines or their receptors, it can play an immunomodulatory role.

[0130] In some embodiments, the second antigen binding portion can specifically recognize and bind to immune cell antigens, especially can specifically recognize and bind to immune cell surface antigens, recruit immune cells to achieve the purpose of tumor killing. Herein, "immune cell" refers to any of the various cells that play a role in the immune system, such as protecting against infection and foreign body invasion. In a specific embodiment, the term can include white blood cells, such as neutrophils, eosinophils, basophils, lymphocytes and monocytes. Immune cells also include immune effector cells. Immune effector cells refer to cells that participate in the immune response, such as promoting immune effector responses. Examples of immune effector cells include but are not limited to T cells, such as CD4+T cells, CD8+T cells, αT cells, βT cells, γT cells and δT cells; B cells; natural killer (NK) cells; natural killer T (NKT) cells; dendritic cells; and mast cells, etc. In some embodiments, the immune cell is a T cell or NK cell. In some embodiments, the immune cell is a cytotoxic T cell (such as CD8+T cell). In some embodiments, the immune cell is a helper T cell, such as CD4+T cell. The immune cell antigens mentioned include but are not limited to CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11, CD11a, CD11b, CD11c, CD11d, CD12w, CD13, CD14, CD15, CD16, CD16a, CD16b, CD17, CD18, CD19, CD20,CD21,CD22,CD23,CD24,CD25,CD26,CD27,CD28,CD29,CD30,CD31,CD32,CD33,CD34,CD35,CD36,CD37,CD38,CD39,CD 40,CD41,CD42,CD42a,CD42b,CD42c,CD42d,CD43,CD44,CD45,CD45RA,CD45RB,CD45RC,CD45RO,CD46,CD47,CD48,CD49a,CD 49b,CD49c,CD49d,CD49e,CD49f,CD50,CD52,CD53,CD54,CD55,CD56,CD57,CD58,CD59,CD60a,CD60b,CD60c,CD61,CD62E, CD62L,CD62P,CD63,CD64,CD65,CD66a,CD66b,CD66c,CD66d,CD66e,CD66f,CD67,CD68,CD69,CD70,CD71,CD72,CD73,CD74,CD75,CD75s,CD77,CD79,CD79a,CD79b,CD80,CD81,CD82,CD83,CD84,CD85,CD85a,CD85b,CD85c,CD85d,CD85e,CD85f,CD85g,CD85h,CD85i,CD85j,CD85k,CD86,CD87,CD88,CD89,CD90,CD91,CD92,CD93,CD94,CD95,CD96,CD97,CD98,CD99,CD100,CD101,CD102,CD103,CD104,CD105,CD106,CD107,CD107a,CD107b,CD108,CD109,CD110,CD11,CD112,CD115,CD116,CD117,CD119,CD120,CD121,CD122,CD123,CD124,CD125,CD126,CD127,CD129,CD130,CD133,CD131,CD132,CD134,CD135,CD137,CD138,CD139,CD141,CD142,CD143,CD144,CD147,CD146,CD148,CD150,CD151,CD152,CD153,CD154,CD155,CD156,CD157,CD158,CD158a,CD158b1,CD158b2,CD158c,CD158d,CD158e,CD158f1,CD158f2,CD158g,CD158h,CD158i,CD158j,CD158k,CD158z,CD159a,CD159c,CD160,CD161,CD162,CD163,CD166,CD165,CD166,CD168,CD169,CD170,CD171,CD172a,CD172b,CD172g,CD173,Cd177,CD178,CD179,CD180,CD181,CD183,CD184,CD185,CD186,CD191,CD192,CD193,CD194,CD195,CD196,CD197,CD198,CD199,CD200,CD203,CD205,CD208,CD209,CD210,CD210b,CD212,CD231a1,CD213a2,CD217,CD218a,CD218b,CD222,CD224,CD225,CD226,CD227,CD229,CD232,CD243,CD244,CD245,CD247,CD252,CD253,CD256,Cd257,CD258,CD261,CD262,CD262,CD263,CD264,CD265,CD268,CD269,CD271,CD272,CD273,CD274,CD275,CD276 ,CD277,CD278,CD279,CD280,CD281,CD282,CD283,CD284,CD286,CD288,CD289,CD290,CD292,CDw293,CD294,CD296,CD29 7. CD300a, CD300b, CD300c, CD300d, CD300e, CD303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD309, CD312, C D314, CD315, CD316, CD317, CD318, CD319, CD320, CD328, CD329, CD335, CD336, CD337, CD352, CD353, CD357, CD361, CD362, etc. ,

[0131] According to a specific embodiment, the second antigen binding portion can specifically recognize and bind to the CD3 antigen. CD3 (T cell surface glycoprotein CD3, signal transduction co-receptor of the T cell receptor) is a differentiation antigen expressed on the surface of all T lymphocytes. It mainly mediates the transduction of T cell activation signals and plays an important role in the anti-infection immunity of the body's immune system. The CD3 mentioned herein includes any natural CD3 of any vertebrate (including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats)), unless otherwise specified. The term "CD3" covers "full-length" unprocessed CD3 and any form of CD3 or any fragment thereof produced by intracellular processing. The term also includes naturally occurring variants of CD3, such as splice variants or allelic variants. In some embodiments, CD3 refers to the full length or fragments thereof from humans and cynomolgus monkeys. In some embodiments, CD3 refers to the full length or fragments thereof from mice or rats.

[0132] In some embodiments, the second antigen-binding portion is capable of specifically recognizing and binding to the CD3 antigen. The anti-CD3 antibody can be any known antibody targeting CD3, or an antibody targeting CD3 currently under development or to be developed in the future. Examples include antibody SP34 (see, e.g., Pessano, S. et al., EMBO J4 (1985) 337-344), antibody OKT3 (see, e.g., Kung, P. et al., Science 206 (1979) 347-349; Salmeron, A. et al., J Immunol 147 (1991) 3047-3052), and antibody UCHT1 (see, e.g., Callard, RE et al., Clin Exp Immunol 43 (1981) 497-505). A second antigen-binding portion that specifically recognizes and targets the CD3 antigen is obtained by sequence optimization and humanization of murine SP34. Moreover, after humanization, it can show high affinity to human and crab-eating macaque CD3 antigens.

[0133] According to a specific embodiment, the second antigen binding portion has the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 35, 36 and 37, and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 38, 39 and 40. The HCDR1, HCDR2 and HCDR3 sequences and LCDR1, LCDR2 and LCDR3 sequences shown are obtained according to the IMGT definition scheme.

[0134] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:42.

[0135] According to specific embodiments, the sequence of the second antigen-binding moiety has been humanized. The humanized second antigen-binding moiety exhibits high affinity for human or cynomolgus monkey CD3 antigens. When combined with the first antigen-binding moiety to form a bispecific antibody, its affinity for the CD3 antigen is reduced compared to that of a humanized CD3 monoclonal antibody. Despite this, by modifying the first antigen-binding moiety, the multispecific antibody maintains a high affinity for the marker polypeptide, effectively achieving tumor targeting and tumor killing. The design and screening of the present invention enables the multispecific antibody to exhibit particularly suitable affinity as a whole. According to specific embodiments, the second antigen-binding moiety has a heavy chain variable region sequence as shown in SEQ ID NO: 43, 44, or 45. According to specific embodiments, the second antigen-binding moiety has a light chain variable region sequence as shown in SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, or 53. The humanized heavy chain variable region or light chain variable region can form a sequence that targets the CD3 antigen with the light chain variable region or heavy chain variable region before humanization. For example, the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO:41 and the light chain variable region sequence set forth in SEQ ID NO:46. For another example, the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO:41 and the light chain variable region sequence set forth in SEQ ID NO:47. For another example, the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO:41 and the light chain variable region sequence set forth in SEQ ID NO:48. For another example, the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO:41 and the light chain variable region sequence set forth in SEQ ID NO:49. For another example, the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO:41 and the light chain variable region sequence set forth in SEQ ID NO:50. For another example, the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO:41 and the light chain variable region sequence set forth in SEQ ID NO:51. For another example, the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO:41 and the light chain variable region sequence set forth in SEQ ID NO:52. For another example, the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO: 41 and the light chain variable region sequence set forth in SEQ ID NO: 53. For another example, the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO: 43 and the light chain variable region sequence set forth in SEQ ID NO: 42. For another example, the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO: 44 and the light chain variable region sequence set forth in SEQ ID NO: 42.As another example, the second antigen-binding portion has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:42.

[0136] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:46.

[0137] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:47.

[0138] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:48.

[0139] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:49.

[0140] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:50.

[0141] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:51.

[0142] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:52.

[0143] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:53.

[0144] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:46.

[0145] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:47.

[0146] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:48.

[0147] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:49.

[0148] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:50.

[0149] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:51.

[0150] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:52.

[0151] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:53.

[0152] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:46.

[0153] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:47.

[0154] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:48.

[0155] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:49.

[0156] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:50.

[0157] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:51.

[0158] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:52.

[0159] According to a specific embodiment, the second antigen binding portion has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:53.

[0160] According to a specific embodiment, the second antigen binding portion and the first antigen binding portion are connected by a connector. According to a specific embodiment, the connector is (G4S)3. The heavy chain variable region of the second antigen binding portion is connected to CH1, CH1 is connected to the Fc region, and the light chain variable region is connected to CL1. The N-terminal amino acid of the first antigen binding portion is connected to the C-terminal amino acid of the Fc region through a connector. The light chain variable region sequence and the heavy chain variable region sequence of the first antigen binding portion are directly connected or connected through a connector. The Fc region mentioned can be a human Fc region sequence. The Fc region sequence can be derived from, but is not limited to, IgA, IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM and variants thereof. The variants mentioned are mutations occurring in the Fc region, including but not limited to K214R, L234F, L235E, P331S, D356E, L358M or any combination thereof, such as L234F / 235E / P331S, K214R / L234F / L235E / P331S, etc.

[0161] According to a specific embodiment, a bispecific antibody is provided, which includes a first antigen-binding portion and a second antigen-binding portion, wherein the heavy chain variable region and the heavy chain constant domain (CH1 and Fc region) of the second antigen-binding portion are directly connected, and the light chain variable region and CL of the second antigen-binding portion are directly connected to form an IgG-like structure, and the C-terminal amino acid of the Fc region and the N-terminal amino acid sequence of the light chain variable region of the first antigen-binding portion are connected by a linker (the linker used is shown in SEQ ID NO: 18), wherein the C-terminal amino acid of the light chain variable region of the first antigen-binding portion is connected to the N-terminal amino acid of the heavy chain variable region of the first antigen-binding portion by a linker (the linker used is shown in SEQ ID NO: 16), as shown in Figure 2a, and the bispecific antibody formed is numbered beginning with "BK-A". According to one embodiment, the provided bispecific antibody is BK-A-101, in which the sequence formed by the light chain variable region and the heavy chain variable region of the first antigen-binding portion is shown in SEQ ID NO:28, the second antigen-binding portion has the heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:42, the heavy chain variable region sequence of the second antigen-binding portion is connected to the heavy chain constant domain CH (sequence shown in SEQ ID NO:57), and the light chain variable region sequence of the second antigen-binding portion is connected to the light chain constant domain CL (sequence shown in SEQ ID NO:55) to form an IgG-type structure; the N-terminal amino acid of the first antigen-binding portion is connected to the C-terminal amino acid of the Fc region.

[0162] According to a specific embodiment, the provided bispecific antibody is BK-A-101-hu1, which is a bispecific antibody formed by humanizing the second antigen-binding portion of the BK-A-101 antibody. The second antigen-binding portion of BK-A-101-hu1 has the heavy chain variable region set forth in SEQ ID NO: 44 and the light chain variable region set forth in SEQ ID NO: 51. The remaining sequences are identical to those of BK-A-101.

[0163] According to a specific embodiment, the provided bispecific antibody is BK-A-101-hu2, which is a bispecific antibody formed by humanizing the second antigen-binding portion of the BK-A-101 antibody. The second antigen-binding portion of BK-A-101-hu2 comprises the heavy chain variable region set forth in SEQ ID NO: 44 and the light chain variable region set forth in SEQ ID NO: 52. The remaining sequences are identical to those of BK-A-101.

[0164] According to a specific embodiment, the provided bispecific antibody is BK-A-101-hu3, which is a bispecific antibody formed by humanizing the second antigen-binding portion of the BK-A-101 antibody. The second antigen-binding portion of BK-A-101-hu3 comprises the heavy chain variable region set forth in SEQ ID NO: 44 and the light chain variable region set forth in SEQ ID NO: 53. The remaining sequences are identical to those of BK-A-101.

[0165] According to a specific embodiment, the provided bispecific antibody is BK-A-101-hu4, which is a bispecific antibody formed by humanizing the second antigen-binding portion of the BK-A-101 antibody. The second antigen-binding portion of BK-A-101-hu4 comprises the heavy chain variable region set forth in SEQ ID NO: 45 and the light chain variable region set forth in SEQ ID NO: 51. The remaining sequences are identical to those of BK-A-101.

[0166] According to a specific embodiment, the provided bispecific antibody is BK-A-101-hu5, which is a bispecific antibody formed by humanizing the second antigen-binding portion of the BK-A-101 antibody. The second antigen-binding portion of BK-A-101-hu5 comprises the heavy chain variable region set forth in SEQ ID NO: 45 and the light chain variable region set forth in SEQ ID NO: 52. The remaining sequences are identical to those of BK-A-101.

[0167] According to a specific embodiment, the provided bispecific antibody is BK-A-101-hu6, which is a bispecific antibody formed by humanizing the second antigen-binding portion of the BK-A-101 antibody. The second antigen-binding portion of BK-A-101-hu6 comprises the heavy chain variable region set forth in SEQ ID NO: 45 and the light chain variable region set forth in SEQ ID NO: 53. The remaining sequences are identical to those of BK-A-101.

[0168] According to a specific embodiment, the provided bispecific antibody comprises a first antigen-binding moiety and a second antigen-binding moiety, wherein the heavy chain variable region and the heavy chain constant domain of the first antigen-binding moiety are directly linked, and the light chain variable region of the first antigen-binding moiety is directly linked to the light chain constant domain to form an IgG-like structure, wherein the carboxyl-terminal amino acid of the Fc region of the IgG-like structure is linked to the N-terminal amino acid of the light chain variable region of the second antigen-binding moiety via a linker, and the N-terminal amino acid of the heavy chain variable region of the second antigen-binding moiety is linked to the C-terminal amino acid of the light chain variable region of the second antigen-binding moiety via a linker, as shown in FIG2 b. The bispecific antibody formed is numbered starting with "BK-D", wherein the linkers used are all (G4S)3. According to a specific embodiment, the provided bispecific antibody is BK-D-01, which comprises the first antigen-binding moiety shown in SEQ ID NO: 31 and SEQ ID NO: 32, and the second antigen-binding moiety shown in SEQ ID NO: 43 and SEQ ID NO: 46. According to a specific embodiment, the bispecific antibody provided is BK-D-02, which comprises a first antigen-binding portion set forth in SEQ ID NO: 31 and SEQ ID NO: 34, and a second antigen-binding portion set forth in SEQ ID NO: 45 and SEQ ID NO: 46. According to a specific embodiment, the bispecific antibody provided is BK-D-03, which comprises a first antigen-binding portion set forth in SEQ ID NO: 29 and SEQ ID NO: 34, and a second antigen-binding portion set forth in SEQ ID NO: 43 and SEQ ID NO: 46. According to a specific embodiment, the bispecific antibody provided is BK-D-04, which comprises a first antigen-binding portion set forth in SEQ ID NO: 29 and SEQ ID NO: 32, and a second antigen-binding portion set forth in SEQ ID NO: 43 and SEQ ID NO: 49. According to a specific embodiment, the bispecific antibody provided is BK-D-05, which comprises a first antigen-binding portion set forth in SEQ ID NO: 29 and SEQ ID NO: 32, and a second antigen-binding portion set forth in SEQ ID NO: 44 and SEQ ID NO: 48. According to one embodiment, the bispecific antibody provided is BK-D-06, which includes a first antigen-binding portion shown in SEQ ID NO: 29 and SEQ ID NO: 32, and a second antigen-binding portion shown in SEQ ID NO: 44 and SEQ ID NO: 50.According to a specific embodiment, the bispecific antibody provided is BK-D-07, which comprises a first antigen-binding portion set forth in SEQ ID NO: 31 and SEQ ID NO: 32, and a second antigen-binding portion set forth in SEQ ID NO: 45 and SEQ ID NO: 46. According to a specific embodiment, the bispecific antibody provided is BK-D-08, which comprises a first antigen-binding portion set forth in SEQ ID NO: 29 and SEQ ID NO: 32, and a second antigen-binding portion set forth in SEQ ID NO: 45 and SEQ ID NO: 46. According to a specific embodiment, the bispecific antibody provided is BK-D-09, which comprises a first antigen-binding portion set forth in SEQ ID NO: 29 and SEQ ID NO: 32, and a second antigen-binding portion set forth in SEQ ID NO: 44 and SEQ ID NO: 47. According to a specific embodiment, the bispecific antibody provided is BK-D-10, which comprises a first antigen-binding portion set forth in SEQ ID NO: 31 and SEQ ID NO: 34, and a second antigen-binding portion set forth in SEQ ID NO: 43 and SEQ ID NO: 46. According to one embodiment, the bispecific antibody provided is BK-D-11, which comprises a first antigen-binding portion as set forth in SEQ ID NO:29 and SEQ ID NO:32, and a second antigen-binding portion as set forth in SEQ ID NO:43 and SEQ ID NO:46. According to one embodiment, the bispecific antibody provided is BK-D-12, which comprises a first antigen-binding portion as set forth in SEQ ID NO:29 and SEQ ID NO:34, and a second antigen-binding portion as set forth in SEQ ID NO:45 and SEQ ID NO:46. According to one embodiment, the bispecific antibody provided is BK-D-13, which comprises a first antigen-binding portion as set forth in SEQ ID NO:29 and SEQ ID NO:32, and a second antigen-binding portion as set forth in SEQ ID NO:45 and SEQ ID NO:50. According to one embodiment, the heavy chain constant domain sequence of the bispecific antibody with the BK-D structure is set forth in SEQ ID NO:54, and the light chain constant domain sequence is set forth in SEQ ID NO:55. The Fc region sequence is set forth in SEQ ID NO:56. The sequence of the heavy chain constant domain in the bispecific antibody with the BK-D structure (SEQ ID NO: 54) is compared to the sequence shown in SEQ ID NO: 57, but the Fc region is mutated.According to specific embodiments, the affinity activity of the provided bispecific antibody for the CD3 antigen is reduced compared to that of a humanized monoclonal antibody without Fc mutation, and the EC50 value of the bispecific antibody for the CD3 antigen is 0.5-200 nM, 1-200 nM, 5-200 nM, 10-200 nM, 15-200 nM, 0.5-150 nM, 1-150 nM, 5-150 nM, 10-150 nM, 15-150 nM, 0.5-120 nM, 1-120 nM, 5-120 nM, 10-120 nM, 15-120 nM, 10-100 nM, 15-100 nM, 10-80 nM, 15-80 nM, 10-60 nM, 15-60 nM, 10-50 nM, or 15-50 nM.

[0169] According to a specific embodiment, a bispecific antibody is provided, which includes a first antigen-binding portion and a second antigen-binding portion, wherein the C-terminal amino acid of the first antigen-binding portion is directly linked to the Fc region sequence (CH2 and CH3), the C-terminal amino acid of the Fc region is linked to the N-terminal amino acid of the light chain variable region of the second antigen-binding portion via a linker, and the C-terminal amino acid of the light chain variable region of the second antigen-binding portion is linked to the N-terminal amino acid of the heavy chain variable region of the second antigen-binding portion via a linker (the linker is shown in SEQ ID NO: 18, depending on the specific embodiment), as shown in Figure 3. According to a specific embodiment, the bispecific antibody provided is BK-B-110, wherein the sequence formed by the light chain variable region and the heavy chain variable region of the first antigen-binding portion is shown in SEQ ID NO: 28, the second antigen-binding portion has the heavy chain variable region sequence shown in SEQ ID NO: 41 and the light chain variable region sequence shown in SEQ ID NO: 42, and the Fc region sequence is shown in SEQ ID NO: 56. According to a specific embodiment, the bispecific antibody provided is BK-B-110-hu1, which is a bispecific antibody formed by humanizing the second antigen-binding portion of the BK-B-110 antibody, wherein the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO: 44 and the light chain variable region sequence set forth in SEQ ID NO: 51. According to a specific embodiment, the bispecific antibody provided is BK-B-110-hu2, which is a bispecific antibody formed by humanizing the second antigen-binding portion of the BK-B-110 antibody, wherein the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO: 44 and the light chain variable region sequence set forth in SEQ ID NO: 52. According to a specific embodiment, the bispecific antibody provided is BK-B-110-hu3, which is a bispecific antibody formed by humanizing the second antigen-binding portion of the BK-B-110 antibody, wherein the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO: 44 and the light chain variable region sequence set forth in SEQ ID NO: 53. According to a specific embodiment, the provided bispecific antibody is BK-B-110-hu4, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of the BK-B-110 antibody, wherein the second antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO: 45 and the light chain variable region set forth in SEQ ID NO: 51. According to a specific embodiment, the provided bispecific antibody is BK-B-110-hu5, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of the BK-B-110 antibody, wherein the second antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO: 45 and the light chain variable region set forth in SEQ ID NO: 52.According to a specific embodiment, the bispecific antibody provided is BK-B-110-hu6, which is a bispecific antibody in which the second antigen-binding portion of the BK-B-110 antibody is humanized, and the second antigen-binding portion has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:53.

[0170] According to a specific embodiment, a bispecific antibody is provided, which includes a first antigen-binding portion and a second antigen-binding portion, the C-terminal amino acid of the first antigen-binding portion is directly linked to the Fc region sequence, the C-terminal amino acid of the Fc region is connected by a linker to the N-terminal amino acid of the heavy chain variable region of the second antigen-binding portion by a linker (according to a specific embodiment, the linker used is as shown in SEQ ID NO: 18), and the C-terminal amino acid of the heavy chain variable region of the second antigen-binding portion is connected to the N-terminal amino acid of the light chain variable region of the second antigen-binding portion by a linker (according to a specific embodiment, the linker used is as shown in SEQ ID NO: 18), as shown in Figure 4. According to a specific embodiment, the bispecific antibody provided is BK-C-120, whose first antigen-binding portion has the sequence shown in SEQ ID NO: 28, the second antigen-binding portion has the heavy chain variable region sequence shown in SEQ ID NO: 41 and the light chain variable region sequence shown in SEQ ID NO: 42, and the Fc region sequence is as shown in SEQ ID NO: 56. According to a specific embodiment, the bispecific antibody provided is BK-C-120-hu1, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of the antibody BK-C-120, wherein the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO: 44 and the light chain variable region sequence set forth in SEQ ID NO: 51. According to a specific embodiment, the bispecific antibody provided is BK-C-120-hu2, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of the antibody BK-C-120, wherein the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO: 44 and the light chain variable region sequence set forth in SEQ ID NO: 52. According to a specific embodiment, the bispecific antibody provided is BK-C-120-hu3, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of the antibody BK-C-120, wherein the second antigen-binding portion has the heavy chain variable region sequence set forth in SEQ ID NO: 44 and the light chain variable region sequence set forth in SEQ ID NO: 53. According to a specific embodiment, the provided bispecific antibody is BK-C-120-hu4, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of the antibody BK-C-120, wherein the second antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO: 45 and the light chain variable region set forth in SEQ ID NO: 51. According to a specific embodiment, the provided bispecific antibody is BK-C-120-hu5, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of the antibody BK-C-120, wherein the second antigen-binding portion comprises the heavy chain variable region set forth in SEQ ID NO: 45 and the light chain variable region set forth in SEQ ID NO: 52.According to a specific embodiment, the bispecific antibody provided is BK-C-120-hu6, which is a bispecific antibody in which the second antigen-binding portion of the antibody BK-C-120 is humanized, and the second antigen-binding portion has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:53.

[0171] The multispecific antibody may further optionally include a third antigen binding portion as needed, which specifically recognizes and binds to a tumor antigen or an immune checkpoint or a second cytokine or its receptor. The tumor antigens mentioned include tumor-associated antigens (TAAs). Tumor-associated antigens are antigenic substances produced by tumor cells that can trigger an immune response in the host. Tumor-associated antigens may be derived from cancer, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, ovarian cancer, breast cancer, and the like. TAAs may be patient-specific. For example, they include but are not limited to p53, Ras, β-catenin, CDK4, α-actinin-4, CD20, PSMA, Her2, EGFR, and the like. According to a specific embodiment, the tumor antigen includes but is not limited to at least one of AFP, BCMA, β-catetin, CD5, CD19, CD20, CD22, CD30, CD38, CD70, CD123, CD133, CD171, CD138, CDK-4, CEA, CA-125, Caspase 8, c-Met, EGFR, Ep-CAM, EphA2, ETA, FAP, GPC3, GP100, GD2, Her2, HPV16, Igk chain, Ley, Mesothlin, MUC-1, MUC16, MAGE, MART1, NKG2D ligand, CLL1, NY-ESO-1, P53, RAS, OSA, PAP, PSCA, ROR1, ROR2, PSMA, SSTR2, Trop-2, TYR, VEGFR, and WT1. The immune checkpoints mentioned include but are not limited to PD-1, PD-L1, CTLA-4, LAG-3, BTLA, TIM-3, TIGIT, VISTA, TIPE2, Galectin-9, NKG2A, KIRs, B7-H3, B7-H7, 2B4, DNAM-1, PTA1, TLISA1, Vstm3, VSIG9, Siglecs family, GITR, OX40, 4-1BB, LAYN, LIRB1-5 or their related receptors.The third antigen binding portion can also specifically recognize and bind to a second cytokine or its receptor, including but not limited to IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-17a, IL-17b, IL-17c, IL-17d, IL-17f, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-37, EPO, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, IFNα, IFNβ, IFNγ, TNFα, TGFβ, VEGF, GM-CSF, GCSF or their related receptors. It should be noted that the first cytokine or its receptor and the second cytokine or its receptor may be the same or different.

[0172] The multispecific antibodies provided can be encoded by polynucleotides. The term "nucleic acid" or "polynucleotide" refers to DNA or RNA and polymers thereof in single-stranded or double-stranded form. Depending on the embodiment, the polynucleotide encodes the multispecific antibody and can be used to express the multispecific antibody in vivo or in vitro. The polynucleotides mentioned are isolatable and include, but are not limited to, DNA, RNA, or cDNA. Conventional methods in the art can be used to obtain isolated polynucleotide sequences for encoding the multispecific antibody.

[0173] In order to prepare the antibodies mentioned above, the polynucleotide sequence encoding the antibody can be inserted into a replicable expression vector and expressed in a host cell or a cell-free expression system. To this end, a construct is also provided, comprising the polynucleotide described above. "Construct" refers to any recombinant polynucleotide molecule (such as a plasmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of integrating with the host genome or autonomously replicating, and completing the expression of the antibody in the host cell. Various methods commonly used in the art can be used to obtain constructs, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. For example, a polynucleotide can be inserted into the multiple cloning site of an expression vector to form a construct. The construct can contain various operating factors such as promoters, terminators, marker genes, etc. as needed, and these operating factors can be operably connected to the polynucleotide. Promoters are generally used to provide a signal to start transcription. Promoters can be selected from lactose promoter (Lac), Trp promoter, Tac promoter, phage PL and PR promoters as needed; terminators provide a signal for transcription termination during transcription, and marker genes on the construct are often used for screening. Of course, enhancers can also be added as needed to enhance protein expression. The expression vector is not particularly limited and can be any commercially available expression vector or an artificially modified expression vector as needed, such as a plasmid, bacteriophage, or virus. The virus can be a plant cell virus or a mammalian cell virus. The construct can express antibodies or proteins in vitro or be transferred into cells for expression of antibodies or proteins.

[0174] For the production or expression of multispecific antibodies for use as components in therapeutic agents. A host cell is also provided, containing the above-mentioned polynucleotide or construct. Any cell suitable for antibody or protein expression of the polynucleotide or construct can be used as a host cell. The host cell can be a prokaryotic cell, such as a bacterial cell; or a eukaryotic cell, such as a yeast cell, a mammalian cell, etc. Commonly used host cells can be yeast cells, Chinese hamster ovary (CHO) cells (such as CHO-K1, CHO DG44, CHO-S, CHO-DXB11, CHO-M, etc.), HEK-293 cells, COS cells, Drosophila S2 or Sf9 insect cells, NS0 cells, Hela cells, 3T3 cells, BHK cells, etc. Host cells containing polynucleotides or constructs can be obtained using methods commonly used in the art, such as microinjection, electroporation, chemical transfection, virus-mediated transformation, etc.

[0175] The present invention also provides a kit of a synergistic combination drug for treating tumors and / or cancers, comprising:

[0176] a first container containing a first composition of a therapeutic agent according to the present invention;

[0177] A second container, the second container containing the second composition of the therapeutic agent according to the present invention, or the multispecific antibody mentioned in the present invention, wherein the first container and the second container are independent; and

[0178] Instructions stating the timing and method of administration.

[0179] The first and second compositions and multispecific antibodies mentioned herein can be prepared into different dosage forms as needed to meet administration requirements, such as injections, powders, liquid preparations, etc. When preparing different dosage forms, the first active ingredient or the second active ingredient can be compounded with different pharmaceutically acceptable carriers.

[0180] The present invention also provides the use of the isolated nucleic acid having the coding sequence of the marker polypeptide according to the present invention in the preparation of a drug for treating or preventing tumors and / or cancers.

[0181] The present invention also provides use of the medicine kit in preparing medicines for treating or preventing tumors and / or cancers.

[0182] The tumors and / or cancers include: breast cancer, head and neck cancer, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, Ureter cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, fibrosarcoma, Paget's disease, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multiple myeloma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymic cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, peritoneal cancer, pleural cancer, and blood cancer.

[0183] The present invention also provides a method for treating tumors and / or cancer, comprising: administering a first composition of the therapeutic agent according to the present invention to a subject; and administering a second composition of the therapeutic agent according to the present invention or administering the multispecific antibody mentioned in the present invention to the subject. "Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. According to a specific embodiment, the subject is a tumor and / or cancer patient. The "treatment" mentioned means being able to result in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease asymptomatic periods, or preventing a reduction in pain caused by the disease. After treatment, the inhibition rate of tumor cells or cancer cells in the subject's body reaches more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or even more than 90% or more than 95% compared with untreated subjects.

[0184] The first and second compositions or multispecific antibodies in the therapeutic agent can be administered simultaneously (e.g., as a mixture), separately but simultaneously (e.g., by intratumoral and intravenous administration, respectively), or sequentially (e.g., the first composition is administered first, followed by the second composition or multispecific antibody).

[0185] Preferably, the method comprises the following steps performed in sequence:

[0186] 1) first administering the first composition to the subject; and

[0187] 2) After administering the first composition, administering a second composition of the therapeutic agent or a multispecific antibody to the subject.

[0188] Preferably, the second composition or multispecific antibody among the therapeutic agents is administered to the subject 1-60 days after the first administration of the first composition.

[0189] Taking the administration of the first composition and the second composition as an example, "administering the second composition of the therapeutic agent to the subject patient on day 1-60 after the first administration of the first composition" means that the time interval between the first administration of the second composition and the first administration of the first composition is 1-60 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13...60 days), or the time interval between the first administration of the second composition and the most recent administration of the first composition is 1-60 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13...60 days). Preferably, the time interval between the first administration of the second composition and the most recent administration of the first composition is 1-30 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13...30 days). More preferably, the time interval between the first administration of the second composition and the most recent previous administration of the first composition is 5-20 days (eg, 5, 6, 7, 8, 9, 10 ... 20 days).

[0190] It should be noted that after the first composition is administered to the subject, the second composition may be administered to the subject more than once, for example, the second composition may be administered twice, three times, four times, or multiple times. The number of administrations of the second composition may be adjusted according to the subject's condition. Similarly, before the second composition is administered to the subject, the first composition may be administered more than once, for example, the first composition may be administered twice, three times, four times, or multiple times. The number of administrations of the first composition may be adjusted according to the subject's condition. Similarly, the first composition may be administered to the subject multiple times, and the second composition may be administered multiple times. The number of administrations of both the first composition and the second composition may be adjusted according to the subject's condition.

[0191] In a preferred embodiment of the present invention, the dosage of the nucleic acid is 0.01-10 mg / day, administered 1-3 times a day, and continuously administered for 1-7 days. The nucleic acid can encode a marker polypeptide in the subject.

[0192] In a preferred embodiment of the present invention, the administration dosage of the recombinant oncolytic poxvirus is: the total dose range for each course of treatment is about 5×10 7 -5×10 12 Virus particles (can be administered singly or multiple times), or any value within the above ranges, or the administration dose of the recombinant oncolytic poxvirus is: the total dose range for each course of treatment is about 5×10 7 -5×10 12PFU (can be administered single or multiple times), or any value within the above range. The exact dosage depends on the judgment of the practitioner and each person's unique situation. In the case of multiple administrations, 1-3 times a day are administered for 1-7 consecutive days.

[0193] In a preferred embodiment of the present invention, the dosage of the multispecific antibody is 0.1-50 mg / Kg body weight, for example, 0.5-50 mg / Kg body weight, 1-50 mg / Kg body weight, 2-49 mg / Kg body weight, 3-48 mg / Kg body weight, 4-47 mg / Kg body weight, 5-46 mg / Kg body weight, 6-45 mg / Kg body weight, 7-44 mg / Kg body weight, 8-43 mg / Kg body weight, 9-42 mg / Kg body weight, 10-41 mg / Kg body weight, 11-40 mg / Kg body weight, 12-39 mg / Kg body weight, g / Kg body weight, 13-38mg / Kg body weight, 14-37mg / Kg body weight, 15-36mg / Kg body weight, 16-35mg / Kg body weight, 17-34mg / Kg body weight, 18-33mg / Kg body weight, 19-32mg / Kg body weight, 20-31mg / Kg, 21-30mg / Kg body weight, 22-29mg / Kg body weight, 23-28mg / Kg body weight, 24-27mg / Kg body weight, 25-26mg / Kg body weight, 10-20mg / Kg body weight, 15-25mg / Kg body weight.

[0194] In certain embodiments, the method for treating tumors and / or cancers further comprises administering to the patient other drugs for treating tumors and / or cancers, and / or drugs for regulating the patient's immune system, to enhance the effect of tumor treatment. The other drugs for treating tumors and / or cancers include, but are not limited to, chemotherapy drugs, such as cyclophosphamide and fludarabine; radiotherapy drugs; immunosuppressants, such as cyclosporine, thioguanine, methotrexate, mycophenolate, and FK50; and antibodies, such as antibodies against CD3, IL-2, IL-6, IL-17, and TNFa.

[0195] The DNA can be formulated for intratumoral or intravenous administration; the mRNA can be formulated for intratumoral or intravenous administration. For example, it can be directly injected into the tumor in the form of a plasmid, packaged in liposomes and then injected intratumorally, or attached to nanoparticles (such as polymers such as polylysine, polyamino acids, polyethyleneimine, and chitosan) and injected intratumorally. Alternatively, electroporation can be used after intratumoral injection to enhance transfection efficiency.

[0196] The recombinant virus can be formulated into different modes of administration, including but not limited to administration by intratumoral injection, intravenous administration, intracranial injection, or intraperitoneal administration, etc. Any mode of administration that can inject the recombinant virus into the subject to exert a therapeutic effect is acceptable. The mode of administration can be adjusted according to the needs of the subject, or a combination of multiple modes of administration can be used. The multispecific antibody can be administered in a variety of ways, such as intratumoral injection, intravenous administration, intraperitoneal injection, etc. The mode of administration of the multispecific antibody can be adjusted according to the needs of the subject, or a combination of multiple modes of administration can be used.

[0197] The present invention also provides a new type of immunotherapy, comprising: administering a therapeutically effective amount of an oncolytic virus to a subject, the genome of the oncolytic virus containing a coding sequence for a marker polypeptide, the marker polypeptide having an operably linked extracellular antigenic determinant, a spacer portion, and a transmembrane portion, the amino acid sequence of the extracellular antigenic determinant containing the amino acid sequence of one or more antigenic epitope polypeptides, and in a natural state, the amino acid sequence of the subject's cell membrane protein or secretory protein does not contain the amino acid sequence of the antigenic epitope polypeptide; and administering a therapeutically effective amount of a multispecific antibody to the subject, the multispecific antibody comprising at least a first antigen binding portion and a second antigen binding portion, the first antigen binding portion being capable of specifically recognizing and binding to the extracellular antigenic determinant of the marker polypeptide, the second antigen binding portion being capable of specifically recognizing and binding to an immune cell antigen or a first cytokine or its receptor; the multispecific antibody may further optionally include a third antigen binding portion, the third antigen binding portion specifically recognizing and binding to a tumor antigen or an immune checkpoint or a second cytokine or its receptor. According to a specific embodiment, the second antigen binding portion is capable of specifically recognizing and binding to an immune cell surface antigen.

[0198] According to a specific embodiment, the method comprises the following steps performed in sequence:

[0199] 1) administering a therapeutically effective amount of an oncolytic virus to the subject; and

[0200] 2) After administering the oncolytic virus, administering a therapeutically effective amount of the multispecific antibody to the subject.

[0201] The present invention also provides an anti-CD3 antibody that, through sequence optimization and humanization of murine SP34, can specifically target the CD3 antigen and exhibit higher affinity for human and cynomolgus monkey CD3 antigens. According to specific embodiments, the anti-CD3 antibody has a heavy chain variable region sequence as shown in SEQ ID NO: 43, 44, or 45. According to specific embodiments, the anti-CD3 antibody has a light chain variable region sequence as shown in SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, or 53. According to specific embodiments, the anti-CD3 antibody has a heavy chain variable region sequence as shown in SEQ ID NO: 44 and a light chain variable region sequence as shown in SEQ ID NO: 51. According to specific embodiments, the anti-CD3 antibody has a heavy chain variable region sequence as shown in SEQ ID NO: 44 and a light chain variable region sequence as shown in SEQ ID NO: 52. According to specific embodiments, the anti-CD3 antibody has a heavy chain variable region sequence as shown in SEQ ID NO: 44 and a light chain variable region sequence as shown in SEQ ID NO: 53. According to a specific embodiment, the anti-CD3 antibody has a heavy chain variable region sequence as set forth in SEQ ID NO:45 and a light chain variable region sequence as set forth in SEQ ID NO:51. According to a specific embodiment, the anti-CD3 antibody has a heavy chain variable region sequence as set forth in SEQ ID NO:45 and a light chain variable region sequence as set forth in SEQ ID NO:52. According to a specific embodiment, the anti-CD3 antibody has a heavy chain variable region sequence as set forth in SEQ ID NO:45 and a light chain variable region sequence as set forth in SEQ ID NO:53. The heavy chain constant domain sequence of the antibody is set forth in SEQ ID NO:57, and the light chain constant domain sequence is set forth in SEQ ID NO:55. According to a specific embodiment, the anti-CD3 antibodies provided bind to human CD3 or monkey (e.g., cynomolgus) CD3 with an EC50 of 10 nM or less, for example, the affinity EC50 value for CD3 is less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.8 nM, less than 0.5 nM, less than 0.3 nM, or less than 0.1 nM, less than 0.05 nM.

[0202] The technical solutions of the present invention are described in detail below with reference to examples. It should be noted that these examples are only intended to facilitate understanding by those skilled in the art and should not be construed as limiting the scope of protection of the present invention. Examples of the present invention are described in detail below. The examples described are illustrative and intended to explain the present invention, and should not be construed as limiting the present invention.

[0203] example

[0204] The cell line information used in the example is as follows:

[0205] Human ovarian cancer cell line SKOV3, human colorectal cancer cell line HCT116, and human pancreatic cancer cell line PANC-1 were purchased from ATCC.

[0206] Human hepatocellular carcinoma cell line SK-HEP-1, human glioma cell line U251, and human melanoma cell line A375 were purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai.

[0207] MC38 cell line was purchased from Nanjing Kebo Biotechnology Co., Ltd.

[0208] Human embryonic kidney 293T cells were obtained from Shanghai GeneCare Gene Medical Technology Co., Ltd.

[0209] Human peripheral blood mononuclear cells (PBMC) were purchased from Shanghai Maishun.

[0210] Jurkat cells were purchased from Ruizhi Chemical.

[0211] SKOV3 and HCT116 were cultured with McCoy 5A (commercially available from Gibco) + 10% FBS (commercially available from PAN), SK-HEP-1 and U251 were cultured with MEM (commercially available from Gibco) + 10% FBS, and PANC-1, A375, and 293T were cultured with DMEM (commercially available from Gibco) + 10% FBS.

[0212] The flow cytometry buffer was 1×DPBS+2% FBS. 1×DPBS was purchased from White Shark Biotechnology Co., Ltd., and FBS was purchased from PAN.

[0213] Packaging of LV-TT3-GFP lentivirus

[0214] 293T cells were digested into single cells with 0.25% trypsin (purchased from Beyotime) and then plated at 2×10 6 / ml was resuspended in packing medium. The formula of packing medium is Opti-MEM (commercially available from Gibco) + 5% FBS (commercially available from Gibco) + Sodium pyruvate (commercially available from Gibco, final concentration is 200mM). 1mL / well was inoculated into 6-well plates. According to the instructions of Lipo3000, a lentiviral plasmid mixture was prepared: (1) 250μL Opti-MEM + 7μL Lipo3000, (2) 250μL Opti-MEM + 6μL p3000 + lentiviral 4 plasmid (molar mass ratio = 1:1:1:1). Then (1) and (2) were mixed and incubated at room temperature for 15 minutes. 500μL of the plasmid mixture was added to each well of the 6-well plate, mixed and placed in an incubator for further culture. 48 hours after transfection, the culture medium was harvested, centrifuged at 3000×rpm for 10 minutes, and the supernatant virus liquid was aliquoted and frozen at -80℃ for later use.

[0215] Construction of HCT116 cell lines stably expressing TT3 (HCT116-TT3) and SK-HEP-1 (SK-HEP-1-TT3)

[0216] Wild-type HCT116 cells were digested into single cells with 0.25% trypsin and plated at 1 × 10 5 / 500μL / well was inoculated in a 48-well plate, and 300μL of lentivirus LV-TT3-GFP and polybrene (commercially available from: Shanghai Yishen Biotechnology Co., Ltd., final concentration 8μg / mL) were added, placed in an incubator for culture and passage amplification. One week later, the infected cells were digested and inoculated into a 96-well plate at 2 cells / well, 100μL per well, and placed in an incubator for continued culture. After continuous culture for about 10 days to grow monoclonal cells, all cells were GFP-positive and there was only one cluster of cell clones. The wells were selected under a microscope and passaged and amplified. The expression of GFP and TT3 was detected by flow cytometry, and the optimal monoclonal clone was finally obtained according to the expression ratio and intensity of GFP and TT3, and a stable cell line (HCT116-TT3) expressing the TT3 antigen was obtained.

[0217] A similar method was used to construct the stable cell line SK-HEP-1-TT3 expressing TT3 antigen.

[0218] Preparation of T cells

[0219] After PBMC cells were recovered, they were added with complete culture medium and incubated in an incubator for 2 h. At the end of the incubation, 5 × 10 5 / 500μL / well were inoculated into 24-well plates, and 10μL Cells were activated with Human T-Activator CD3 / CD28 (purchased from Gibco). Complete medium was then replenished every other day depending on cell growth status. Culture was continued until day 10, at which time the magnetic beads were removed and the cells were frozen for future use. The complete medium consisted of AIM-V + 5% FBS + hIL-2 (final concentration 100 IU / ml). AIM-V was purchased from Gibco, and human recombinant interleukin-2 (hIL-2) was purchased from Jiangsu Jinsili Pharmaceutical Co., Ltd.

[0220] Poxvirus (DDVV-TT3) Construction: The poxvirus DDVV-TT3 can be obtained by referring to the methods described in PCT International Publication No. WO2020038490A (International Application No. PCT / CN2019 / 102480). It should be noted that in WO2020038490A, the poxvirus is represented as vvDD-TT3. Both represent the same recombinant poxvirus.

[0221] The humanized anti-CD3 monoclonal antibody and its activity characterization results are as follows:

[0222] Based on the SP34 antibody sequence, humanized sequence templates were searched in the database, and the importance of different amino acids was analyzed based on the 3D structure. If necessary, key amino acids that affect the structure were back-mutated to determine the corresponding humanized antibody. The humanized antibody was fused with a human constant region sequence and constructed into the pcDNA3.1 mammalian expression vector to construct a chimeric antibody. The obtained different antibodies were then expressed and purified using a mammalian cell system to obtain different antibodies with a purity of at least 90%. The expressed antibody sequences were CD3 Ab01 to CD3 Ab15, respectively. The expressed antibodies were IgG antibodies, the heavy chain constant domain of the antibody was shown in SEQ ID NO: 57, and the light chain constant domain of the antibody was shown in SEQ ID NO: 55. The sequences of the heavy chain variable region and light chain variable region of each antibody are numbered herein as follows: the heavy chain variable region of antibody CD3 Ab01 is shown in SEQ ID NO: 43, and the light chain variable region is shown in SEQ ID NO: 46. The heavy chain variable region of antibody CD3 Ab02 is set forth in SEQ ID NO:43, and the light chain variable region is set forth in SEQ ID NO:47. The heavy chain variable region of antibody CD3 Ab03 is set forth in SEQ ID NO:43, and the light chain variable region is set forth in SEQ ID NO:48. The heavy chain variable region of antibody CD3 Ab04 is set forth in SEQ ID NO:43, and the light chain variable region is set forth in SEQ ID NO:49. The heavy chain variable region of antibody CD3 Ab05 is set forth in SEQ ID NO:43, and the light chain variable region is set forth in SEQ ID NO:50. The heavy chain variable region of antibody CD3 Ab06 is set forth in SEQ ID NO:44, and the light chain variable region is set forth in SEQ ID NO:46. The heavy chain variable region of antibody CD3 Ab07 is set forth in SEQ ID NO:44, and the light chain variable region is set forth in SEQ ID NO:47. The heavy chain variable region of antibody CD3 Ab08 is set forth in SEQ ID NO:44, and the light chain variable region is set forth in SEQ ID NO:48. The heavy chain variable region of antibody CD3 Ab09 is set forth in SEQ ID NO:44, and the light chain variable region is set forth in SEQ ID NO:49. The heavy chain variable region of anti-CD3 Ab10 is set forth in SEQ ID NO:44, and the light chain variable region is set forth in SEQ ID NO:50. The heavy chain variable region of antibody CD3 Ab11 is set forth in SEQ ID NO:45, and the light chain variable region is set forth in SEQ ID NO:46. The heavy chain variable region of antibody CD3 Ab12 is set forth in SEQ ID NO:45, and the light chain variable region is set forth in SEQ ID NO:47. The heavy chain variable region of antibody CD3 Ab13 is set forth in SEQ ID NO:45, and the light chain variable region is set forth in SEQ ID NO:48.The heavy chain variable region of antibody CD3 Ab14 is shown in SEQ ID NO: 45, and the light chain variable region is shown in SEQ ID NO: 49. The heavy chain variable region of antibody CD3 Ab15 is shown in SEQ ID NO: 45, and the light chain variable region is shown in SEQ ID NO: 50.

[0223] The activity of the prepared humanized antibody was assessed by ELISA. The assay procedure was as follows: hCD3 (human CD3 antigen) was diluted to 0.5 μg / mL using coating buffer, and 100 μL / well was added to a 96-well plate and incubated overnight at 4°C. The plate was washed with PBST, and 200 μL of blocking buffer was added to each well. The plate was blocked at 37°C for 2 hours. The plate was washed with PBST, and then 100 μL / well of diluted antibody (initial concentration 40 nM, serially diluted 1:6) was added. The plate was incubated at 37°C for 2 hours. The plate was washed with PBST, and then 100 μL / well of HRP-conjugated goat anti-human IgG was added. The plate was incubated at 37°C for 1 hour. The plate was washed with PBST, and then 100 μL / well of TMB was added. The plate was developed at 37°C for 10-20 minutes. The reaction was terminated by adding 50 μL of 2M H2SO4 to each well. The readings were read at 450 nm. The EC50 results of antibodies CD3 Ab01, CD3 Ab02, CD3 Ab03, CD3 Ab04, CD3 Ab05, CD3 Ab06, CD3 Ab07, CD3 Ab08, CD3 Ab09, CD3 Ab10, CD3 Ab11, CD3 Ab12, CD3 Ab13, CD3 Ab14, and CD3 Ab15 were 0.0096nM, 0.0117nM, 0.0042nM, 0.0068nM, 0.0099nM, 0.0126nM, 0.0129nM, 0.0116nM, 0.0080nM, 0.0020nM, 0.0118nM, 0.0095nM, 0.0099nM, 0.0087nM, and 0.0099nM, respectively.

[0224] The binding activity of the prepared humanized antibodies was tested using FACS technology. The experimental process is as follows: Jurkat cells or 293T-CD3 cells were plated, washed with FACS buffer, and centrifuged at 1200 rpm for 3-5 minutes. Then, different diluted concentrations of antibody were added, incubated at 4°C for 0.5 hours, centrifuged at 1200 rpm for 3-5 minutes, and washed with FACS buffer. Then, goat anti-human IgG secondary antibody (manufacturer: Abcam, ab98596) was added at 100 uL / well, incubated at 4°C for 0.5 hours, centrifuged at 1200 rpm for 3-5 minutes, washed with FACS buffer, and resuspended in 50 uL of FACS buffer for analysis by flow cytometry. Among them, the EC50 results of FACs assay of antibodies CD3 Ab01, CD3 Ab02, CD3 Ab03, CD3 Ab04, CD3 Ab05, CD3 Ab06, CD3 Ab07, CD3 Ab08, CD3 Ab09, CD3 Ab10, CD3 Ab11, CD3 Ab12, CD3 Ab13, CD3 Ab14, and CD3 Ab15 on Jurkat cells were 3.299nM, 2.642nM, 2.284nM, 1.182nM, 1.024nM, 3.309nM, 2.415nM, 1.823nM, 0.862nM, 0.784nM, 4.451nM, 3.518nM, 3.194nM, 1.142nM, and 1.108nM, respectively; The EC50 results of FACs detection of 93T-CD3 cells were 22.600nM, 12.300nM, 10.060nM, 5.234nM, 4.267nM, 12.990nM, 9.020nM, 4.238nM, 4.401nM, 2.438nM, 21.550nM, 15.770nM, 12.360nM, 5.728nM, and 3.479nM, respectively.

[0225] The humanized anti-TT3 monoclonal antibody and its activity characterization results are as follows:

[0226] The antibody (numbered Ab-TT3, its heavy chain variable region is SEQ ID NO: 26, and its light chain variable region is SEQ ID NO: 27) was humanized. Specifically, based on the Ab-TT3 antibody sequence, a humanized sequence template was retrieved from the database. In order to maintain the conformation of the CDR region, three types of residues were focused on, including residues located at the VL and VH binding interface, residues close to the CDR region and embedded in the protein, and residues that directly interact with the CDR region. The interactions include: hydrophobic interactions, hydrogen bonds, and salt bridges. The 3D structure was analyzed for the importance of different amino acids, and if necessary, the key amino acids that affect the structure were reverse mutated to determine the corresponding humanized antibody.

[0227] The humanized antibody was fused with a human constant region sequence and constructed into the pcDNA3.1 mammalian expression vector to form a chimeric antibody. The obtained different antibodies were then expressed and purified using a mammalian cell system to obtain different antibodies with a purity of at least 90%. The expressed antibodies were TT3 Ab-1, TT3 Ab-2, and TT3 Ab-3, respectively, wherein the heavy chain constant domain of the antibody is shown in SEQ ID NO:57, and the light chain constant domain of the antibody is shown in SEQ ID NO:55. TT3 Ab-1 has a heavy chain variable region shown in SEQ ID NO:29 and a light chain variable region shown in SEQ ID NO:32. TT3 Ab-2 has a heavy chain variable region shown in SEQ ID NO:30 and a light chain variable region shown in SEQ ID NO:33. TT3 Ab-3 has a heavy chain variable region shown in SEQ ID NO:31 and a light chain variable region shown in SEQ ID NO:34. At the same time, the Fc region of the TT3 Ab-3 antibody was mutated. The mutated antibody was numbered TT3 Ab-3mu, the heavy chain constant domain thereof was shown in SEQ ID NO: 54, and the light chain constant domain thereof was shown in SEQ ID NO: 55.

[0228] The activity of the prepared humanized antibodies was tested by ELISA. The experimental procedure was as follows: TT3 antigen was diluted to 0.5 μg / mL using coating buffer and 100 μL / well was added to a 96-well plate. The plate was incubated overnight at 4°C. The plate was washed with PBST, and then 200 μL of antibody blocking buffer was added to each well and blocked at 37°C for 2 hours. The plate was washed with PBST, and different antibodies (starting at 100 nM, serially diluted 1:6) were added at 100 μL / well and incubated at 37°C for 2 hours. The plate was washed with PBST, and HRP-conjugated goat anti-human / mouse IgG antibody was added at 100 μL / well and incubated at 37°C for 1 hour. The plate was washed with PBST, and TMB was added at 100 μL / well. The color was developed at 37°C for 10-20 minutes. The reaction was terminated by adding 50 μL of 2M sulfuric acid to each well. The OD value was read at a wavelength of 450 nm. The EC50 values ​​of antibodies TT3 Ab-1, TT3 Ab-2, TT3 Ab-3, and Ab-TT3 were 0.0104 nM, 0.0105 nM, 0.0140 nM, and 0.0878 nM, respectively.

[0229] The activity of the prepared humanized antibodies was tested using the FACS method. The experimental process is as follows: different cells (HCT116-TT3, SK-HEP-1-TT3, MC38-TT3) were plated, then washed with FACS buffer, centrifuged at 1200 rpm for 3-5 minutes, and different concentrations of diluted antibodies were added (starting at 100 nM, with a 1:5 serial dilution), incubated at 4°C for 0.5 hours, and centrifuged at 1200 rpm for 3-5 minutes. After washing with FACS buffer, goat anti-human IgG secondary antibody (Abcam, ab98596) was diluted 1:250 and added to the plate at 100 uL / well. The cells were incubated at 4°C for 0.5 hours, centrifuged at 1200 rpm for 3-5 minutes, washed with FACS buffer, and then resuspended in 50 uL of FACS buffer for analysis by flow cytometry. Among them, the EC50 results of FACs affinity activity of antibodies TT3 Ab-1, TT3 Ab-2, and TT3 Ab-3 with HCT116-TT3 cells were 0.568nM, 0.958nM, and 0.692nM, respectively; the EC50 results of FACs affinity activity with SK-HEP-1-TT3 cells were 0.546nM, 1.527nM, and 0.738nM, respectively; and the EC50 results of FACs affinity activity with MC38-TT3 cells were 1.201nM, 2.327nM, and 0.588nM, respectively.

[0230] The same method was used to detect the antibody after Fc mutation (TT3 Ab-3mu), and it was found that both before and after Fc mutation could bind to TT3 antigen and had biological activity, and the binding activity of the two was not much different.

[0231] Example 1

[0232] The bispecific antibody BK-A-101 was prepared using genetic engineering methods. Specifically, the nucleotide sequences encoding the heavy and light chains of the bispecific antibody were synthesized and cloned into the pcDNA3.1 expression vector (purchased from GenScript) according to conventional techniques in the art. The bispecific antibody protein was then transiently transfected into CHO-S cells using electroporation for expression. The supernatant was collected and purified using a protein A affinity column. The purity of the purified antibody was determined by HPLC and SDS-PAGE, and the purity of the bispecific antibody BK-A-101 was determined to be above 90%. The binding activity of the bispecific antibody BK-A-101 to the target antigen was then assayed by flow cytometry. Similar methods were used to obtain other bispecific antibodies with the BK-A structure (including BK-A-101-hu1, BK-A-101-hu2, BK-A-101-hu3, BK-A-101-hu4, BK-A-101-hu5, and BK-A-101-hu6).

[0233] A similar method was used to prepare bispecific antibodies with BK-B structure (BK-B-110, BK-B-110-hu1, BK-B-110-hu2, BK-B-110-hu3, BK-B-110-hu4, BK-B-110-hu5, and BK-B-110-hu6), and bispecific antibodies with BK-C structure (BK-C-120, BK-C-121, BK-C-122). BK-D-01, BK-D-02, BK-D-03, BK-D-04, BK-D-05, BK-D-06, BK-D-07, BK-D-08, BK-D-09, BK-D-10, BK-D-11, BK-D-12, and BK-D-13 bispecific antibodies.

[0234] The binding activity of bispecific antibodies to T cells was assessed by flow cytometry (FACS). The following steps were performed: T cells were resuspended in flow cytometry buffer and aliquoted into 96-well plates. Different antibodies were then incubated for incubation. Group 1 served as a blank control group, without any antibody staining. Group 2 added an isotype control antibody (negative control, human IgG1, purchased from Biolegend) to a final concentration of 50 nM. Group 3 added an anti-CD3 monoclonal antibody (L2K antibody, purchased from Kangyuan Broad Group; the same anti-CD3 monoclonal antibody used hereafter) to a final concentration of 50 nM. Group 4 added the BK-A-101 bispecific antibody to a final concentration of 50 nM. After incubation at 2–8°C for 30 min, the cells were diluted, washed, and resuspended in flow cytometry buffer. A PE-conjugated anti-human IgG secondary antibody (purchased from ABCAM, diluted 1:200) was added to each group and incubated at 2–8°C for 30 min. After dilution and washing with flow cytometry buffer, the cells were resuspended in 200 μL of flow cytometry buffer and analyzed on a Novocyte flow cytometer. The results are shown in Figure 5. The blank and negative controls were negative by flow cytometry. The positive control showed a 99.45% positive rate by flow cytometry, while the bispecific antibody showed a 99.44% positive rate. These results demonstrate that the bispecific antibody BK-A-101 has high T cell binding activity.

[0235] The binding activity of the bispecific antibody BK-A-101 to HCT116 cells or HCT116-TT3 cells was detected by flow cytometry (FACS). The steps are as follows: HCT116 and HCT116-TT3 cells were digested into single cells with 0.25% trypsin and resuspended in flow cytometry buffer. BK-A-101 bispecific antibody was added to a final concentration of 0.1nM, 1nM, 10nM, and 100nM, respectively. After mixing, the cells were incubated at 2-8°C for 30 minutes. After dilution and washing with flow cytometry buffer, the cells were resuspended with PE-labeled anti-human IgG secondary antibody (1:200 dilution) and incubated at 2-8°C for 30 minutes. After dilution and washing with flow cytometry buffer, the cells were resuspended in 200μL flow cytometry buffer and detected by Novocyte. The results are shown in Figures 6 and 7.

[0236] Figure 6 shows the results of the bispecific antibody binding activity test with the HCT116 cell line. The results show that even with increasing bispecific antibody concentration, it does not show binding activity with the HCT116 cell line. Figure 7 shows the binding activity of the bispecific antibody BK-A-101 with HCT116-TT3 cells. The results show that with increasing bispecific antibody concentration, the positive rate of flow cytometry detection shows an increasing trend. The results of Figures 6 and 7 demonstrate the specificity of the bispecific antibody for TT3 binding.

[0237] Similar methods were used to test the binding activity of the bispecific antibody to SK-HEP-1 and SK-HEP-1-TT3 cell lines. The results showed that the bispecific antibody did not bind to the SK-HEP-1 cell line, but did bind to SK-HEP-1-TT3. Furthermore, the binding rate increased with increasing bispecific antibody concentration, further demonstrating the specificity of the bispecific antibody for TT3 binding.

[0238] The affinity (i.e., affinity) of bispecific antibodies (BK-D-01, BK-D-02, BK-D-03, BK-D-04, BK-D-05, BK-D-06, BK-D-07, BK-D-08, BK-D-09, BK-D-10, BK-D-11, and BK-D-12) to the CD3 and TT3 ends was tested by FACs. The detection methods and results are as follows:

[0239] Jurkat cells were plated; washed with FACS buffer and centrifuged at 1200 rpm for 3-5 minutes; then 100 ul / well of diluted antibodies of different concentrations were added and incubated at 4°C for 0.5 h; centrifuged at 1200 rpm for 3-5 minutes and washed with FACS buffer; then diluted goat anti-human IgG (FITC, Abcam / ab97224) antibody was added and incubated at 4°C for 0.5 h; centrifuged at 1200 rpm for 3-5 minutes, washed with FACS buffer, and 50 uL of FACS buffer was added to resuspend the cells for detection by flow cytometry.

[0240] Similarly, the affinity activity of the bispecific antibody to the TT3 end was detected. The experimental steps were as follows: HCT116-TT3 cells were plated; washed with FACS buffer and centrifuged at 1200 rpm for 3-5 minutes; then different concentrations of diluted antibodies were added; incubated at 4°C for 0.5 hours; centrifuged at 1200 rpm for 3-5 minutes and washed with FACS buffer; diluted goat anti-human IgG Fc (Dylight 650, Abcam / ab98622) antibody was added and incubated at 4°C for 0.5 hours; centrifuged at 1200 rpm for 3-5 minutes and washed with FACS buffer; 50 uL of FACS buffer was added to resuspend the cells and detected by flow cytometry.

[0241] The corresponding test results are shown in the following table and Figure 8:

[0242] Table 1 FACs detection of affinity activity of bispecific antibodies to CD3 and TT3

[0243] In Table 1, “-” means that the experiment was not performed.

[0244] The experimental results showed that the prepared bispecific antibody exhibited high affinity activity with the TT3 end and the CD3 end. A similar method was used to detect the affinity of the bispecific antibody with the TT3 end using SKHEP1-TT3 cells or MC38-TT3 cells. The results showed that the bispecific antibody exhibited high affinity activity with the TT3 end.

[0245] Other BK-B and BK-D structured bispecific antibodies also showed similar affinity activities with the CD3 end and TT3 end.

[0246] Furthermore, non-specific binding between bispecific antibodies and wild-type tumor cell lines was detected by FACS. The steps are as follows: HCT116 and SK-HEP-1 cells were digested into single cells with 0.25% trypsin and resuspended in flow cytometry buffer. BK-A-101 and BK-D-01~09 bispecific antibodies were added to final concentrations of 10nM and 100nM, respectively, mixed and incubated at 2~8°C for 30min. After dilution and washing with flow cytometry buffer and resuspending, APC-labeled anti-human IgG secondary antibody (1:200 dilution) was added to each group and incubated at 2~8°C for 30min. After dilution and washing with flow cytometry buffer, the cells were resuspended in 200μL flow cytometry buffer and detected by Novocyte. The results are shown in Figure 9. The non-specific binding ratio of the bispecific antibody BK-A-101 and BK-D-01~09 groups was low at a general concentration of 10nm. The bispecific antibody groups BK-D-01, 02, 03, 04, 05, 06, and 08 showed low nonspecific binding rates even at an extremely high concentration of 100 nM. This indicates that in the absence of a specific target, the bispecific antibodies of the present invention exhibit low nonspecific binding and have a good safety profile.

[0247] Example 2

[0248] Example 2 provides the detection of the killing effect of the combination of bispecific antibodies and pre-activated and expanded T cells on HCT116 and HCT116-TT3 cell lines using RTCA real-time killing instrument.

[0249] First, 50 μL McCoy 5A + 10% FBS medium was used to equilibrate a 96E-Plate (purchased from Agilent Technologies). Then, wild-type HCT116 and HCT116-TT3 were digested into single cells using 0.25% trypsin and resuspended in McCoy 5A + 10% FBS medium. The cells were plated at a rate of 5 × 10 3Tumor cells were seeded into a 96E-Plate at 50 μL / well. Five hours after plating, T cells were added to the corresponding wells of the 96E-Plate at an E:T ratio of 2:1 (E:T stands for effector-to-target ratio, representing the ratio of effector cells to target cells) at 50 μL / well. Simultaneously, a bispecific antibody (BK-A-101) or anti-CD3 monoclonal antibody (CD3 Ab) was serially diluted and added to the corresponding wells of the 96E-Plate. The final concentrations of BK-A-101 were 10 nM, 1 nM, and 0.1 nM, respectively, and the final concentrations of CD3 Ab were 10 nM, 1 nM, and 0.1 nM, respectively. The final volume per well was 200 μL, and data were collected every 15 minutes.

[0250] The results are shown in Figure 10. T cells acting alone, and various concentrations of CD3 Ab or BK-A-101 combined with T cells, did not show significant killing effects on wild-type HCT116; T cells acting alone, and various concentrations of CD3 Ab combined with T cells, also did not show significant killing effects on HCT116-TT3; however, various concentrations of BK-A-101 combined with T cells showed very significant killing effects on HCT116-TT3, and showed a dose-related effect with the concentration of BK-A-101. Specifically, 1nM and 1nM BK-A-101 combined with T cells can almost completely eliminate HCT116-TT3 with good sustainability, while 0.1nM BK-A-101 combined with T cells can almost completely eliminate HCT116-TT3 before 40 hours.

[0251] A similar method was used to obtain the detection results of the bispecific antibody for the BK-D structure. The difference was that after the tumor cell lines were plated overnight, effector cells and bispecific antibodies were added, and the ratio of effector cells to target cells was 5:1. The results are shown in Figure 11. Figure 11 A and B respectively show the killing effect of the bispecific antibody at different concentrations (0.1nM or 1nM) and pre-activated and expanded T cells on the HCT116-TT3 cell line, where labels 1-9 represent HCT116-TT3+T cells+bispecific antibodies BK-D-01~BK-D-09 groups, label 10 represents HCT116-TT3+T cells+BK-A-101 group, label 11 represents HCT116-TT3 cells only, without the addition of bispecific antibodies and T cell treatment, and label 12 represents HCT116-TT3 cells with only T cell treatment and no bispecific antibodies. The results show that the combination of the bispecific antibody and T cells of the present invention can almost completely eliminate specific cancer cells at all concentrations with good persistence, showing an excellent tumor cell killing effect.

[0252] We used a similar method to test the killing effect of bispecific antibodies on wild-type tumor cell lines HCT116 (Figure 12A) and SK-HEP-1 (Figure 12B) at high concentrations. Numbers 1-6 represent wild-type tumor cell lines + T cells + bispecific antibodies BK-D-01, 04, 05, 06, 07, and 08, respectively; number 7 represents wild-type tumor cell lines + T cells + TT3 monoclonal antibody-1; number 8 represents wild-type tumor cell lines + T cells + CD3 monoclonal antibody SP34; number 9 represents wild-type tumor cell lines alone without antibody and T cell treatment; and number 10 represents wild-type tumor cell lines treated with only T cells without antibody. In Figure 12A, the bispecific antibody concentration was 100 nM; in Figure 12B, the bispecific antibody concentration was 10 nM. The results are shown in Figure 12. Even at extremely high concentrations, the bispecific antibodies BK-D-04, 05, 06, 07, and 08 of the present invention did not exhibit significant killing effects on wild-type cell lines, indicating that these bispecific antibodies have good killing specificity.

[0253] Example 3

[0254] Example 3 provides a method for detecting the killing effect of a combination of bispecific antibodies and PBMC cells on the HCT116 cell line using an RTCA real-time killing instrument.

[0255] After PBMC recovery, resuspend them in complete culture medium and recover overnight in an incubator. The next day, equilibrate a 96E-Plate (Agilent) with 50 μL McCoy 5A + 10% FBS culture medium. Then, HCT116-TT3 cells were digested into single cells with 0.25% trypsin and resuspended in McCoy 5A + 10% FBS culture medium. 3 Tumor cells were seeded into a 96E-plate at 50 μL / well. Five hours after plating, PBMCs were added to the corresponding wells of the 96E-plate at an E:T ratio of 2:1 (50 μL / well). Simultaneously, BK-A-101 was serially diluted and added to the corresponding wells of the 96E-plate, with final concentrations of 10 nM and 1 nM, respectively. The final volume per well was 200 μL, and data were acquired with a scan every 15 minutes.

[0256] The results are shown in Figure 13. PBMC cells alone did not show a significant killing effect on HCT116-TT3; however, BK-A-101 at various concentrations combined with PBMC cells showed a very significant killing effect on HCT116-TT3. The combination of 10nM and 1nM BK-A-101 and PBMC cells can almost completely eliminate HCT116-TT3 with good sustainability.

[0257] Example 4

[0258] Example 4 Flow cytometry (FACS) was used to detect the binding of BK-A-101 to human tumor cell lines infected with DDvv-TT3. The steps were as follows:

[0259] SKOV3, PANC1, A375 and U251 were digested into single cells with 0.25% trypsin and resuspended in their respective complete culture medium. 5 / 2mL / well The cells were seeded in a 6-well plate. The next day, the 6-well plate was removed, the culture medium was discarded, and the DDvv-TT3 recombinant oncolytic poxvirus was serially diluted with the corresponding culture medium supplemented with 5% FBS and added at 500μL / well, with MOI = 2, 0.2, and 0.02. After gently shaking and incubating in a 37°C incubator for 2 hours, the 6-well plate was removed, 1.5mL of culture medium containing 5% FBS was added to each well to 2mL, and after gently shaking, the well was placed in the incubator and continued to be cultured. 48 hours after viral infection, the cells were harvested and resuspended in flow cytometry buffer, the following antibodies were added, and incubated at 2-8°C for 30min.

[0260] Group 1: isotype control antibody (isotype, human IgG1, negative control, final concentration 100 nM),

[0261] Group 2: bispecific antibody BK-A-101 (final concentration 100 nM),

[0262] Group 3: anti-Strep tag II antibody (positive control, anti-TT3 monoclonal antibody, purchased from GenScript, final concentration of 10 μg / mL).

[0263] After washing and resuspending in flow cytometry buffer, PE-labeled anti-human IgG secondary antibodies (commercially available from ABCAM, 1:200 dilution) were added to Groups 1 and 2, respectively, and PE-labeled anti-mouse IgG antibodies (commercially available from ABCAM, 1:200 dilution) were added to Group 3. The cells were incubated at 2-8°C for 30 min. After washing with dilution buffer, the cells were resuspended in 200 μl of buffer and analyzed by a Novocyte analyzer.

[0264] The results are shown in Figure 14. As the MOI value increases, the flow cytometry results of the isotype control group are all negative, while the flow cytometry results of the positive control and the bispecific antibody staining group show an increasing trend. The experimental results demonstrate that after infecting human tumor cell lines with DDvv-TT3, the addition of bispecific antibodies can specifically bind to tumor cells infected with the oncolytic virus, and the percentage of binding increases with increasing MOI.

[0265] Example 5 (Experiment on stimulating T cell proliferation and activation)

[0266] Example 5: Flow cytometry (FACS) was used to detect T cell activation after 3 days of co-incubation of bispecific antibodies with in vitro cultured T cells (shown in FIG. 15A ) or PBMC (shown in FIG. 15B ). The steps were as follows:

[0267] The pre-activated and expanded T cells or PBMCs were resuspended in complete culture medium at a concentration of 1×10 5 Cells were seeded in 48-well plates at 150 μL / well. After seeding, 150 μL of bispecific antibodies diluted to various concentrations were added to the corresponding wells. The final concentrations of the bispecific antibodies were 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, and 250 nM. In the blank group, no bispecific antibodies were added, and only the same volume of blank culture medium was added. After 3 days of culture, the 48-well plates were removed, and the cells from each group were harvested, diluted, washed, and resuspended in flow cytometry buffer. FITC anti-human CD3, PE anti-human CD4, APC-Cy7 anti-human CD25, and APC anti-human CD127 were added to each group, mixed, and incubated at 2-8°C for 30 minutes. After washing with flow cytometry buffer, the cells were resuspended in 200 μL of flow cytometry buffer and analyzed on a Novocyte analyzer. The results, as shown in Figure 15 , show that the bispecific antibodies BK-D-01-09 exhibited low expression rates of the activation marker CD25 on the T cell surface at standard concentrations, such as 0.01 nm, 0.1 nm, 1 nm, and 10 nm. The bispecific antibodies BK-D-04-09 exhibited low expression rates of the activation marker CD25 on the T cell surface even at extremely high concentrations, such as 100 nm and 250 nm. These results demonstrate that, even in the absence of a specific target, the bispecific antibodies of the present invention exhibit low T cell activation and a favorable safety profile.

[0268] Example 5 also examined the release of the cytokine IFN-γ in the culture supernatant after 3 days of co-incubation of the bispecific antibody with PBMC and SK-HEP-1 cells by ELISA (Figure 16). The final concentrations of the bispecific antibody were 0.1 nM, 1 nM, 10 nM, 100 nM, and 250 nM. Figure 16A shows the results of 3-day co-culture of PBMC and the bispecific antibody, Figure 16B shows the results of 3-day co-culture of PBMC with the bispecific antibody and wild-type SK-HEP-1 cells, and Figure 16C shows the results of 3-day co-culture of PBMC with the bispecific antibody and SK-HEP-1-TT3 cells. The results showed that when the bispecific antibody was co-cultured with PBMC, or with PBMC and SK-HEP-1, the IFN-γ secretion levels of the bispecific antibody BK-D-01 and 04-08 groups were low at typical concentrations, such as 0.1 nM, 1 nM, and 10 nM. When the bispecific antibodies were co-cultured with PBMCs, the bispecific antibody groups BK-D-04, 05, and 06 showed low IFN-γ secretion even at extremely high concentrations, such as 100 nM and 250 nM. These results indicate that, in the absence of a specific target, the bispecific antibodies of the present invention have a low ability to activate T cells and a good safety profile. When the bispecific antibodies were co-cultured with PBMCs and SK-HEP-1-TT3, all bispecific antibody groups secreted significant amounts of IFN-γ, demonstrating that the bispecific antibodies of the present invention can strongly activate T cells in the presence of a specific target.

[0269] Example 6

[0270] Example 6 provides the detection of the killing effect of the bispecific antibody, recombinant oncolytic poxvirus DDvv-TT3, and pre-activated and expanded T cells on the HCT116 cell line in vitro using an RTCA real-time killing instrument (as shown in Figure 17).

[0271] First, 50 μL McCoy 5A + 10% FBS medium was used to equilibrate a 96E-Plate (purchased from Agilent Technologies). Wild-type HCT116 cells were then digested into single cells using 0.25% trypsin and resuspended in McCoy 5A + 10% FBS medium. 3Tumor cells were inoculated into 96E-Plate at 50 μl / well. After plating overnight, DDvv-TT3 was used to inoculate tumor cells into 96E-Plate at 50 μl / well (MOI = 1). After adding oncolytic poxvirus overnight, T cells were added to the corresponding wells of 96E-Plate at a cell ratio of E:T = 10:1, 50 μL / well; at the same time, bispecific antibodies BK-A-101, BK-D-04, BK-D-06, and BK-D-07 were added to the corresponding wells of 96E-Plate at 50 μl / well. The final concentration of the bispecific antibody was 1 nM, the final volume of each well was 250 μL, and the data was scanned every 15 minutes. In Figure 17A, number 1 represents the HCT116 group without T cells, poxvirus, or bispecific antibodies; number 2 represents the HCT116+T cell group; numbers 3 to 6 represent HCT116+T+ bispecific antibodies BK-D-04, 06, 07, or BK-A-101. In Figure 17C, number 1 represents the HCT116 group without T cells, poxvirus, or bispecific antibodies; number 2 represents the HCT116+DDvv-TT3 group; number 3 represents the HCT116+DDvv-TT3+T cell group; numbers 4 to 7 represent HCT116+DDvv-TT3+T+ bispecific antibodies BK-D-04, 06, 07, or BK-A-101. The cell index at 80 hours in Figure 17A was converted into tumor growth inhibition rate (IR, %) to obtain Figure 17B; the cell index at 80 hours in Figure 17C was converted into tumor growth inhibition rate to obtain Figure 17D.

[0272] The results showed that without the addition of recombinant oncolytic poxvirus DDvv-TT3, tumor cells showed continuous growth (as shown in Figure 17A). After the addition of recombinant oncolytic poxvirus DDvv-TT3, the bispecific antibodies BK-D-04, 06, 07 or BK-A-101 combined with T cells all showed significant killing effects (as shown in Figure 17C). Combined with the results shown in B and D in Figure 17, the growth inhibition rate of HCT116 cells in the bispecific antibody BK-D-04, 06, 07+T cell group was less than <10%; in the DDvv-TT3+T cell group, the growth inhibition rate of HCT116 cells was approximately 40%; in the bispecific antibody BK-D-04, 06, 07 combined with DDvv-TT3 and T cell group, the growth inhibition rate of HCT116 cells was less than >80%, indicating that the bispecific antibodies BK-D-04, 06, 07 combined with DDvv-TT3 and T cells showed a synergistic killing effect.

[0273] In the description of the present invention, the term "plurality" means at least two, for example, two, three, etc., unless otherwise clearly and specifically defined.

[0274] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0275] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A therapeutic agent, characterized in that Include: (a) a first composition comprising a first active ingredient, the first active ingredient comprising or containing a nucleic acid having a marker polypeptide encoding sequence for introduction into tumor cells and / or cancer cells; the marker polypeptide having an operably linked extracellular antigenic determinant, a spacer portion, and a transmembrane portion; the amino acid sequence of the extracellular antigenic determinant containing the amino acid sequence of one or more antigenic epitope polypeptides; and the amino acid sequence of a mammalian cell membrane protein or secretory protein not containing the amino acid sequence of the antigenic epitope polypeptide in a natural state; and (b) a second composition comprising a second active ingredient, wherein the second active ingredient comprises a multispecific antibody, wherein the multispecific antibody comprises at least a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion can specifically recognize and bind to the extracellular antigenic determinant region of the marker polypeptide; The second antigen binding portion is capable of specifically recognizing and binding to an immune cell antigen or a first cytokine or its receptor; The multispecific antibody may also optionally include a third antigen binding portion, which is capable of specifically recognizing and binding to a tumor antigen or an immune checkpoint or a second cytokine or its receptor.

2. The therapeutic agent according to claim 1, characterized in that The immune cells include at least one of T cells, NK cells, DC cells, B cells, macrophages, natural killer T cells, and neutrophils.

3. The therapeutic agent according to claim 1, characterized in that The immune cell antigens include CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11, CD11a, CD11b, CD11c, CD11d, CD12w, CD13, CD14, CD15, CD16, CD16a, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD85a, CD85b, CD85c, CD85d, CD85e, CD85f, CD85g, CD85h, CD85i, CD85j, CD85k, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD107a, CD107b, CD108, CD109, CD110, CD11, CD112, CD115, CD116, CD117, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD133, CD131, CD132, CD134, CD135CD137,CD138,CD139,CD141,CD142,CD143,CD144,CD147,CD146,CD148,CD150,CD151,CD152,CD153,CD154,CD155,CD156, CD157,CD158,CD158a,CD158b1,CD158b2,CD158c,CD158d,CD158e,CD158f1,CD158f2,CD158g,CD158h,CD158i,CD158j,CD158k,CD158z,CD159a,CD159c,CD160,CD161,CD162,CD163,CD166,CD165,CD166,CD168,CD169,CD170,CD171,CD172a,CD172b,CD172g,CD173,Cd177,CD178,CD179,CD180,CD181,CD183,CD184,CD185,CD186,CD191,CD192,CD193,CD194,CD195,CD196,CD197,CD198,CD199,CD200,CD203,CD205,CD208,CD209,CD210,CD210b,CD212,CD231a1,CD213a2,CD217,CD218a,CD218b,CD222,CD224,CD225,CD226,CD227,CD229,CD232,CD243,CD244,CD245,CD247,CD252,CD253,CD256,Cd257,CD258,CD261,CD262,CD262,CD263,CD264,CD265,CD268,CD269,CD271,CD272,CD273,CD274,CD275,CD276,CD277,CD278,CD279,CD280,CD281,CD282,CD283,CD284,CD286,CD288,CD289,CD290,CD292,CD293,CD294,CD296,CD297,CD300a,CD300b,CD300c,CD300d,CD300e,CD303,CD304,CD305,CD306,CD307a,CD307b,CD307c,CD307d,CD309,CD312,CD314,CD315,CD316,CD317,CD318,CD319,CD320,CD328,CD329,CD335,CD336,CD337,CD352,CD353,CD357,CD361,At least one of CD362; The first cytokine or its receptor, the second cytokine or its receptor each independently include IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-17a, IL-17b, IL-17c, IL-17d, IL-17f, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-37, EPO, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CC At least one of L7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, IFNα, IFNβ, IFNγ, TNFα, TGFβ, VEGF, GM-CSF, GCSF, or their related receptors.

4. The therapeutic agent according to claim 1, characterized in that The tumor antigen is selected from at least one of AFP, BCMA, β-catetin, CD5, CD19, CD20, CD22, CD30, CD38, CD70, CD123, CD133, CD171, CD138, CDK-4, CEA, CA-125, Caspase 8, c-Met, EGFR, Ep-CAM, EphA2, ETA, FAP, GPC3, GP100, GD2, Her2, HPV16, Igk chain, Ley, Mesothlin, MUC-1, MUC16, MAGE, MART1, NKG2D ligand, CLL1, NY-ESO-1, P53, RAS, OSA, PAP, PSCA, ROR1, ROR2, PSMA, SSTR2, Trop-2, TYR, VEGFR, and WT1; The immune checkpoints are selected from PD-1, PD-L1, CTLA-4, LAG-3, BTLA, TIM-3, TIGIT, VISTA, TIPE2, Galectin-9, NKG2A, KIRs, B7-H3, B7-H7, 2B4, DNAM-1, PTA1, TLISA1, Vstm3, At least one of VSIG9, Siglecs family, GITR, OX40, 4-1BB, LAYN, LIRB1-5 or their related receptors.

5. The therapeutic agent according to claim 1, characterized in that The second antigen binding portion can specifically recognize and bind to CD3, and the second antigen binding portion includes at least one selected from OKT3, SP34, L2K, UCHT1 and variants thereof.

6. The therapeutic agent according to claim 1, characterized in that The second antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are the CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ ID NO:41, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are the CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ ID NO:42; Optionally, the second antigen binding portion has the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 35, 36 and 37; and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 38, 39 and 40; or the second antigen-binding portion has a heavy chain variable region sequence as shown in SEQ ID NO: 41, 43, 44 or 45 and a light chain variable region sequence as shown in SEQ ID NO: 42, 46, 47, 48, 49, 50, 51, 52 or 53; Optionally, the second antigen binding moiety is selected from: (a-1) has the heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:42; or (a-2) having the heavy chain variable region sequence shown in SEQ ID NO: 41 and the light chain variable region sequence shown in SEQ ID NO: 46; or (a-3) having the heavy chain variable region sequence shown in SEQ ID NO: 41 and the light chain variable region sequence shown in SEQ ID NO: 47; or (a-4) having the heavy chain variable region sequence shown in SEQ ID NO: 41 and the light chain variable region sequence shown in SEQ ID NO: 48; or (a-5) has the heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:49; or (a-6) having the heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:50; or (a-7) has the heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:51; or (a-8) has the heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:52; or (a-9) has the heavy chain variable region sequence shown in SEQ ID NO: 41 and the light chain variable region shown in SEQ ID NO: 53 sequence; or (b-1) has the heavy chain variable region sequence shown in SEQ ID NO: 43 and the light chain variable region sequence shown in SEQ ID NO: 42; or (b-2) having the heavy chain variable region sequence shown in SEQ ID NO: 44 and the light chain variable region sequence shown in SEQ ID NO: 42; or (b-3) having the heavy chain variable region sequence shown in SEQ ID NO: 45 and the light chain variable region sequence shown in SEQ ID NO: 42; or (c-1) has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:46; or (c-2) having the heavy chain variable region sequence shown in SEQ ID NO: 43 and the light chain variable region sequence shown in SEQ ID NO: 47; or (c-3) having the heavy chain variable region sequence shown in SEQ ID NO: 43 and the light chain variable region sequence shown in SEQ ID NO: 48; or (c-4) having the heavy chain variable region sequence shown in SEQ ID NO: 43 and the light chain variable region sequence shown in SEQ ID NO: 49; or (c-5) has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:50; or (c-6) has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:51; or (c-7) has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:52; or (c-8) has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:53; or (d-1) has the heavy chain variable region sequence shown in SEQ ID NO: 44 and the light chain variable region sequence shown in SEQ ID NO: 46; or (d-2) having the heavy chain variable region sequence shown in SEQ ID NO: 44 and the light chain variable region sequence shown in SEQ ID NO: 47; or (d-3) having the heavy chain variable region sequence shown in SEQ ID NO: 44 and the light chain variable region sequence shown in SEQ ID NO: 48; or (d-4) has the heavy chain variable region sequence shown in SEQ ID NO: 44 and the light chain variable region sequence shown in SEQ ID NO: 49; or (d-5) has the heavy chain variable region sequence shown in SEQ ID NO: 44 and the light chain variable region shown in SEQ ID NO: 50 sequence; or (d-6) has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:51; or (d-7) has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:52; or (d-8) has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:53; or (e-1) has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:46; or (e-2) having the heavy chain variable region sequence shown in SEQ ID NO: 45 and the light chain variable region sequence shown in SEQ ID NO: 47; or (e-3) having the heavy chain variable region sequence shown in SEQ ID NO: 45 and the light chain variable region sequence shown in SEQ ID NO: 48; or (e-4) has the heavy chain variable region sequence shown in SEQ ID NO: 45 and the light chain variable region sequence shown in SEQ ID NO: 49; or (e-5) has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:50; or (e-6) has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:51; or (e-7) has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:52; or (e-8) has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:

53.

7. The therapeutic agent according to claim 1, characterized in that The multispecific antibody is produced by a host cell containing a construct containing a nucleotide encoding the multispecific antibody.

8. The therapeutic agent according to claim 7, characterized in that The host cell is selected from at least one of CHO series cell lines, 293 series cell lines, NS0 cells, Hela cells, 3T3 cells, and BHK cells.

9. The therapeutic agent according to claim 1, characterized in that The amino acid sequence of the antigenic epitope polypeptide is derived from the amino acid sequence of a protein existing in nature, or is an artificially synthesized amino acid sequence that does not exist in nature.

10. The therapeutic agent according to claim 1, characterized in that The amino acid sequence of the antigenic epitope polypeptide includes the amino acid sequence of the following tags: Myc tag, HA tag, Strep tag I, Strep tag II, Flag tag, HAT tag, S tag, S1 tag, protein C tag, tag-100 tag, E2 tag, TAP tag, HSV tag, KT3 tag, V5 tag, VSV-G tag, His tag or RFP tag.

11. The therapeutic agent according to claim 1, characterized in that The amino acid sequence of the extracellular antigenic determinant region is shown in SEQ ID NO: 1, 2, 3, 4 or 5.

12. The therapeutic agent according to claim 1, characterized in that The spacer portion is derived from the hinge region of CD8α, the hinge region of IgG, or the hinge region of IgD; preferably, the amino acid sequence of the spacer portion is as shown in SEQ ID NO: 6; The transmembrane portion is derived from the transmembrane region of CD8, CD3ζ, CD4 or CD28; preferably, the amino acid sequence of the transmembrane portion is as shown in SEQ ID NO:

7.

13. The therapeutic agent according to claim 1, characterized in that The amino acid sequence of the marker polypeptide is shown in SEQ ID NO: 11, 12, 13, 14 or 15.

14. The therapeutic agent according to claim 1, characterized in that The first antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the HCDRs, HCDR2 and HCDR3 are the CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 27, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are the CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ ID NO: 26; Optionally, the first antigen binding portion has the HCDR1, HCDR and HCDR3 sequences shown in SEQ ID NOs: 20, 21 and 22; and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 23, 24 and 25; or the first antigen-binding portion has a heavy chain variable region sequence as shown in SEQ ID NO: 27, 29, 30 or 31 and a light chain variable region sequence as shown in SEQ ID NO: 26, 32, 33 or 34; Optionally, the first antigen binding moiety is selected from: (1-1) has the heavy chain variable region sequence shown in SEQ ID NO: 27 and the heavy chain variable region sequence shown in SEQ ID NO: 26; or (1-2) having the heavy chain variable region sequence shown in SEQ ID NO: 27 and the heavy chain variable region sequence shown in SEQ ID NO: 32; or (1-3) having the heavy chain variable region sequence shown in SEQ ID NO: 27 and the heavy chain variable region sequence shown in SEQ ID NO: 33; or (1-4) having the heavy chain variable region sequence shown in SEQ ID NO: 27 and the heavy chain variable region sequence shown in SEQ ID NO: 34; or (1-5) having the heavy chain variable region sequence shown in SEQ ID NO: 29 and the heavy chain variable region sequence shown in SEQ ID NO: 26; or (1-6) has the heavy chain variable region sequence shown in SEQ ID NO: 30 and the heavy chain variable region sequence shown in SEQ ID NO: 26; or (1-7) has a heavy chain variable region sequence shown in SEQ ID NO: 31 and a heavy chain variable region sequence shown in SEQ ID NO: 26 region sequence; or (1-8) has the heavy chain variable region sequence shown in SEQ ID NO: 29 and the light chain variable region sequence shown in SEQ ID NO: 32; or (1-9) has the heavy chain variable region sequence shown in SEQ ID NO: 29 and the light chain variable region sequence shown in SEQ ID NO: 33; or (1-10) has the heavy chain variable region sequence shown in SEQ ID NO: 29 and the light chain variable region sequence shown in SEQ ID NO: 34; or (1-11) has the heavy chain variable region sequence shown in SEQ ID NO: 30 and the light chain variable region sequence shown in SEQ ID NO: 32; or (1-12) has the heavy chain variable region sequence shown in SEQ ID NO: 30 and the light chain variable region sequence shown in SEQ ID NO: 33; or (1-13) has the heavy chain variable region sequence shown in SEQ ID NO: 30 and the light chain variable region sequence shown in SEQ ID NO: 34; or (1-14) has the heavy chain variable region sequence shown in SEQ ID NO: 31 and the light chain variable region sequence shown in SEQ ID NO: 32; (1-15) has the heavy chain variable region sequence shown in SEQ ID NO: 31 and the light chain variable region sequence shown in SEQ ID NO: 33; or (1-16) has the heavy chain variable region sequence shown in SEQ ID NO:31 and the light chain variable region sequence shown in SEQ ID NO:

34.

15. The therapeutic agent according to claim 1, characterized in that The nucleic acid includes DNA or RNA; the RNA includes mRNA transcribed from DNA.

16. The therapeutic agent according to claim 15, characterized in that The first composition includes a therapeutically effective amount of the DNA, or a therapeutically effective amount of the mRNA.

17. The therapeutic agent according to claim 15, characterized in that The DNA is formulated for administration by intratumoral injection, intravenous injection, intraperitoneal injection or intracranial injection; the mRNA is formulated for administration by intratumoral injection, intravenous injection, intraperitoneal injection or intracranial injection.

18. The therapeutic agent according to claim 1, characterized in that The first active ingredient is a recombinant virus, the genome of which has a marker polypeptide coding sequence; the recombinant virus includes a selective replication-type recombinant oncolytic virus or a replication-deficient recombinant virus.

19. The therapeutic agent according to claim 18, characterized in that The first composition includes a therapeutically effective amount of the recombinant virus.

20. The therapeutic agent according to claim 18, characterized in that The recombinant oncolytic virus is derived from a genetically mutated virus with oncolytic activity and a wild-type virus with oncolytic activity; preferably, the recombinant oncolytic virus is derived from an adenovirus, poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus and Maraba virus with oncolytic activity.

21. The therapeutic agent according to claim 18, characterized in that The recombinant virus is configured for administration by intratumoral injection, intravenous administration, intraperitoneal injection or intracranial injection.

22. The therapeutic agent according to claim 1, characterized in that The first composition and the second composition are each independently present in the therapeutic agent without mixing with each other.

23. The therapeutic agent according to claim 1, characterized in that The therapeutic agent consists of the first composition and the second composition.

24. The therapeutic agent according to claim 1, characterized in that The first antigen binding moiety and the second antigen binding moiety are linked by: 1) the heavy chain variable region and light chain variable region of the second antigen-binding moiety are linked to the heavy chain constant region and light chain constant region, respectively, to form an IgG-like structure, the carboxyl-terminal amino acid of the Fc region of the IgG-like structure is linked to the amino-terminal amino acid of the light chain variable region of the first antigen-binding moiety, and the carboxyl-terminal amino acid of the light chain variable region of the first antigen-binding moiety is linked to the amino-terminal amino acid of the heavy chain variable region of the first antigen-binding moiety; or 2) the heavy chain variable region and light chain variable region of the first antigen-binding moiety are respectively linked to the heavy chain constant region and the light chain constant region to form an IgG-like structure, the carboxyl-terminal amino acid of the Fc region of the IgG-like structure is linked to the amino-terminal amino acid of the light chain variable region of the second antigen-binding moiety, and the carboxyl-terminal amino acid of the light chain variable region of the second antigen-binding moiety is linked to the amino-terminal amino acid of the heavy chain variable region of the second antigen-binding moiety; or 3) the carboxyl-terminal amino acid of the light chain variable region of the first antigen-binding moiety is linked to the amino-terminal amino acid of the heavy chain variable region of the first antigen-binding moiety, the carboxyl-terminal amino acid of the first antigen-binding moiety is directly linked to the Fc region, the carboxyl-terminal amino acid of the Fc region is linked to the amino-terminal amino acid of the second antigen-binding moiety, and the carboxyl-terminal amino acid of the light chain variable region of the second antigen-binding moiety is linked to the amino-terminal amino acid of the heavy chain variable region of the second antigen-binding moiety; or 4) the carboxyl-terminal amino acid of the light chain variable region of the first antigen-binding portion is linked to the amino-terminal amino acid of the heavy chain variable region of the first antigen-binding portion, the carboxyl-terminal amino acid of the first antigen-binding portion is directly linked to the Fc region, the carboxyl-terminal amino acid of the Fc region is linked to the amino-terminal amino acid of the second antigen-binding portion, and the carboxyl-terminal amino acid of the heavy chain variable region of the second antigen-binding portion is linked to the amino-terminal amino acid of the light chain variable region of the second antigen-binding portion.

25. The therapeutic agent according to claim 24, characterized in that The Fc region is a humanized sequence or a mutated humanized sequence.

26. The therapeutic agent according to claim 1, characterized in that The second antigen binding portion comprises one or more domains capable of specifically recognizing and binding to CD3, and the first antigen binding portion comprises one or more domains capable of specifically recognizing and binding to the antigen epitope polypeptide, wherein the amino acid sequence of the antigen epitope polypeptide comprises the amino acid sequence of Strep tag I or Strep tag II.

27. A multispecific antibody, characterized in that At least comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion comprises one or more domains capable of specifically recognizing and binding to the amino acid sequence of Strep tag I or Strep tag II, and the second antigen-binding portion comprises one or more domains capable of specifically recognizing and binding to an immune cell antigen or a first cytokine or its receptor; The multispecific antibody may also optionally include a third antigen-binding portion, which comprises one or more domains capable of specifically recognizing and binding to a tumor antigen or an immune checkpoint or a second cytokine or its receptor.

28. The multispecific antibody according to claim 27, characterized in that The immune cells include at least one of T cells, NK cells, DC cells, B cells, macrophages, natural killer T cells, and neutrophils.

29. The multispecific antibody according to claim 27, wherein The immune cell antigens include CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11, CD11a, CD11b, CD11c, CD11d, CD12w, CD13, CD14, CD15, CD16, CD16a, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD\alpha, CD\beta, CD\gamma, CD\delta, CD\epsilon, CD\zeta, CD\eta, CD\theta, CD\iota, CD\kappa, CD\lambda, CD\mu, CD\nu, CD\xi, CD\omicron, CD\pi, CD\rho, CD\sigma, CD\tau, CD\upsilon, CD\phi, CD\chi, CD\psi, CD\omega, CD112, CD115, CD116, CD117, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD133, CD131, CD132, CD134, CD135CD137,CD138,CD139,CD141,CD142,CD143,CD144,CD147,CD146,CD148,CD150,CD151,CD152,CD153,CD154,CD155,CD156,CD157,CD158,CD158a,CD158b1,CD158b2,CD158c,CD158d,CD158e,CD158f1,CD158f2,CD158g,CD158h,CD158i,CD158j,CD158k,CD158z,CD159a,CD159c,CD160, CD161,CD162,CD163,CD166,CD165,CD166,CD168,CD169,CD170,CD171,CD172a,CD172b,CD172g,CD173,Cd177,CD178,CD179,CD180,CD181,CD183,CD184,CD185,CD186,CD191,CD192,CD193,CD194,CD195,CD196,CD197,CD198,CD199,CD200,CD203,CD205,CD208,CD209,CD210,CD210b,CD212,CD231a1,CD213a2,CD217,CD218a,CD218b,CD222,CD224,CD225,CD226,CD227,CD229,CD232,CD243,CD244,CD245,CD247,CD252,CD253,CD256,Cd257,CD258,CD261,CD262,CD262,CD263,CD264,CD265,CD268,CD269,CD271,CD272,CD273,CD274,CD275,CD276,CD277,CD278,CD279,CD280,CD281,CD282,CD283,CD284,CD286,CD288,CD289,CD290,CD292,CD293,CD294,CD296,CD297,CD300a,CD300b,CD300c,CD300d,CD300e,CD303,CD304,CD305,CD306,CD307a,CD307b,CD307c,CD307d,CD309,CD312,CD314,CD315,CD316,CD317,CD318,CD319,CD320,CD328,CD329,CD335,CD336,CD337,CD352,CD353,CD357,CD361,At least one of CD362; The first cytokine or its receptor, the second cytokine or its receptor each independently include IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-17a, IL-17b, IL-17c, IL-17d, IL-17f, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-37, EPO, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CC At least one of L7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, IFNα, IFNβ, IFNγ, TNFα, TGFβ, VEGF, GM-CSF, GCSF, or their related receptors.

30. The multispecific antibody according to claim 27, wherein The tumor antigen is selected from at least one of AFP, BCMA, β-catetin, CD5, CD19, CD20, CD22, CD30, CD38, CD70, CD123, CD133, CD171, CD138, CDK-4, CEA, CA-125, Caspase 8, c-Met, EGFR, Ep-CAM, EphA2, ETA, FAP, GPC3, GP100, GD2, Her2, HPV16, Igk chain, Ley, Mesothlin, MUC-1, MUC16, MAGE, MART1, NKG2D ligand, CLL1, NY-ESO-1, P53, RAS, OSA, PAP, PSCA, ROR1, ROR2, PSMA, SSTR2, Trop-2, TYR, VEGFR, and WT1; The immune checkpoint is selected from at least one of PD-1, PD-L1, CTLA-4, LAG-3, BTLA, TIM-3, TIGIT, VISTA, TIPE2, Galectin-9, NKG2A, KIRs, B7-H3, B7-H7, 2B4, DNAM-1, PTA1, TLISA1, Vstm3, VSIG9, Siglecs family, GITR, OX40, 4-1BB, LAYN, LIRB1-5 or their related receptors.

31. The multispecific antibody according to claim 27, wherein The second antigen binding portion comprises one or more domains capable of specifically recognizing and binding to CD3, and the first antigen binding portion comprises one or more domains capable of specifically recognizing and binding to the amino acid sequence of Strep tag I or Strep tag II.

32. The multispecific antibody according to claim 27, wherein The second antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are the CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 41, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are the CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ ID NO: 42; and / or The first antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the HCDRs, HCDR2 and HCDR3 are the CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 27, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are the CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ ID NO: 26; Optionally, the second antigen binding portion has the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 35, 36 and 37; and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 38, 39 and 40; the first antigen binding portion has the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 20, 21 and 22; and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 23, 24 and 25; Optionally, the second antigen-binding portion has a heavy chain variable region sequence as shown in SEQ ID NO: 41, 43, 44 or 45 and a light chain variable region sequence as shown in SEQ ID NO: 42, 46, 47, 48, 49, 50, 51, 52 or 53; the first antigen-binding portion has a heavy chain variable region sequence as shown in SEQ ID NO: 27, 29, 30 or 31 and a light chain variable region sequence as shown in SEQ ID NO: 26, 32, 33 or 34; Optionally, the second antigen binding moiety is selected from: (a-1) has the heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:42; or (a-2) having the heavy chain variable region sequence shown in SEQ ID NO: 41 and the light chain variable region sequence shown in SEQ ID NO: 46; or (a-3) having the heavy chain variable region sequence shown in SEQ ID NO: 41 and the light chain variable region sequence shown in SEQ ID NO: 47; or (a-4) having the heavy chain variable region sequence shown in SEQ ID NO: 41 and the light chain variable region sequence shown in SEQ ID NO: 48; or (a-5) has the heavy chain variable region sequence shown in SEQ ID NO: 41 and the light chain variable region shown in SEQ ID NO: 49 sequence; or (a-6) having the heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:50; or (a-7) has the heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:51; or (a-8) has the heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:52; or (a-9) has the heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:53; or (b-1) has the heavy chain variable region sequence shown in SEQ ID NO: 43 and the light chain variable region sequence shown in SEQ ID NO: 42; or (b-2) having the heavy chain variable region sequence shown in SEQ ID NO: 44 and the light chain variable region sequence shown in SEQ ID NO: 42; or (b-3) having the heavy chain variable region sequence shown in SEQ ID NO: 45 and the light chain variable region sequence shown in SEQ ID NO: 42; or (c-1) has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:46; or (c-2) having the heavy chain variable region sequence shown in SEQ ID NO: 43 and the light chain variable region sequence shown in SEQ ID NO: 47; or (c-3) having the heavy chain variable region sequence shown in SEQ ID NO: 43 and the light chain variable region sequence shown in SEQ ID NO: 48; or (c-4) having the heavy chain variable region sequence shown in SEQ ID NO: 43 and the light chain variable region sequence shown in SEQ ID NO: 49; or (c-5) has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:50; or (c-6) has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:51; or (c-7) has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:52; or (c-8) has the heavy chain variable region sequence shown in SEQ ID NO:43 and the light chain variable region sequence shown in SEQ ID NO:53; or (d-1) has the heavy chain variable region sequence shown in SEQ ID NO: 44 and the light chain variable region shown in SEQ ID NO: 46 sequence; or (d-2) having the heavy chain variable region sequence shown in SEQ ID NO: 44 and the light chain variable region sequence shown in SEQ ID NO: 47; or (d-3) having the heavy chain variable region sequence shown in SEQ ID NO: 44 and the light chain variable region sequence shown in SEQ ID NO: 48; or (d-4) has the heavy chain variable region sequence shown in SEQ ID NO: 44 and the light chain variable region sequence shown in SEQ ID NO: 49; or (d-5) has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:50; or (d-6) has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:51; or (d-7) has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:52; or (d-8) has the heavy chain variable region sequence shown in SEQ ID NO:44 and the light chain variable region sequence shown in SEQ ID NO:53; or (e-1) has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:46; or (e-2) having the heavy chain variable region sequence shown in SEQ ID NO: 45 and the light chain variable region sequence shown in SEQ ID NO: 47; or (e-3) having the heavy chain variable region sequence shown in SEQ ID NO: 45 and the light chain variable region sequence shown in SEQ ID NO: 48; or (e-4) has the heavy chain variable region sequence shown in SEQ ID NO: 45 and the light chain variable region sequence shown in SEQ ID NO: 49; or (e-5) has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:50; or (e-6) has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:51; or (e-7) has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:52; or (e-8) has the heavy chain variable region sequence shown in SEQ ID NO:45 and the light chain variable region sequence shown in SEQ ID NO:53; Optionally, the first antigen binding moiety is selected from: (1-1) has the heavy chain variable region sequence shown in SEQ ID NO: 27 and the heavy chain variable region sequence shown in SEQ ID NO: 26; or (1-2) having the heavy chain variable region sequence shown in SEQ ID NO: 27 and the heavy chain variable region sequence shown in SEQ ID NO: 32; or (1-3) having the heavy chain variable region sequence shown in SEQ ID NO: 27 and the heavy chain variable region sequence shown in SEQ ID NO: 33; or (1-4) having the heavy chain variable region sequence shown in SEQ ID NO: 27 and the heavy chain variable region sequence shown in SEQ ID NO: 34; or (1-5) having the heavy chain variable region sequence shown in SEQ ID NO: 29 and the heavy chain variable region sequence shown in SEQ ID NO: 26; or (1-6) has the heavy chain variable region sequence shown in SEQ ID NO: 30 and the heavy chain variable region sequence shown in SEQ ID NO: 26; or (1-7) has the heavy chain variable region sequence shown in SEQ ID NO: 31 and the heavy chain variable region sequence shown in SEQ ID NO: 26; or (1-8) has the heavy chain variable region sequence shown in SEQ ID NO: 29 and the light chain variable region sequence shown in SEQ ID NO: 32; or (1-9) has the heavy chain variable region sequence shown in SEQ ID NO: 29 and the light chain variable region sequence shown in SEQ ID NO: 33; or (1-10) has the heavy chain variable region sequence shown in SEQ ID NO: 29 and the light chain variable region sequence shown in SEQ ID NO: 34; or (1-11) has the heavy chain variable region sequence shown in SEQ ID NO: 30 and the light chain variable region sequence shown in SEQ ID NO: 32; or (1-12) has the heavy chain variable region sequence shown in SEQ ID NO: 30 and the light chain variable region sequence shown in SEQ ID NO: 33; or (1-13) has the heavy chain variable region sequence shown in SEQ ID NO: 30 and the light chain variable region sequence shown in SEQ ID NO: 34; or (1-14) has the heavy chain variable region sequence shown in SEQ ID NO: 31 and the light chain variable region sequence shown in SEQ ID NO: 32; (1-15) has the heavy chain variable region sequence shown in SEQ ID NO: 31 and the light chain variable region sequence shown in SEQ ID NO: 33; or (1-16) has the heavy chain variable region sequence shown in SEQ ID NO: 31 and the light chain variable region sequence shown in SEQ ID NO:

34.

33. The multispecific antibody according to claim 32, characterized in that The first antigen binding moiety and the second antigen binding moiety are linked by: 1) the heavy chain variable region and light chain variable region of the second antigen-binding moiety are linked to the heavy chain constant region and light chain constant region, respectively, to form an IgG-like structure, the carboxyl-terminal amino acid of the Fc region of the IgG-like structure is linked to the amino-terminal amino acid of the light chain variable region of the first antigen-binding moiety, and the carboxyl-terminal amino acid of the light chain variable region of the first antigen-binding moiety is linked to the amino-terminal amino acid of the heavy chain variable region of the first antigen-binding moiety; or 2) the heavy chain variable region and light chain variable region of the first antigen-binding moiety are respectively linked to the heavy chain constant region and the light chain constant region to form an IgG-like structure, the carboxyl-terminal amino acid of the Fc region of the IgG-like structure is linked to the amino-terminal amino acid of the light chain variable region of the second antigen-binding moiety, and the carboxyl-terminal amino acid of the light chain variable region of the second antigen-binding moiety is linked to the amino-terminal amino acid of the heavy chain variable region of the second antigen-binding moiety; or 3) the carboxyl-terminal amino acid of the light chain variable region of the first antigen-binding moiety is linked to the amino-terminal amino acid of the heavy chain variable region of the first antigen-binding moiety, the carboxyl-terminal amino acid of the first antigen-binding moiety is directly linked to the Fc region, the carboxyl-terminal amino acid of the Fc region is linked to the amino-terminal amino acid of the second antigen-binding moiety, and the carboxyl-terminal amino acid of the light chain variable region of the second antigen-binding moiety is linked to the amino-terminal amino acid of the heavy chain variable region of the second antigen-binding moiety; or 4) the carboxyl-terminal amino acid of the light chain variable region of the first antigen-binding moiety is linked to the amino-terminal amino acid of the heavy chain variable region of the first antigen-binding moiety, the carboxyl-terminal amino acid of the first antigen-binding moiety is directly linked to the Fc region, the carboxyl-terminal amino acid of the Fc region is linked to the amino-terminal amino acid of the second antigen-binding moiety, and the carboxyl-terminal amino acid of the heavy chain variable region of the second antigen-binding moiety is linked to the amino-terminal amino acid of the light chain variable region of the second antigen-binding moiety.

34. The multispecific antibody according to claim 33, characterized in that The Fc region is a humanized sequence or a mutated humanized sequence.

35. A polynucleotide, characterized in that The polynucleotide encodes the multispecific antibody of any one of claims 27-34.

36. A construct, characterized in that The construct comprises the polynucleotide of claim 35.

37. A host cell, characterized in that The host cell comprises the construct of claim 36; Optionally, the host cell is a prokaryotic cell or a eukaryotic cell.

38. A method for producing the multispecific antibody according to any one of claims 27 to 34, characterized in that: include: Cultivating the host cell of claim 37, and The multispecific antibody is collected from the culture.

39. Use of the therapeutic agent according to any one of claims 1 to 26 or the multispecific antibody according to any one of claims 27 to 34 in the preparation of a medicament for treating tumors and / or cancer.

40. The use according to claim 39, wherein the tumor and / or cancer comprises: Breast cancer, head and neck cancer, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, Liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, von At least one of the following: Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown primary site, carcinoid tumor, fibrosarcoma, Paget's disease, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multiple myeloma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymic cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, peritoneal cancer, pleural cancer, and blood cancer.

41. A kit of synergistically effective combination drugs for treating tumors and / or cancers, comprising: a first container containing a first composition of the therapeutic agent according to any one of claims 1 to 26; a second container containing a second composition of the therapeutic agent according to any one of claims 1 to 26 or a multispecific antibody according to any one of claims 27 to 34, wherein the first container and the second container are separate; and Instructions stating the timing and method of administration.

42. A method for treating tumors and / or cancers, comprising: administering to a subject a first composition of the therapeutic agent of any one of claims 1-26; and The subject is administered a second composition of the therapeutic agent of any one of claims 1-26, or administered a multispecific antibody of any one of claims 27-34.

43. The method according to claim 42, comprising the following steps performed in sequence: 1) administering the first composition to the subject; and 2) After administering the first composition, administering the second composition or the multispecific antibody to the subject.

44. The method of claim 43, wherein the tumor and / or cancer comprises: Breast cancer, head and neck cancer, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, Liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown primary site, carcinoid tumor, fibrosarcoma, Paget's disease, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multiapical myeloma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymic cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, At least one of vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, peritoneal cancer, pleural cancer, and blood cancer.

45. An immunotherapy, characterized in that include: Administering a therapeutically effective amount of an oncolytic virus to a subject, wherein the genome of the oncolytic virus contains a coding sequence for a marker polypeptide, wherein the marker polypeptide has an operably linked extracellular antigenic determinant, a spacer portion, and a transmembrane portion, wherein the amino acid sequence of the extracellular antigenic determinant contains the amino acid sequence of one or more antigenic epitope polypeptides, and in a natural state, the amino acid sequence of the cell membrane protein or secretory protein of the subject does not contain the amino acid sequence of the antigenic epitope polypeptide; and administering to the subject a therapeutically effective amount of a multispecific antibody, wherein the multispecific antibody comprises at least a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion is capable of specifically recognizing and binding to the extracellular antigenic determinant region of the marker polypeptide, and the second antigen-binding portion is capable of specifically recognizing and binding to an immune cell antigen or a first cytokine or a receptor thereof; The multispecific antibody may also optionally include a third antigen binding portion, which is capable of specifically recognizing and binding to a tumor antigen or an immune checkpoint or a second cytokine or its receptor.

46. ​​The method according to claim 45, characterized in that The method includes the following steps: 1) administering a therapeutically effective amount of the oncolytic virus to the subject; and 2) After administering the oncolytic virus, administering a therapeutically effective amount of the multispecific antibody to the subject.

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