Therapeutic agent containing a multispecific antibody and its use in tumor treatment

Abstract

The present invention provides a therapeutic agent comprising a labeled polypeptide to be introduced into tumor cells and a multispecific antibody capable of recognizing and binding to the labeled polypeptide, the multispecific antibody, a kit comprising the therapeutic agent, and the use of the therapeutic agent or the multispecific antibody in the manufacture of a therapeutic agent for tumors and / or cancers.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically, to a therapeutic agent containing a multispecific antibody and its use in tumor treatment.

Background Art

[0002] According to data released by the World Health Organization (WHO), tumors are one of the most serious diseases threatening human health, with the number of affected people continuing to increase and the mortality rate being high. However, there are still many unmet clinical needs in conventional tumor treatment methods. Oncolytic virus therapy provides a novel tumor immunotherapy. Oncolytic viruses selectively replicate in tumor cells, directly lyse and destroy tumor cells, and stimulate the host's anti-tumor immune response to kill tumor cells, while not affecting the growth of normal cells, bringing hope to many patients with malignant tumors (Shi T et al., Front Immunol 2020; 11: 683). Compared with other tumor immunotherapies, oncolytic virus therapy exhibits various advantages such as high tumor killing efficiency, good targeting, low drug resistance, and low cost (Heo et al., 2012).

[0003] Similar to other anti-tumor therapies, oncolytic virus therapy also has some limitations, such as viral delivery to the target, penetration into the tumor mass, and induction of a strong anti-viral immune response, and is restricted by physical barriers and the host's immunity. Therefore, in oncolytic virus therapy, generally, it is necessary to combine chemotherapy or immune drugs to improve the therapeutic effect. Worldwide, most of the oncolytic virus drugs that have been launched or are under development are conducting combination administration experiments, such as in combination with immune checkpoint or CAR-T cell therapy.

[0004] Bispecific antibody (BsAb) is an antibody that can specifically bind to two antigens or epitopes simultaneously. Since Nisonoff and his co-researchers first proposed the concept of BsAb in 1960, bispecific antibodies have evolved rapidly as recombinant antibody technology has gradually matured. Because BsAbs can target multiple antigens or epitopes, they not only exhibit more advantages compared to monoclonal antibodies but also gradually show higher efficacy than combination therapies with monoclonal antibodies. For example, by redirecting specific effector cells to adjacent tumor cells, tumor killing can be enhanced, and the binding specificity can be increased through the interaction of two different cell surface antigens. Also, compared to the combination of single antibodies, the development cost and clinical trials can be reduced. Bispecific antibodies are currently a hot spot in antibody engineering research and are expected to be widely used in fields such as tumor treatment and autoimmune diseases. Moreover, as technology progresses, antibodies targeting more antigens or more epitopes, such as trispecific antibodies or multifunctional fusion antibodies, have emerged.

[0005] Although various therapies are emerging one after another, clinically, unmet needs still exist, and there is an urgent need to develop new immunotherapies.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The present invention provides a therapeutic agent comprising a multispecific antibody and its use in tumor treatment. By utilizing the first active ingredient in the therapeutic agent, a labeled polypeptide is carried on tumor cells or cancer cells, and the labeled polypeptide labeled on the tumor cells or cancer cells is recognized by using the multispecific antibody. Another antigen-binding portion of the multispecific antibody can mobilize immune cells (e.g., T cells, NK cells, etc.) to achieve the killing of tumor cells or cancer cells. The therapeutic effect of tumor treatment can be significantly improved by using the therapeutic agent provided by the present invention. Furthermore, by administering an oncolytic virus to carry a labeled polypeptide on tumor cells or cancer cells, the tumor killing effect can be achieved before the administration of the multispecific antibody, and the purpose of significantly improving tumor treatment after the administration of the multispecific antibody can be achieved. Therefore, the present invention also provides a new immunotherapy, which includes 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 the combined use of oncolytic virus therapy and multispecific antibody therapy. The immunotherapy provided by the present invention provides a novel therapy with progressiveness in the treatment of tumors.

Means for Solving the Problems

[0007] Specifically speaking, the present invention provides the following technical solutions. The first aspect of the present invention provides a therapeutic agent, which (a) a first composition comprising a first active ingredient, the first active ingredient comprising or containing a nucleic acid having a labeled polypeptide coding sequence to be introduced into tumor cells and / or cancer cells, the labeled polypeptide having an extracellular antigen determinant, a spacer portion, and a transmembrane portion operably linked thereto, the amino acid sequence of the extracellular antigen determinant containing the amino acid sequence of one or more epitope polypeptides, and in the natural state, the amino acid sequence of a mammalian cell membrane protein or secreted protein not containing the amino acid sequence of the epitope polypeptide; and (b) A second composition comprising a second active ingredient, wherein the second active ingredient comprises a multispecific antibody, the multispecific antibody comprises at least a first antigen-binding portion and a second antigen-binding portion, and the first antigen-binding portion can specifically recognize and bind to the extracellular antigen determinant of the labeled polypeptide; and the second composition. The second antigen-binding portion can specifically recognize and bind to an immune cell antigen or a first cytokine or its receptor. The multispecific antibody may further selectively comprise a third antigen-binding portion, and the third antigen-binding portion can specifically recognize and bind to a tumor antigen or an immune checkpoint or a second cytokine or its receptor.

[0008] The second aspect of the present invention provides a multispecific antibody, which comprises at least a first antigen-binding portion and a second antigen-binding portion. The first antigen-binding portion comprises one or more domains that can specifically recognize and bind to the amino acid sequence of Strep-tag I or Strep-tag II. The second antigen-binding portion comprises one or more domains that can specifically recognize and bind to an immune cell antigen or a first cytokine or its receptor. The multispecific antibody may further selectively comprise a third antigen-binding portion, and the third antigen-binding portion comprises one or more domains that can specifically recognize and bind 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, and it has been proven that these tags do not exist or hardly exist in the mammalian body. However, by carrying tags corresponding to mammalian, especially human, tumor cells, the first antigen-binding portion can specifically target these tags, thereby enabling the multispecific antibody to specifically target tumor cells. Next, by mobilizing immune cells or cytokines or their receptors with the second antigen-binding portion, tumor cells are specifically killed, thereby obtaining the effect of specifically targeting and killing tumors. The multispecific antibody may optionally further comprise a third antigen-binding portion that can specifically target a tumor antigen or an immune checkpoint or a cytokine or its receptor, further exerting the effect of targeting and killing tumors.

[0009] The third aspect of the present invention provides a polynucleotide encoding the multispecific antibody according to the second aspect of the present invention.

[0010] The fourth aspect of the present invention provides a construct comprising the polynucleotide according to the third aspect of the present invention.

[0011] The fifth aspect of the present invention provides a host cell comprising the construct according to the fourth aspect of the present invention.

[0012] A sixth aspect of the present invention provides a method for producing a multispecific antibody according to the second aspect, which includes culturing the host cell according to the fifth aspect and recovering the multispecific antibody from the culture.

[0013] A seventh aspect of the present invention provides the use of a therapeutic agent or a multispecific antibody in the manufacture of a drug for treating a tumor and / or cancer, wherein the therapeutic agent mentioned 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.

[0014] An eighth aspect of the present invention provides a kit having a synergistic combination agent for treating a tumor and / or cancer, which includes a first container containing the first composition in the therapeutic agent according to the first aspect, and a second container containing the second composition in the therapeutic agent according to the first aspect or the multispecific antibody described in the second aspect, wherein the first container and the second container are independent, and an instruction manual indicating the timing and method of administration.

[0015] A ninth aspect of the present invention provides a method for treating a tumor and / or cancer, which includes administering the first composition in the therapeutic agent according to any one of the first aspects to a subject, and administering the second composition in the therapeutic agent according to the first aspect or the multispecific antibody described in the second aspect to the subject.

[0016] A tenth aspect of the present invention provides a novel immunotherapy, which is administering a therapeutically effective amount of an oncolytic virus to a subject, wherein the genome of the oncolytic virus contains a labeled polypeptide coding sequence, the labeled polypeptide has an extracellular antigen determinant, a spacer portion, and a transmembrane portion that are operably linked, the amino acid sequence of the extracellular antigen determinant contains the amino acid sequence of one or more epitope polypeptides, and in the natural state, the amino acid sequence of the cell membrane protein or secreted protein of the subject does not contain the amino acid sequence of the epitope polypeptide. Administering a therapeutically effective amount of a multispecific antibody to the subject, wherein the multispecific antibody comprises 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 antigen determinant of the labeled polypeptide, and 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 optionally further comprise a third antigen-binding portion, the third antigen-binding portion being capable of specifically recognizing and binding to a tumor antigen or an immune checkpoint or a second cytokine or its receptor.

Advantages of the Invention

[0017] The present invention has obtained the following beneficial technical effects. The present invention provides a therapeutic agent containing a first composition and a second composition. The first composition contains a first active ingredient that labels the surface of a tumor and / or cancer cells with an exogenous labeled polypeptide. The multivalent antibody in the second composition recognizes the labeled polypeptide, and by using both in combination, tumor cells can be effectively killed. Further, when an oncolytic virus is used as a vector to mediate the expression of an exogenous labeled polypeptide in tumor cells, in addition to the oncolytic killing effect inherent to the oncolytic virus, the multivalent antibody targeting the labeled polypeptide can effectively remove the remaining tumor cells infected by the oncolytic virus. Further, since the oncolytic virus disrupts the tumor microenvironment, the killing ability of the multivalent antibody against tumors is further improved, thereby significantly improving the effectiveness of tumor treatment, particularly the treatment effect against solid tumors. Specifically, the present invention utilizes the amino acid sequence of a labeled polypeptide having an extracellular antigen determinant, a spacer portion, and a transmembrane portion, and a nucleic acid encoding the labeled polypeptide, transfects the nucleic acid into tumor cells and / or cancer cells, or inserts the nucleic acid into a viral genome, and after infecting the obtained recombinant virus into tumor cells and / or cancer cells, the tumor cells and / or cancer cells can express the labeled polypeptide, and the labeled polypeptide is finally modified on the surface of the tumor cells and / or cancer cells. Since the amino acid sequence of the extracellular antigen determinant of the labeled polypeptide contains the amino acid sequence of one or more epitope polypeptides, the problems of uneven expression of solid tumor antigens and tumor immune surveillance evasion can be effectively solved. Further, in combination with a multivalent antibody capable of recognizing the extracellular antigen determinant, by using the multivalent antibody to recognize and bind to immune cells, the connection between immune cells and tumor cells is realized, and tumor cells are specifically killed. Further, compared with the killing by immune cells modified with CAR, the killing by immune cells mediated by a multivalent antibody can greatly reduce the cost, toxicity, and side effects of the drug.In particular, CAR-T cells have a long survival time in the body. The continuous presence of CAR-T cells targeting the labeled polypeptide may interfere with multiple administrations of the nucleic acid or recombinant virus encoding the labeled polypeptide later. However, bispecific antibodies have a certain half-life and do not continue to exist in the body. Therefore, by adjusting the administration period and frequency, the drug concentration of bispecific antibodies in the body can be maintained more flexibly.

[0018] Furthermore, using oncolytic virus as a vector and taking advantage of the ability of oncolytic virus to specifically replicate in tumor cells, the nucleic acid encoding the labeled polypeptide is introduced into tumor cells and / or cancer cells. Oncolytic virus can not only kill tumor cells and / or cancer cells, but also significantly enhance the expression of the above-mentioned epitope peptide on the surface of tumor cells. Moreover, in combination with immune cells mobilized by bispecific antibodies, it shows a synergistic therapeutic effect. More importantly, it avoids the on-target off-tumor effect on normal tissues. Oncolytic virus destroys the tumor microenvironment and further improves the killing ability of bispecific antibodies against tumor cells, thereby improving the effectiveness of tumor (especially solid tumor) treatment. In addition, after being infected with oncolytic virus, bispecific antibodies can effectively remove lysed tumor cells without completing the replication phase and generating a sufficient number of progeny viruses, thus realizing a further synergistic effect. Also, the antigen released from tumor cells lysed by oncolytic virus can further activate the anti-tumor immunity in the body, thereby achieving a better tumor killing effect than the single use of oncolytic virus or bispecific antibodies alone and realizing a synergistic therapeutic effect.

[0019] In a further invention, the present invention has improved the amino acid sequence and / or protein structure of the multispecific antibody targeting the labeled polypeptide, which includes humanized modifications to the first antigen-binding portion, the second antigen-binding portion, and the heavy chain / light chain constant domains for reducing the immunogenicity of the therapeutic agent of the present invention. Also, by modifying the first antigen-binding portion and the second antigen-binding portion of the multispecific antibody, the affinity of at least these two portions for the target antigen has been improved, whereby the multispecific antibody can not only effectively achieve an antitumor effect, but also avoid safety problems such as an immune rampage caused by excessive affinity for immune cell antigens. For example, the humanized modification to the first antigen-binding portion targeting the labeled polypeptide ensures the affinity of the labeled polypeptide for the target antigen and effectively reduces the possible immunogenicity, thereby alleviating the risk of occurrence in the human body. Also, it maintains a very high level of affinity for the target antigen of the labeled polypeptide, thereby ensuring the tumor-killing ability of the multispecific antibody. Further, by the humanized modification to the second antigen-binding portion targeting the immune cell antigen, when the multispecific antibody mobilizes immune cells, it can maintain the affinity for the target antigen on the immune cells at an appropriate level, thereby realizing the killing of tumors and avoiding the occurrence of safety problems. Through the design and screening of the present invention, the entire multispecific antibody exhibits particularly appropriate affinity. The modified multispecific antibody has excellent antitumor effects and safety.

Brief Description of the Drawings

[0020]

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[0021] Note that the same numerical numbers (for example, 1, 2, 3, etc.) in Figures 11, 12, 3, 17, etc. do not exactly correspond to the same content, and the meanings represented by the numerical numbers in each figure are as described above.

Modes for Carrying Out the Invention

[0022] Hereinafter, the technical solution of the present invention will be described in detail using specific examples. Also, for the convenience of those skilled in the art, some terms of the present invention will be interpreted and explained. Note that these interpretations and explanations are only for the further understanding of those skilled in the art and cannot be regarded as a limitation on the protection scope of the present invention.

[0023] As used in this specification and the claims, the terms "one" and "said" include cases of multiple referents unless otherwise clearly indicated in the context. The terms "tumor", "cancer", "tumor cell", and "cancer cell" cover their general meanings in the art.

[0024] As used herein, the term "oncolytic virus" refers to a virus that can selectively replicate in tumor cells and lyse tumor cells.

[0025] As used herein, the term "therapeutically effective amount" refers to an amount of a drug, formulation, or active ingredient that can exhibit a measurable therapeutic or inhibitory effect. The measurable therapeutic or inhibitory effect mentioned may be measured by any measurement method known in the art.

[0026] The terms "first", "second", and "third" are used only for description and cannot be understood as indicating or suggesting relative importance or implicitly indicating the number of technical features of the subject, nor do they represent an order. For example, a first composition and a second composition, a first active ingredient and a second active ingredient, etc. neither represent an order nor importance, but are only used for distinction.

[0027] As used herein, the term "MOI" or "Multiplicity of infection" refers to the ratio of virus to the number of cells and indicates the number of virus particles infecting per cell. MOI = pfu / cell. PFU (Plaque forming unit) is a plaque forming unit and is used to describe the number of virus particles with infectious activity.

[0028] The present invention provides a therapeutic agent, which (a) A first composition comprising a first active ingredient, wherein the first active ingredient comprises or contains a nucleic acid having a labeled polypeptide coding sequence to be introduced into tumor cells and / or cancer cells, and the labeled polypeptide has an extracellular antigen determinant, a spacer portion, and a transmembrane portion operably linked thereto, and the amino acid sequence of the extracellular antigen determinant contains the amino acid sequence of one or more epitope polypeptides, and in the natural state, the amino acid sequence of a mammalian cell membrane protein or secreted protein does not contain the amino acid sequence of the epitope polypeptide. A first composition; (b) A second composition comprising a second active ingredient, wherein the second active ingredient comprises a multispecific antibody, the multispecific antibody comprises at least a first antigen-binding portion and a second antigen-binding portion, and the first antigen-binding portion can specifically recognize and bind to the extracellular antigen determinant of the labeled polypeptide. A second composition; The second antigen-binding portion can specifically recognize and bind to an immune cell antigen or a first cytokine or its receptor; The multispecific antibody may further selectively include a third antigen-binding portion, and the third antigen-binding portion can specifically recognize and bind to a tumor antigen or an immune checkpoint or a second cytokine or its receptor.

[0029] For easy understanding, FIG. 1 shows a schematic diagram of the therapeutic effect exerted by the provided therapeutic agent. In FIG. 1, an IgG-like bispecific antibody is taken as an example, and TT3 is taken as an example of the labeled polypeptide for explanation. The artificially synthesized epitope polypeptide (the epitope polypeptide forms a spacer part, a transmembrane part and a TT3-labeled polypeptide as at least one part of the extracellular antigen determinant) that is not contained in the amino acid sequence of mammalian cell membrane proteins or secreted proteins is carried to tumor cells and labeled on the tumor cell surface. The bispecific antibody can specifically recognize and bind to TT3, while it can specifically recognize and bind to immune cells (for example, T cells and NK cells shown in the figure). By connecting immune cells and tumor cells with the bispecific antibody, redirected tumour lysis is realized, the immune cells are activated to generate cytotoxic molecules such as perforin and granzymes, and the tumor cells are killed, thus exerting a specific killing effect. It should be noted that FIG. 1 only takes the IgG-like bispecific antibody as an example, and the multispecific antibodies mentioned in this specification do not necessarily represent a specific IgG-type structure.

[0030] When a tumor-lytic virus is used as a vector and the labeled polypeptide is expressed in tumor cells or cancer cells by utilizing the specific replication of the tumor-lytic virus in tumors, the tumor-lytic virus not only exerts a killing effect on tumor cells or cancer cells, but also significantly enhances the expression of the epitope peptide on the tumor cell surface. Furthermore, it shows a synergistic therapeutic effect in combination with immune cells connected by the antibody.

[0031] (Labeled polypeptide) The labeled polypeptides referred to in this specification are described in the patent application with international application number PCT / CN2019 / 102480 and the PCT patent application with international publication number WO2020038490A, and since this is necessary, all or part of it is cited herein. The labeled polypeptides referred to are for modifying the surface of tumor cells and / or cancer cells, have an extracellular antigen determinant, a spacer portion, and a transmembrane portion operably linked, and can be modified on the surface of tumor cells and / or cancer cells by expression. The amino acid sequence of the extracellular antigen determinant contains the amino acid sequence of one or more epitope polypeptides. In addition, in the natural state, the amino acid sequence of mammalian cell membrane proteins or secreted proteins does not contain the amino acid sequence of the epitope polypeptides.

[0032] As used herein, the term "extracellular antigen determinant" refers to the portion containing the epitope polypeptide located outside the cell membrane when the labeled polypeptide is expressed on the cell surface.

[0033] According to an embodiment of the present invention, the amino acid sequence of the epitope polypeptide is derived from the amino acid sequence of a naturally occurring protein or is a synthetic amino acid sequence that does not naturally exist. Among them, the naturally occurring proteins include proteins in mammalian cells and proteins derived from organisms other than mammals. Proteins derived from organisms other than mammals include viral proteins, bacterial proteins, fungal proteins, protozoan proteins, plant proteins, insect proteins, and proteins derived from animals other than mammals.

[0034] In some embodiments of the present invention, the amino acid sequence of the epitope polypeptide is derived from the amino acid sequences such as Myc tag, HA tag, Strep tag I, Strep tag II, Flag tag, HAT tag, S tag, Sl 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. The amino acid sequences and nucleotide sequences of these tags are all known and can be searched in publicly available databases commonly used in the art.

[0035] In a preferred embodiment of the present invention, the amino acid sequence of the epitope polypeptide is derived from the amino acid sequence of a human Myc tag (the corresponding labeled polypeptide is denoted as TT1), an influenza virus HA tag (the corresponding labeled polypeptide is denoted as TT2), a Strep tag II (the corresponding labeled polypeptide is denoted as TT3), or a Strep tag I. The Strep tag I or Strep tag II mentioned herein has an amino acid sequence length of at least 3 amino acids, 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, 15 amino acids, and may contain amino acids derived from the sequence represented by NWSHPQFEK (SEQ ID NO: 59) or AWRHPQFGG (SEQ ID NO: 60), and may be a partial sequence or a combination of partial sequences of the sequence represented by SEQ ID NO: 59 or SEQ ID NO: 60. More specifically, the amino acid sequence of Strep tag II may be, for example, the one represented by WSHPQFEK (SEQ ID NO: 58), NWSHPQFEK (SEQ ID NO: 59). The amino acid sequence of the Strep tag I mentioned herein may be, for example, the one represented by AWRHPQFGG (SEQ ID NO: 60). Preferably, the amino acid sequence of the epitope polypeptide coincides with the amino acid sequence at positions 410-419 of the human Myc protein, or the amino acid sequence of the epitope polypeptide coincides with the amino acid sequence at positions 119-127 of the influenza virus HA protein, or the amino acid sequence of the epitope polypeptide coincides with Strep tag II (for a detailed description of Strep tag II, refer to the literature "Molecular Interaction Between the Strep-tag Affinity Peptide and its Cognate Target, Streptavidin, Thomas G. M. Schmidt, Jurgen Koepke, Ronald Frank and Arne Skerra"). In some embodiments of the present invention, the amino acid sequences of the epitope polypeptides of TTl and TT2 may be extended by no more than 10 amino acids upstream and downstream of the above sequences.The amino acid sequence of the human Myc protein may be the amino acid sequence of No. P0l106 isoforml in UniProtKB. The amino acid sequence of the influenza virus HA protein may be the amino acid sequence of No. Q03909 in UniProtKB.

[0036] In a specific embodiment, the amino acid sequence of the extracellular antigen determinant of the labeled polypeptide includes the amino acid sequence of one or more of the above epitope polypeptides. Here, when the amino acid sequence of the extracellular antigen determinant of the labeled polypeptide includes the amino acid sequences of a plurality of the above epitope polypeptides, two adjacent epitope polypeptides are operably linked, for example, they may be linked using a linker or directly linked without using a linker. The amino acid sequence of the linker may be, for example, G (used in labeled polypeptide Cl&2a and Cl&2b), GGS (used in Cl&2a and Cl&2b), GGGGSGGGGS (used in TT1 to TT3).

[0037] In order to enhance the immunogenicity of the labeled polypeptide, the extracellular antigenic determinant of the labeled polypeptide preferably contains n epitope polypeptides, where n is an integer of 1 or more. For example, n is 1, 2, 3, 4, etc. Preferably, n is an integer between 1 and 10, more preferably, n is an integer between 2 and 5, and even more preferably, n is 2 or 3. For example, the extracellular antigenic determinant of the labeled polypeptide may contain three repeats of an epitope polypeptide derived from the Myc tag (see, for example, TT1), or three repeats of an epitope polypeptide derived from the HA tag (see, for example, TT2), or three repeats of an epitope polypeptide derived from the Strep tag II (see, for example, TT3), or three repeats of an epitope polypeptide derived from the Myc tag and three repeats of an epitope polypeptide derived from the HA tag (see, for example, Cl&2a), or two repeats of an epitope polypeptide derived from the Myc tag and two repeats of an epitope polypeptide derived from the HA tag (see, for example, Cl&2b).

[0038] In one embodiment of the present invention, the amino acid sequence of the extracellular antigenic determinant is represented by 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).

[0039] Preferably, the transmembrane portion is derived from the transmembrane region of CD8, CD3ζ, CD4 or CD28, the full-length amino acid sequence and nucleotide sequence of which are both known and can be searched in publicly available databases commonly used in the art. Preferably, the transmembrane portion is derived from the transmembrane region of human CD8α. More preferably, the amino acid sequence of the transmembrane portion includes the amino acid sequence shown in SEQ ID NO: 7. CD8 is a transmembrane glycosylated membrane protein composed of two subunits, α and β, and acts in conjunction with the T cell surface receptor to bind a specific antigen to the T cell and specifically bind CD8 to MHC I, mediating the killing effect of cytotoxic T cells. The transmembrane region is generally a hydrophobic α-helix spanning the cell membrane.

[0040] In the labeled polypeptide of the present invention, the transmembrane portion and the extracellular antigen determinant may be linked by a spacer portion. The structure of this region preferably has flexibility, whereby the extracellular antigen determinant can be oriented in various directions, promoting the recognition and binding of the corresponding multispecific antibody. The simplest form of the spacer portion is the hinge region of immunoglobulin IgGl, immunoglobulin CH 2 CH 3It may be a part of the region. Through research and experiments, the following findings were obtained. The spacer portion 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 portion is shown by SEQ ID NO: 6. More preferably, the spacer portion and the transmembrane portion constitute a spacer transmembrane portion, and the amino acid sequence of the spacer transmembrane portion is identical to the amino acid sequence at positions Y to 210 of CD8α, where 118 ≤ Y ≤ 128 and Y is an integer. Here, the UniProtKB number of the amino acid sequence of CD8α may be P01732. That is, the amino acid sequence of the spacer transmembrane portion is preferably selected from the amino acids at positions 118 to 210 of CD8α and includes the amino acids at positions 128 to 210. For example, the amino acid sequence of the spacer transmembrane portion is the amino acid sequence of the amino acids at positions 118 to 210 of CD8α, 119 to 210 of CD8α, 120 to 210 of CD8α, 121 to 210 of CD8α, 122 to 210 of CD8α, 123 to 210 of CD8α, 124 to 210 of CD8α, 125 to 210 of CD8α, 126 to 210 of CD8α, 127 to 210 of CD8α, or the amino acids at positions 128 to 210 of CD8α, and is represented by any amino acid sequence in the amino acid sequence group.

[0041] The definitions of the terms "spacer portion" and "transmembrane portion" used in this specification are known in the art. Specifically, reference may be made to "Introduction to Immunology, Yu Shanqian, Higher Education Press, 2008" and "Immunobiology, Seventh Edition, Kenneth Murphy, Paul Travers, Mark Walport et al.".

[0042] A nucleic acid having the labeled polypeptide coding sequence of the present invention, preferably further comprising a signal peptide coding sequence before the 5' end of the labeled polypeptide coding sequence, wherein the signal peptide has a function of inducing the secretion of the target protein to the cell surface. The following has been found in the present invention. When the extracellular antigen determinant is combined with a signal peptide derived from the GM-CSFα chain, the labeled polypeptide can be expressed on the surface of tumor cells. The GM-CSFα chain signal peptide is a leader sequence that targets the labeled polypeptide of the present invention to the secretory pathway, and its coding sequence is first translated as a protein intracellularly together with the labeled polypeptide coding sequence, and the synthesized protein is induced to enter the intracellular secretory pathway. Before the labeled polypeptide is expressed on the cell surface, the signal peptide is removed. The full-length amino acid sequence and nucleotide sequence of the GM-CSFα chain are both known and can be searched in publicly available databases commonly used in the art. Preferably, the amino acid sequence of the signal peptide is selected from the amino acids at positions 1 to 22 of the human GM-CSFα chain. More preferably, the amino acid sequence of the signal peptide is the one represented by SEQ ID NO: 8. The amino acid sequence of the GM-CSFα chain is derived from UniProtKB-P15509.

[0043] In a specific embodiment of the present invention, the labeled polypeptide comprises an extracellular antigen determinant (including the amino acid sequence of one or more of the epitope polypeptides), a spacer portion, and a transmembrane portion, which are operably linked and sequentially connected. In a more specific embodiment of the present invention, the amino acid sequence of the epitope polypeptide of the extracellular antigen determinant is derived from the Myc tag derived from the human intracellular protein Myc, the HA tag derived from the influenza virus HA protein, and the epitope polypeptide in the polypeptide which is the 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.

[0044] As described above, the signal peptide, extracellular antigen determinant, extracellular antigen determinant and spacer portion, and spacer portion and transmembrane portion are operably linked, and may be linked, for example, using a linker, or may be directly linked without using a linker. In one embodiment of the present invention, the signal peptide and the extracellular antigen determinant are linked using a linker, for example, GAHADITS (used in TT1 to TT3), GAHAAQLTLTKGNK (used in Cl&2a), GAHA (used in Cl&2b). The extracellular antigen determinant and the spacer portion are linked using a linker, for example, -Ala-Ser-, G, and the spacer portion and the transmembrane portion are directly linked without using a linker.

[0045] In the present invention, the following has further been found. The labeled polypeptide can simultaneously express two types of epitope polypeptides. Specifically, in the present invention, a labeled polypeptide Cl&2a in which the antigen determinant contains three repeats of each of the Myc tag and the HA tag is designed. In the present invention, further, a labeled polypeptide Cl&2b in which the antigen determinant contains two repeats of each of the Myc tag and the HA tag is designed. In order to further stabilize the expression of the labeled polypeptide on the cell membrane surface, the spacer region of TIGIT is added to the spacer portion of Cl&2b. Here, the amino acid sequence of the added spacer region corresponds to the amino acid sequence at positions 24 to 140 of TIGIT. Here, the amino acid sequence of TIGIT is derived from UniProtKB-Q495Al. More preferably, the amino acid sequence of the added TIGIT spacer is shown by SEQ ID NO: 9, and the amino acid sequence of the spacer transmembrane portion of Cl&2b is shown by SEQ ID NO: 10.

Table 1

[0046] Preferably, the amino acid sequence of the labeled polypeptide is represented by 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).

[0047] SEQ ID NO: 11:

Chemical Structure

[0048] SEQ ID NO: 12:

Chemical Structure

[0049] SEQ ID NO: 13:

Chemical Structure

[0050] SEQ ID NO: 14:

Chemical Structure

[0051] SEQ ID NO: 15:

Chemical Structure

[0052] (Nucleic acid having a labeled polypeptide coding sequence) When obtaining the above-mentioned labeled polypeptide coding sequence, according to the general rules in the art, the coding sequences encoding these labeled polypeptides may be obtained. The nucleic acid having the labeled polypeptide coding sequence includes DNA or RNA, and the RNA includes mRNA transcribed from the DNA. The nucleic acid contains a promoter operably linked and the labeled polypeptide coding sequence in this order. Examples of the promoter include any promoter known in the art for initiating the transcription of downstream DNA sequences, such as the CMV promoter, T7 promoter, etc. Since the above-mentioned labeled polypeptide coding sequence is described in detail in the patent application with international application number PCT / CN2019 / 102480 and the PCT patent application with international publication number WO2020038490A, no detailed description is provided here.

[0053] (Recombinant virus) In one embodiment of the present invention, the first active ingredient is a recombinant virus, and the genome of the recombinant virus has a promoter, a signal peptide coding sequence, and the labeled polypeptide coding sequence operably linked in sequence. Among them, the recombinant virus includes a selectively replicating recombinant oncolytic virus or a replication-deficient recombinant virus. The labeled polypeptide coding sequence encodes the labeled polypeptide.

[0054] Preferably, the recombinant oncolytic virus is derived from a genetically mutated virus having oncolytic activity and a wild-type virus having oncolytic activity. Preferably, the recombinant oncolytic virus is 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 having oncolytic activity.

[0055] Oncolytic virus therapy has a long history, and many types of viruses, such as adenovirus, herpesvirus, poxvirus, picornavirus, paramyxovirus, reovirus, parvovirus, and rhabdovirus, have been modified for use in the study of oncolytic viruses. So far, several oncolytic viruses (H101, T-VEC, Delytact) have been approved for clinical treatment of tumors. Oncolytic viruses are artificially modified viruses. Since these viruses have their pathogenic genes knocked out, they basically cannot replicate in normal tissues or cells, but can selectively replicate in tumor cells and exert an oncolytic effect. Taking poxvirus as an example, oncolytic poxvirus is generally constructed by using homologous recombination technology through genetic engineering means to knock out the endogenous thymidine kinase (TK) gene of poxvirus or double knock out the TK and vaccinia growth factor (VGF) genes. TK is one of the important enzymes in DNA synthesis, and poxvirus needs to form a high-concentration nucleic acid pool through the action of TK during the replication process so that the replication of offspring can be successfully completed. Poxvirus generally realizes its own replication and growth by promoting the growth of host and surrounding cells through the VGF gene product. However, poxvirus lacking VGF has significantly weakened damage to normal cells. When the TK and VGF genes are deleted, the replication ability of the virus is limited in normal cells, and the expression of exogenous genes inserted into the oncolytic virus vector is also greatly restricted. However, in most tumor cell environments, the TK gene is highly expressed, which provides a favorable environment for the replication and packaging of poxvirus. Poxvirus has selective replication in tumor cells, an oncolytic function, and highly expresses exogenous genes inserted into the oncolytic virus vector. In addition to the direct oncolytic effect, oncolytic viruses can release completely new tumor antigens into tumor cells to activate the endogenous antitumor immunity in the body, and also have a certain regulatory effect on the tumor microenvironment.

[0056] In the present invention, a nucleic acid encoding the labeled polypeptide is introduced into tumor cells and / or cancer cells by inserting the labeled polypeptide coding sequence into the genome of a oncolytic virus and utilizing the ability of the oncolytic virus to specifically replicate in tumor cells. While the oncolytic virus exerts a killing effect on tumor cells and / or cancer cells, an exogenous epitope peptide is expressed on the surface of tumor cells, achieving a synergistic therapeutic effect in combination with a multispecific antibody. Since the oncolytic virus destroys the tumor microenvironment, 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 effectively remove tumor cells lysed after infecting the oncolytic virus and ending the replication period without generating a sufficient number of progeny viruses by mobilizing immune cells, thereby realizing a further synergistic effect. Moreover, the antigen released from tumor cells lysed by the oncolytic virus can further activate antitumor immunity in the body, thereby achieving a better tumor killing effect than the single use of the oncolytic virus or the multispecific antibody and realizing a synergistic therapeutic effect.

[0057] In one embodiment of the present invention, a nucleic acid of the above-described labeled polypeptide coding sequence may be inserted into the genome of the obtained selectively replicating recombinant oncolytic poxvirus.

[0058] Preferably, the recombinant oncolytic virus is a recombinant oncolytic poxvirus lacking the functions of the TK gene and the VGF gene. The function of the TK gene may be deleted by inserting an exogenous nucleotide sequence. The function of the VGF gene may be deleted by gene knockout or insertion of an exogenous nucleotide sequence, but it is preferable to knockout the VGF gene.

[0059] As used herein, the term "function loss" 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, the function is lost, and the purpose may be achieved, for example, by inserting an exogenous fragment into the gene or knocking out the gene. Further, preferably, the recombinant oncolytic poxvirus is the Dryvax strain or WR strain manufactured by Wyeth.

[0060] The labeled polypeptide coding sequence may be inserted into the TK gene, whereby the recombinant oncolytic virus described in the present invention is obtained. The labeled polypeptide coding sequence may be inserted into the VGF gene, whereby the recombinant oncolytic virus described in the present invention is obtained.

[0061] In a preferred embodiment, the recombinant oncolytic poxvirus is obtained by genetic modification of the VSC20 poxvirus. The VSC20 poxvirus is a poxvirus lacking the VGF gene, and the production method may refer to the technical 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 labeled polypeptide coding sequence into the TK gene of the VSC20 poxvirus, thereby deleting the function of the TK gene.

[0062] Other exogenous genes, such as, for example, exogenous screening genes, may be further incorporated into the genome of the recombinant virus, and the exogenous screening genes include a puromycin gene, a gpt gene and / or a LacZ gene. The exogenous screening gene may be knocked out by a gene knockout system (for example, LoxP) when purifying those containing an exogenous labeled polypeptide coding gene.

[0063] In some embodiments, the present invention controls an exogenous screening gene by using the poxvirus early / late promoter p7.5, respectively, and controls a signal peptide sequence and an exogenous labeled polypeptide coding sequence by using the synthetic poxvirus early promoter pSEL, and inserts an exogenous screening gene coding sequence, a signal peptide coding sequence, and a labeled polypeptide coding sequence into the TK gene region of the poxvirus VSC20 strain by using an in vitro intracellular recombination technique, thus constructing an oncolytic virus, and the two promoters initiate the expression of their respective regulatory genes as if they were back to back.

[0064] In another embodiment of the present invention, a recombinant oncolytic poxvirus is constructed by using the Crisper-Cas9 gene editing technology. Preferably, the Crisper-Cas9 gene editing method can achieve the purpose of simultaneously cleaving and homologous recombining at two sites of one gene to complete the knockout of one sequence segment and the insertion of a transgene at one time.

[0065] Preferably, the Crisper-Cas9 gene editing method can simultaneously cleave the target sequence and the donor sequence to improve the efficiency of homologous recombination. Since the design of cleavage and homologous recombination of the Crisper-Cas9 gene editing method is described in detail in the PCT patent application with international publication number WO2020038490A (application number PCT / CN2019 / 102480), no further detailed description will be given here.

[0066] (Multispecific antibody) As used herein, the term "antibody" is used in the broadest sense to refer to a protein or polypeptide that includes an antigen-binding site or antigen-binding portion or antigen-binding fragment, covering natural and artificial antibodies of various structures, including intact antibody forms or antigen-binding fragments of antibodies, but not limited thereto.

[0067] As used herein, "intact antibody" or "whole antibody" is used interchangeably and refers to a protein comprising at least two heavy chains (H) and two light chains (L) linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (also called the heavy chain constant domain and abbreviated as CH). The heavy chain constant region includes the heavy chain constant domain CH1, the heavy chain constant domain CH2, and the heavy chain constant domain CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region (also called the light chain constant domain and abbreviated as CL). VH and VL may further be divided into complementarity-determining regions (also called hypervariable regions and abbreviated as CDR or HVR), with conserved framework regions (FR) sandwiched therebetween. Each VH and VL contains three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus (N-terminus) to the carboxy terminus (C-terminus). The CDRs of the heavy chain variable region are called HCDR1, HCDR2, and HCDR3, respectively, from the amino terminus, and the CDRs of the light chain variable region are called LCDR1, LCDR2, and LCDR3, respectively, from the amino terminus.

[0068] "Antigen-binding fragment", "antigen-binding portion", or "antigen-binding site" refers to a portion contained in a full-length antibody that exhibits binding specificity to an antigen. As described above, the hypervariable regions of the heavy and light chains of an intact antibody exhibit specific binding to an antigen. Examples of antigen-binding fragments or antigen-binding portions are Fab, Fab’, F(ab’) 2, including but not limited to bispecific Fab’ and Fv fragments, linear antibodies, single-chain antibodies, single-domain antibodies, etc. Two completely identical antigen-binding fragments generated by digesting intact antibodies with papain are called Fab fragments, each containing a heavy-chain and a light-chain variable region, a light-chain constant domain, and a heavy-chain constant domain CH1. The Fab’ fragment is different from the Fab fragment in that several residues, such as one or more cysteines derived from the antibody hinge region, are added to the carboxyl terminus of the heavy-chain constant domain CH1. Digesting intact antibodies with pepsin yields F(ab’) 2 fragments. The F(ab’) 2 fragment has two antigen-binding F(ab) moieties linked by disulfide bonds, and the F(ab’) 2 fragment is a bivalent antibody. A single-chain antibody is a fusion protein in which the antibody heavy-chain variable region and the light-chain variable region are linked by a flexible short peptide consisting of about 10 to 25 amino acids. A single-domain antibody is an antibody fragment consisting of a single monomer variable region. Single-domain antibodies are generally derived from the variable region of the heavy chain of camelid or shark antibodies and are often called nanobodies.

[0069] The term "multispecific antibody (also known as a multispecific antibody)" refers to one that contains antigen-binding portions 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 may be a bispecific antibody (also called a bispecific antibody), or a trispecific antibody, etc. "Multi" means two or more. Also, for the sake of convenience in description, a quantifier is attached to each antigen-binding portion for distinction. For example, in some cases, they may be described as "first antigen-binding portion", "second antigen-binding portion", "third antigen-binding portion", etc. respectively. "First", "second", and "third" do not represent the order of binding nor the importance. The following is an interpretation and explanation regarding the "first antigen-binding portion", "second antigen-binding portion", and "third antigen-binding portion". Unless otherwise specified, there is no limitation on the order of binding of the listed multispecific antibody to the antigen. Naturally, when a multispecific antibody specifically binds to different biomolecules, it may bind to one or more epitopes of the specific biomolecule. According to a preferred embodiment of the present invention, the multispecific antibody mentioned contains antigen-binding portions that bind to different epitopes of at least two different biomolecules.

[0070] The term "and / or" is understood to mean any one of the options or any two or more of the options when used to connect two or more options.

[0071] The term "comprising" or "comprises" means including the recited element or step, but not excluding other elements or steps. Naturally, unless otherwise specified, when "comprises" is used, it also covers the case of consisting of the recited element or step. For example, when an antibody variable region comprising a specific sequence is mentioned, it is also intended to cover an antibody variable region consisting of the specific sequence.

[0072] As used herein, "affinity" or "binding affinity" is understood in the ordinary sense in the art and is used when reflecting the strength and / or stability between an antigen and an antibody or a binding site on an antigen-binding fragment.

[0073] "Specifically binds to" or "is specifically bound by" or "binds to" or "specifically targets" a particular antigen or epitope, or "has specificity for" or "is capable of binding to" a particular antigen or epitope means that, unlike non-specific interactions, such specific binding can be measured by several methods commonly used in the art. The ability of an antibody to bind to an antigen may be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art. For example, it may be measured by a flow cytometer against cells carrying the antigen, and by measuring an index such as the positive rate of the cells, the competitive binding situation between the test antibody and the labeled antibody is measured. Since the antigen on the cell surface has a spatial structure closer to the form existing in the body, the actual situation can be more accurately reflected by this method. According to a specific embodiment of the present invention, the provided bispecific antibody has an EC 50 value of 0.5 to 200 nM, 1 to 200 nM, 5 to 200 nM, 10 to 200 nM, 15 to 200 nM, 0.5 to 150 nM, 1 to 150 nM, 5 to 150 nM, 10 to 150 nM, 15 to 150 nM, 0.5 to 120 nM, 1 to 120 nM, 5 to 120 nM, 10 to 120 nM, 15 to 120 nM, 10 to 100 nM, 15 to 100 nM, 10 to 80 nM, 15 to 80 nM, 10 to 60 nM, 15 to 60 nM, 10 to 50 nM, or 15 to 50 nM with respect to an immune cell antigen (e.g., a T cell antigen (e.g., CD3 antigen)), and an EC 50 value of 0.01 to 20 nM, 0.05 to 20 nM, 0.01 to 16 nM, 0.05 to 16 nM, 0.01 to 15 nM, 0.05 to 15 nM, 0.01 to 10 nM, 0.05 to 10 nM, 0.1 to 10 nM, 0.3 to 5 nM with respect to a labeled polypeptide (e.g., TT3 antigen). Also, the binding activity of the antibody to the antigen may be measured by surface plasmon resonance technology (SPR) or biolayer interferometry (BLI).

[0074] Multispecific antibodies or polynucleotides referred to in this specification are generally separable or recombinable. "Separable" means that it can be identified from a cell or cell culture expressing the polypeptide or protein and can be separated and / or recovered. Generally, a separated polypeptide is produced by at least one purification step. "Separated antibody" refers to an antibody that substantially does not contain other antibodies or antigen-binding fragments thereof having different antigen specificities. "Recombinant" means that antibodies can be generated in exogenous host cells using genetic recombination techniques.

[0075] "Humanized" antibodies generally refer to antibodies consisting of an antigen-binding portion derived from a non-human species, a partial structure and sequence of a human immunoglobulin molecule. For example, in a humanized antibody, the entire antibody except for the CDRs is encoded by a polynucleotide derived from a human, the antigen-binding activity is retained, and the immunogenicity is reduced.

[0076] The CDR sequences shown in this specification are obtained by analyzing with reference to conventional schemes. For example, the CDR sequences of the antibodies shown, the CDR sequences of the first antigen-binding portion of the multispecific antibody, and the CDR sequences of the second antigen-binding portion are obtained with reference to the IMGT 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)). They may also be obtained by schemes such as Kabat (for example, referring to U.S. Dept. of Health and Human Servies, "Sequences of Proteins of Immunological Interest" (1983)), Chothia (for example, referring to J. Mol. Biol. 196:901-917 (1987)). It is known to those skilled in the art that the differences in CDRs shown by the definition methods are also included in the protection scope of the present invention.

[0077] A bispecific antibody (BsAb, also known as a bivalent antibody) is an antibody that can specifically bind to two antigens or different epitopes of the same antigen simultaneously. Since BsAb can target multiple antigens or epitopes, it not only shows more advantages than monoclonal antibodies but also gradually shows higher efficacy than combination therapies with monoclonal antibodies. For example, by redirecting specific effector cells to adjacent tumor cells, tumor killing can be enhanced, and the binding specificity can be increased by the interaction of two different cell surface antigens. Also, compared to the combination of single antibodies, the development cost and clinical trials can be reduced. Bispecific antibodies are expected to be widely used in fields such as tumor treatment and autoimmune diseases, and they have been evolving rapidly as recombinant antibody technology gradually matures.

[0078] The second composition contains a second active ingredient, and the second active ingredient contains a multispecific antibody. The second composition may contain a pharmaceutically acceptable carrier, if any, except for the multispecific antibody mentioned therein. Some pharmaceutically acceptable salts are, for example, acid addition salts or base addition salts (e.g., as described in Berge, S.M. et al (1977) J. Pharma. Sci. 66:1-19). The multispecific antibody may be a bispecific antibody containing a first antigen-binding portion and a second antigen-binding portion. The first antigen-binding portion can specifically recognize and bind to the extracellular antigen determinant of the labeled polypeptide. The second antigen-binding portion can specifically recognize and bind to an immune cell antigen or a first cytokine or its receptor. By mobilizing or connecting immune cells or cytokines or their receptors, the effect of immunotherapy is exerted. The mentioned first antigen-binding portion and second antigen-binding portion are fused, and the first antigen-binding portion and the second antigen-binding portion each target different epitopes of different antigens. The mentioned first antigen-binding portion and second antigen-binding portion are fused and may be directly linked (e.g., directly linked by the terminal amino acids of both), indirectly linked by a linker, or other functional region sequences such as an Fc region, CH1, CL sequence, etc. may be linked between them. It should be noted that the terms "first", "second", and "third" used in the specification are only for description purposes and cannot be understood as indicating or suggesting relative importance or implicitly indicating the number of target technical features, nor are they for representing an order. The terms "first antigen-binding portion", "second antigen-binding portion", and "third antigen-binding portion" each mean a portion that can bind to different epitopes of different target antigens or different epitopes of the same target antigen, and may be an intact antibody, for example, a heavy chain variable region, Fab, Fab’, F(ab’) 2 , a single-chain antibody (ScFv), or a single-domain antibody (sdAb), etc., and may be a part of an intact antibody. According to a preferred embodiment of the present invention, the "first antigen-binding portion", "second antigen-binding portion", and "third antigen-binding portion" each specifically bind to different epitopes of different antigens.

[0079] In this specification, "linker" and "linking fragment" have the same meaning. The linker may be several oligopeptides or polypeptides, or any amino acid sequence that can provide flexibility. Available linkers are those commonly used in the art and include, but are not limited to, the group consisting of GS, SG, GGS, GSG, SGG, GGG, GGGS, SGGG, GGGGS, GGGGGGSGS, GGGGSGS, GGGGSGGS, GGGGSGGGGSGGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGSGGGSGGGS, etc.

[0080] The provided first antigen-binding portion can specifically recognize and bind to the extracellular antigen determinant of the labeled polypeptide. According to a specific embodiment, the provided first antigen-binding portion can specifically recognize and bind to the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, or 5 above. According to a specific embodiment, the provided first antigen-binding portion includes one or more domains that can specifically recognize and bind to the epitope polypeptide, provided that the amino acid sequence of the epitope polypeptide includes the amino acid sequence of Strep tag II.

[0081] Preferably, the amino acid sequence of the light chain of the antigen-binding domain targeting TT1 is identical to the amino acid sequence at positions 1 to 111 of the light chain of an anti-Myc antibody (e.g., clone 9e10) (in some embodiments of the present invention, the amino acid sequence of the light chain of the antigen-binding domain targeting TT1 may be extended by 10 or fewer amino acids downstream of the above sequence), and the amino acid sequence of the heavy chain is identical to the amino acid sequence at positions 23 to 143 of the heavy chain of an anti-Myc antibody (e.g., clone 9e10) (in some embodiments of the present invention, the amino acid sequence of the heavy chain of the antigen-binding domain targeting TT1 may be extended by 10 or fewer amino acids upstream and downstream of the above sequence).

[0082] Preferably, the amino acid sequence of the light chain of the antigen-binding domain targeting TT2 is identical to the amino acid sequence of positions 2 to 121 of the light chain of an anti-HA antibody (e.g., clone 12ca5) (in some embodiments of the present invention, the amino acid sequence of the light chain of the antigen-binding domain targeting TT2 may be extended by 10 or fewer amino acids downstream of the above sequence), and the amino acid sequence of the heavy chain is identical to the amino acid sequence of positions 2 to 114 of the heavy chain of an anti-HA antibody (e.g., clone 12ca5) (in some embodiments of the present invention, the amino acid sequence of the heavy chain of the antigen-binding domain targeting TT2 may be extended by 10 or fewer amino acids downstream of the above sequence).

[0083] Preferably, the amino acid sequence of the light chain of the antigen-binding domain targeting TT3 is identical to the light chain of an anti-Strep tag II antibody (see, for example, Patent Document EP2871189A1), and the amino acid sequence of the heavy chain is identical to the heavy chain of an anti-Strep tag II antibody (see, for example, Patent Document EP2871189A1).

[0084] Here, the amino acid sequence of the light chain of the anti-Myc antibody (clone 9e10) may be derived from the amino acid sequence with the number PDB:2ORB_L (https: / / www.ncbi.nlm.nih.gov / protein / 2ORB_L), and the amino acid sequence of the heavy chain of the anti-Myc antibody (clone 9e10) may be derived from the amino acid sequence with the number GenBank:CAA73271.1 (https: / / www.ncbi.nlm.nih.gov / protein / CAA73271.1?report=genbank&log$=protalign&blast_rank=1&RID=P597FX1S014). The amino acid sequence of the linker that connects the light chain and the heavy chain is the one 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 light chain of the anti-HA antibody (clone 12ca5) may be derived from the amino acid sequence with the number PDB:5XCS_B (https: / / www.ncbi.nlm.nih.gov / protein / 1258501213), and the amino acid sequence of the heavy chain of the anti-HA antibody (clone 12ca5) may be derived from the amino acid sequence with the number PDB:5XCS_A (https: / / www.ncbi.nlm.nih.gov / protein / 5XCS_A). The amino acid sequence of the linker that connects the light chain and the heavy chain may be the one 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.

[0085] The amino acid sequences of the light and heavy chains of the anti-Strep tag II antibody may be derived from the amino acid sequences disclosed in Patent Document EP2871189A1, and the amino acid sequence of the linker for linking the light and heavy chains may be the one shown in SEQ ID NO: 16. According to a specific embodiment, the first antigen-binding portion has the HCDR1, HCDR2, HCDR3 sequences shown in SEQ ID NO: 20, 21, 22 and the LCDR1, LCDR2, LCDR3 sequences shown in SEQ ID NO: 23, 24, 25. The shown HCDR1, HCDR2, HCDR3 sequences and LCDR1, LCDR2, LCDR3 sequences are obtained based on the IMGT scheme. According to a specific embodiment, the first antigen-binding portion 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 linked by a linker, and according to a specific embodiment, they may be linked by the linker shown in SEQ ID NO: 16. According to a specific embodiment, the first antigen-binding portion has the sequence shown in SEQ ID NO: 28. According to a specific embodiment, the first antigen-binding portion reduces immunogenicity and retains the binding activity with TT3 by humanizing the light chain variable region shown in SEQ ID NO: 26 and the heavy chain variable region shown in SEQ ID NO: 27. According to a specific embodiment, the first antigen-binding portion has the heavy chain variable region shown in SEQ ID NO: 29 and the light chain variable region shown in SEQ ID NO: 32. According to a specific embodiment, the first antigen-binding portion has the heavy chain variable region shown in SEQ ID NO: 29 and the light chain variable region shown in SEQ ID NO: 33. According to a specific embodiment, the first antigen-binding portion has the heavy chain variable region shown in SEQ ID NO: 29 and the light chain variable region shown in SEQ ID NO: 34. According to a specific embodiment, the first antigen-binding portion has the heavy chain variable region shown in SEQ ID NO: 30 and the light chain variable region shown in SEQ ID NO: 32. According to a specific embodiment, the first antigen-binding portion has the heavy chain variable region shown in SEQ ID NO: 30 and the light chain variable region shown in SEQ ID NO: 33. According to a specific embodiment, the first antigen-binding portion has the heavy chain variable region shown in SEQ ID NO: 30 and the light chain variable region shown in SEQ ID NO: 34.According to a specific embodiment, the first antigen-binding portion has a heavy chain variable region represented by SEQ ID NO: 31 and a light chain variable region represented by SEQ ID NO: 32. According to a specific embodiment, the first antigen-binding portion has a heavy chain variable region represented by SEQ ID NO: 31 and a light chain variable region represented by SEQ ID NO: 33. According to a specific embodiment, the first antigen-binding portion has a heavy chain variable region represented by SEQ ID NO: 31 and a light chain variable region represented by SEQ ID NO: 34.

Table 2-1

Table 2-2

[0086] The first antigen-binding portion and the second antigen-binding portion can be linked by a linker to form a multispecific antibody. Any flexible oligopeptide commonly used in the art for linking amino acid fragments may be used as the linker between the first antigen-binding portion and the second antigen-binding portion. According to a specific embodiment, the amino-terminal (also referred to as the N-terminal) amino acid of the first antigen-binding portion may be linked to the carboxyl-terminal (also referred to as the C-terminal) amino acid of the second antigen-binding portion. According to a specific embodiment, the C-terminal amino acid of the first antigen-binding portion may be linked to the N-terminal amino acid of the second antigen-binding portion. When the first antigen-binding portion and the second antigen-binding portion are linked, they may be directly linked, linked by a linker, or another functional amino acid sequence may be linked between the two. The other functional amino acid sequence mentioned may be, for example, an Fc sequence, which can thereby mediate functions such as ADCC (antibody-dependent cell-mediated cytotoxicity), ADCP (antibody-dependent cell-mediated phagocytosis), or CDC (complement-mediated cytotoxicity). In some cases, CH 1 , CH 2, etc. such as the CL sequence may be linked. According to a specific embodiment, the C-terminal amino acid of the first antigen-binding portion is linked to the Fc sequence and is linked to the second antigen-binding portion by a linker. According to a specific embodiment, the C-terminal amino acid of the second antigen-binding portion forms a CH1, CL sequence, Fc region and an IgG-like antibody structure, and the C-terminal amino acid of the Fc region is linked to the N-terminal amino acid of the first antigen-binding portion by a linker. According to a specific embodiment, the light chain variable region and the heavy chain variable region sequence of the first antigen-binding portion may be directly linked by a linker to form a single-chain antibody (scFv) form, or may be directly linked.

[0087] In some specific 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 may be the full length, fragment or variant of the cytokine. In some embodiments, the first cytokine mentioned is 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-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 at least one of its related receptors, but not limited thereto. By binding to the cytokine or its receptor, an immunomodulatory effect can be achieved.

[0088] In some specific embodiments, the second antigen-binding portion specifically recognizes and binds to an immune cell antigen, particularly, specifically recognizes and binds to an immune cell surface antigen, and can mobilize immune cells to achieve the purpose of tumor killing. As used herein, the term "immune cell" refers to, for example, any one of various cells that play a role in the immune system to protect against infection and foreign substances. In specific embodiments, the term may include white blood cells (e.g., neutrophils, eosinophils, basophils), lymphocytes, and monocytes. Immune cells further include effector cells. Effector cells refer to cells involved in the immune response, such as promoting the immune response of effector cells. Examples of effector cells include T cells (e.g., CD4+ T cells, CD8+ T cells, α T cells, β T cells, γ T cells, δ T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, mast cells, etc., but are not limited thereto. In some embodiments, the immune cell is a T cell or an NK cell. In some embodiments, the immune cell is a cytotoxic T cell (e.g., CD8+ T cell). In some embodiments, the immune cell is a helper T cell (e.g., CD4+ T cell). The immune cell antigens mentioned 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, 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, CD210Including, but not limited to, 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, 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, CD362, etc.

[0089] According to specific embodiments, the second antigen-binding portion can specifically recognize and bind to the CD3 antigen. CD3 (T cell surface glycoprotein CD3, a signal transduction coreceptor of the T cell receptor) is a differentiation antigen expressed on the surface of all T lymphocytes, and mainly mediates the transmission of T cell activation signals and plays an important role in the body's immune system immunity against infections. CD3 referred to in this specification includes any natural CD3 of any vertebrate (including mammals, such as primates like humans, and rodent animals such as mice and rats) unless otherwise specified. The term "CD3" covers CD3 that has not been processed in "full length", and any form of CD3 or any fragment thereof generated by intracellular processing. The term further includes naturally occurring variants of CD3 (e.g., splice variants or allelic variants). In some specific embodiments, CD3 refers to the full length or a fragment thereof derived from human and cynomolgus monkey CD3. In some specific embodiments, CD3 refers to the full length or a fragment thereof derived from mouse or rat CD3.

[0090] In some embodiments, the second antigen-binding portion can specifically recognize and bind to the CD3 antigen. The anti-CD3 antibody may be derived from any known antibody targeting CD3, or may be an antibody targeting CD3 that is under development or will be developed in the future. For example, antibody SP34 (see, e.g., Pessano, S. et al., EMBO J 4 (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), antibody UCHT1 (see, e.g., Callard, R.E. et al., Clin Exp Immunol 43 (1981) 497-505), etc. By performing sequence optimization and humanization modification on mouse SP34, a second antigen-binding portion that specifically recognizes and targets the CD3 antigen can be obtained. Also, through humanization, it can show high affinity for human and cynomolgus monkey CD3 antigens.

[0091] According to a specific embodiment, the second antigen-binding portion has HCDR1, HCDR2, and HCDR3 sequences represented by SEQ ID NOs: 35, 36, and 37, and LCDR1, LCDR2, and LCDR3 sequences represented by SEQ ID NOs: 38, 39, and 40. The shown HCDR1, HCDR2, HCDR3 sequences, and LCDR1, LCDR2, LCDR3 sequences are obtained based on the IMGT scheme.

Table 3

[0092] According to a specific embodiment, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 41 and a light chain variable region sequence represented by SEQ ID NO: 42.

[0093] According to a specific embodiment, after the sequence of the second antigen-binding portion has undergone humanization modification, the humanized second antigen-binding portion exhibits high affinity for human or cynomolgus CD3 antigen. When forming a bispecific antibody with the first antigen-binding portion, the affinity for CD3 antigen is lower compared to the humanized CD3 monoclonal antibody. Nevertheless, by modifying the first antigen-binding portion, the multispecific antibody maintains a high level of affinity for the labeled polypeptide and can effectively achieve tumor targeting and tumor killing effects. Through the design and screening of the present invention, the entire multispecific antibody exhibits particularly appropriate affinity. According to a specific embodiment, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 43, 44, or 45. According to a specific embodiment, the second antigen-binding portion has a light chain variable region sequence represented by SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, or 53. The humanized heavy chain variable region or light chain variable region after humanization modification can form a sequence targeting the CD3 antigen with the light chain variable region or heavy chain variable region before humanization modification. For example, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 41 and a light chain variable region sequence represented by SEQ ID NO: 46. As another example, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 41 and a light chain variable region sequence represented by SEQ ID NO: 47. As another example, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 41 and a light chain variable region sequence represented by SEQ ID NO: 48. As another example, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 41 and a light chain variable region sequence represented by SEQ ID NO: 49. As another example, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 41 and a light chain variable region sequence represented by SEQ ID NO: 50. As another example, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 41 and a light chain variable region sequence represented by SEQ ID NO: 51. As another example, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 41 and a light chain variable region sequence represented by SEQ ID NO: 52. As another example, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 41 and a light chain variable region sequence represented by SEQ ID NO: 53.As another example, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 43 and a light chain variable region sequence represented by SEQ ID NO: 42. As another example, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 44 and a light chain variable region sequence represented by SEQ ID NO: 42. As another example, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 45 and a light chain variable region sequence represented by SEQ ID NO: 42.

[0094] According to a specific embodiment, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 43 and a light chain variable region sequence represented by SEQ ID NO: 46.

[0095] According to a specific embodiment, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 43 and a light chain variable region sequence represented by SEQ ID NO: 47.

[0096] According to a specific embodiment, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 43 and a light chain variable region sequence represented by SEQ ID NO: 48.

[0097] According to a specific embodiment, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 43 and a light chain variable region sequence represented by SEQ ID NO: 49.

[0098] According to a specific embodiment, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 43 and a light chain variable region sequence represented by SEQ ID NO: 50.

[0099] According to a specific embodiment, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 43 and a light chain variable region sequence represented by SEQ ID NO: 51.

[0100] According to a specific embodiment, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 43 and a light chain variable region sequence represented by SEQ ID NO: 52.

[0101] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 43 and a light-chain variable region sequence represented by SEQ ID NO: 53.

[0102] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 44 and a light-chain variable region sequence represented by SEQ ID NO: 46.

[0103] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 44 and a light-chain variable region sequence represented by SEQ ID NO: 47.

[0104] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 44 and a light-chain variable region sequence represented by SEQ ID NO: 48.

[0105] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 44 and a light-chain variable region sequence represented by SEQ ID NO: 49.

[0106] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 44 and a light-chain variable region sequence represented by SEQ ID NO: 50.

[0107] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 44 and a light-chain variable region sequence represented by SEQ ID NO: 51.

[0108] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 44 and a light-chain variable region sequence represented by SEQ ID NO: 52.

[0109] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 44 and a light-chain variable region sequence represented by SEQ ID NO: 53.

[0110] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 45 and a light-chain variable region sequence represented by SEQ ID NO: 46.

[0111] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 45 and a light-chain variable region sequence represented by SEQ ID NO: 47.

[0112] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 45 and a light-chain variable region sequence represented by SEQ ID NO: 48.

[0113] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 45 and a light-chain variable region sequence represented by SEQ ID NO: 49.

[0114] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 45 and a light-chain variable region sequence represented by SEQ ID NO: 50.

[0115] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 45 and a light-chain variable region sequence represented by SEQ ID NO: 51.

[0116] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 45 and a light-chain variable region sequence represented by SEQ ID NO: 52.

[0117] According to a specific embodiment, the second antigen-binding portion has a heavy-chain variable region sequence represented by SEQ ID NO: 45 and a light-chain variable region sequence represented by SEQ ID NO: 53.

Table 4-1

Table 4-2

[0118] According to a specific embodiment, the second antigen-binding portion and the first antigen-binding portion are linked by a linker. According to a specific embodiment, the linker is (G 4 S) 3 . The heavy chain variable region of the second antigen-binding portion is linked to CH1, CH1 is linked to the Fc region, and the light chain variable region is linked to CL1. The N-terminal amino acid of the first antigen-binding portion is linked to the C-terminal amino acid of the Fc region by a linker. The light chain variable region sequence and the heavy chain variable region sequence of the first antigen-binding portion are directly linked or linked by a linker. The Fc region mentioned may be a human Fc region sequence. The Fc region sequence may be derived from, but not limited to, IgA, IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM and variants thereof. The variants mentioned include, but are not limited to, K214R, L234F, L235E, P331S, D356E, L358M or any combination thereof (e.g., L234F / 235E / P331S, K214R / L234F / L235E / P331S, etc.), which are mutations occurring in the Fc region.

[0119] According to a specific embodiment, the bispecific antibody provided includes a first antigen-binding portion and a second antigen-binding portion. The heavy chain variable region of the second antigen-binding portion is directly linked to the heavy chain constant domains (CH1 and Fc region), and the light chain variable region of the second antigen-binding portion is directly linked to CL, forming an IgG-like structure. The C-terminal amino acid of the Fc region is linked by a linker (the linker used is shown by SEQ ID NO: 18) to the N-terminal amino acid sequence of the light chain variable region of the first antigen-binding portion. Here, as shown in a of FIG. 2, the C-terminal amino acid of the light chain variable region of the first antigen-binding portion is linked by a linker (the linker used is shown by SEQ ID NO: 16) to the N-terminal amino acid of the heavy chain variable region of the first antigen-binding portion. The formed bispecific antibody number starts with "BK-A". According to a specific embodiment, the bispecific antibody provided is BK-A-101. The sequences formed by the light chain variable region and the heavy chain variable region of its first antigen-binding portion are shown by SEQ ID NO: 28. The second antigen-binding portion has a heavy chain variable region sequence shown by SEQ ID NO: 41 and a light chain variable region sequence shown by SEQ ID NO: 42. The heavy chain variable region sequence of the second antigen-binding portion is linked to the heavy chain constant domain CH (the sequence shown by SEQ ID NO: 57), and the light chain variable region sequence of the second antigen-binding portion is linked to the light chain constant domain CL (the sequence shown by SEQ ID NO: 55) to constitute an IgG-type structure. The N-terminal amino acid of the first antigen-binding portion is linked to the C-terminal amino acid of the Fc region.

[0120] According to a specific embodiment, the bispecific antibody provided is BK-A-101-hu1, which is a bispecific antibody formed through humanization modification of the second antigen-binding portion sequence of the BK-A-101 antibody. The second antigen-binding portion of BK-A-101-hu1 has a heavy chain variable region shown by SEQ ID NO: 44 and a light chain variable region sequence shown by SEQ ID NO: 51. Other sequences are the same as those of BK-A-101.

[0121] According to a specific embodiment, the bispecific antibody provided is BK-A-101-hu2, which is a bispecific antibody formed through humanization modification of the second antigen-binding partial sequence of the BK-A-101 antibody. The second antigen-binding part of BK-A-101-hu2 has a heavy chain variable region represented by SEQ ID NO: 44 and a light chain variable region sequence represented by SEQ ID NO: 52. Other sequences are the same as those of BK-A-101.

[0122] According to a specific embodiment, the bispecific antibody provided is BK-A-101-hu3, which is a bispecific antibody formed through humanization modification of the second antigen-binding partial sequence of the BK-A-101 antibody. The second antigen-binding part of BK-A-101-hu3 has a heavy chain variable region represented by SEQ ID NO: 44 and a light chain variable region sequence represented by SEQ ID NO: 53. Other sequences are the same as those of BK-A-101.

[0123] According to a specific embodiment, the bispecific antibody provided is BK-A-101-hu4, which is a bispecific antibody formed through humanization modification of the second antigen-binding partial sequence of the BK-A-101 antibody. The second antigen-binding part of BK-A-101-hu4 has a heavy chain variable region represented by SEQ ID NO: 45 and a light chain variable region sequence represented by SEQ ID NO: 51. Other sequences are the same as those of BK-A-101.

[0124] According to a specific embodiment, the bispecific antibody provided is BK-A-101-hu5, which is a bispecific antibody formed through humanization modification of the second antigen-binding partial sequence of the BK-A-101 antibody. The second antigen-binding part of BK-A-101-hu5 has a heavy chain variable region represented by SEQ ID NO: 45 and a light chain variable region sequence represented by SEQ ID NO: 52. Other sequences are the same as those of BK-A-101.

[0125] According to a specific embodiment, the bispecific antibody provided is BK-A-101-hu6, which is a bispecific antibody formed through humanization modification of the second antigen-binding portion sequence of the BK-A-101 antibody. The second antigen-binding portion of BK-A-101-hu6 has a heavy chain variable region represented by SEQ ID NO: 45 and a light chain variable region sequence represented by SEQ ID NO: 53. Other sequences are the same as those of BK-A-101.

[0126] According to a specific embodiment, the bispecific antibody provided includes a first antigen-binding portion and a second antigen-binding portion. The heavy chain variable region of the first antigen-binding portion is directly linked to the heavy chain constant domain, and the light chain variable region of the first antigen-binding portion is directly linked to the light chain constant domain to form an IgG-like structure. As shown in b of FIG. 2, the carboxy-terminal amino acid of the Fc region of the IgG-like structure is linked by a linker to the N-terminal amino acid of the light chain variable region of the second antigen-binding portion, and the N-terminal amino acid of the heavy chain variable region of the second antigen-binding portion is linked by a linker to the C-terminal amino acid of the light chain variable region of the second antigen-binding portion. The bispecific antibody number formed starts with "BK-D". All the linkers used here are (G 4 S) 3It is as follows. According to specific embodiments, the bispecific antibody provided is BK-D-01, which comprises a first antigen-binding portion represented by SEQ ID NO: 31 and SEQ ID NO: 32, and a second antigen-binding portion represented by SEQ ID NO: 43 and SEQ ID NO: 46. According to specific embodiments, the bispecific antibody provided is BK-D-02, which comprises a first antigen-binding portion represented by SEQ ID NO: 31 and SEQ ID NO: 34, and a second antigen-binding portion represented by SEQ ID NO: 45 and SEQ ID NO: 46. According to specific embodiments, the bispecific antibody provided is BK-D-03, which comprises a first antigen-binding portion represented by SEQ ID NO: 29 and SEQ ID NO: 34, and a second antigen-binding portion represented by SEQ ID NO: 43 and SEQ ID NO: 46. According to specific embodiments, the bispecific antibody provided is BK-D-04, which comprises a first antigen-binding portion represented by SEQ ID NO: 29 and SEQ ID NO: 32, and a second antigen-binding portion represented by SEQ ID NO: 43 and SEQ ID NO: 49. According to specific embodiments, the bispecific antibody provided is BK-D-05, which comprises a first antigen-binding portion represented by SEQ ID NO: 29 and SEQ ID NO: 32, and a second antigen-binding portion represented by SEQ ID NO: 44 and SEQ ID NO: 48. According to specific embodiments, the bispecific antibody provided is BK-D-06, which comprises a first antigen-binding portion represented by SEQ ID NO: 29 and SEQ ID NO: 32, and a second antigen-binding portion represented by SEQ ID NO: 44 and SEQ ID NO: 50. According to specific embodiments, the bispecific antibody provided is BK-D-07, which comprises a first antigen-binding portion represented by SEQ ID NO: 31 and SEQ ID NO: 32, and a second antigen-binding portion represented by SEQ ID NO: 45 and SEQ ID NO: 46. According to specific embodiments, the bispecific antibody provided is BK-D-08, which comprises a first antigen-binding portion represented by SEQ ID NO: 29 and SEQ ID NO: 32, and a second antigen-binding portion represented by SEQ ID NO: 45 and SEQ ID NO: 46. According to specific embodiments, the bispecific antibody provided is BK-D-09, which comprises a first antigen-binding portion represented by SEQ ID NO: 29 and SEQ ID NO: 32, and a second antigen-binding portion represented by SEQ ID NO: 44 and SEQ ID NO: 47. According to specific embodiments, the bispecific antibody provided is BK-D-10, which comprises a first antigen-binding portion represented by SEQ ID NO: 31 and SEQ ID NO: 34, and a second antigen-binding portion represented by SEQ ID NO: 43 and SEQ ID NO: 46.According to specific embodiments, the bispecific antibody provided is BK-D-11, which includes a first antigen-binding portion represented by SEQ ID NO: 29 and SEQ ID NO: 32, and a second antigen-binding portion represented by SEQ ID NO: 43 and SEQ ID NO: 46. According to specific embodiments, the bispecific antibody provided is BK-D-12, which includes a first antigen-binding portion represented by SEQ ID NO: 29 and SEQ ID NO: 34, and a second antigen-binding portion represented by SEQ ID NO: 45 and SEQ ID NO: 46. According to specific embodiments, the bispecific antibody provided is BK-D-13, which includes a first antigen-binding portion represented by SEQ ID NO: 29 and SEQ ID NO: 32, and a second antigen-binding portion represented by SEQ ID NO: 45 and SEQ ID NO: 50. According to specific embodiments, the heavy chain constant domain sequence in the bispecific antibody of the BK-D structure is the one represented by SEQ ID NO: 54, and the light chain constant domain sequence is the one represented by SEQ ID NO: 55. The Fc region sequence is the one represented by SEQ ID NO: 56. The heavy chain constant domain sequence (SEQ ID NO: 54) in the bispecific antibody of the BK-D structure has a mutation in the Fc region compared to the sequence represented by SEQ ID NO: 57. According to specific embodiments, the affinity of the provided bispecific antibody for the CD3 antigen is reduced compared to that of a humanized monoclonal antibody without Fc mutation, and the EC of the bispecific antibody for the CD3 antigen. 50 The value 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.

[0127] According to a specific embodiment, the provided bispecific antibody comprises a first antigen-binding portion and a second antigen-binding portion. As shown in Figure 3, the C-terminal amino acid of the first antigen-binding portion is directly linked to the Fc region sequence (CH2, CH3), the C-terminal amino acid of the Fc region is linked by a linker to the N-terminal amino acid of the light chain variable region of the second antigen-binding portion, and the C-terminal amino acid of the light chain variable region of the second antigen-binding portion is linked by a linker (according to a specific embodiment, the linker is the one represented by SEQ ID NO: 18) to the N-terminal amino acid of the heavy chain variable region of the second antigen-binding portion. According to a specific embodiment, the provided bispecific antibody is BK-B-110, provided that the sequence formed by the light chain variable region and the heavy chain variable region of the first antigen-binding portion is the one represented by SEQ ID NO: 28, the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 41 and a light chain variable region sequence represented by SEQ ID NO: 42, and the Fc region sequence is the one represented by SEQ ID NO: 56. According to a specific embodiment, the provided bispecific antibody is BK-B-110-hu1, which is a bispecific antibody formed through humanization modification of the second antigen-binding portion of the BK-B-110 antibody, and its second antigen-binding portion has a heavy chain variable region represented by SEQ ID NO: 44 and a light chain variable region sequence represented by SEQ ID NO: 51. According to a specific embodiment, the provided bispecific antibody is BK-B-110-hu2, which is a bispecific antibody formed through humanization modification of the second antigen-binding portion of the BK-B-110 antibody, and its second antigen-binding portion has a heavy chain variable region represented by SEQ ID NO: 44 and a light chain variable region sequence represented by SEQ ID NO: 52. According to a specific embodiment, the provided bispecific antibody is BK-B-110-hu3, which is a bispecific antibody formed through humanization modification of the second antigen-binding portion of the BK-B-110 antibody, and its second antigen-binding portion has a heavy chain variable region represented by SEQ ID NO: 44 and a light chain variable region sequence represented by SEQ ID NO: 53. According to a specific embodiment, the provided bispecific antibody is BK-B-110-hu4, which is a bispecific antibody formed through humanization modification of the second antigen-binding portion of the BK-B-110 antibody, and its second antigen-binding portion has a heavy chain variable region represented by SEQ ID NO: 45 and a light chain variable region sequence represented by SEQ ID NO: 51.According to a specific embodiment, the bispecific antibody provided is BK-B-110-hu5, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of the BK-B-110 antibody, and its second antigen-binding portion has a heavy-chain variable region represented by SEQ ID NO: 45 and a light-chain variable region sequence represented by SEQ ID NO: 52. According to a specific embodiment, the bispecific antibody provided is BK-B-110-hu6, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of the BK-B-110 antibody, and the second antigen-binding portion has a heavy-chain variable region represented by SEQ ID NO: 45 and a light-chain variable region sequence represented by SEQ ID NO: 53.

[0128] According to specific embodiments, the provided bispecific antibody comprises a first antigen-binding portion and a second antigen-binding portion. As shown in Figure 4, the C-terminal amino acid of the first antigen-binding portion is directly linked to the Fc region sequence, and the C-terminal amino acid of the Fc region is linked by a linker (according to specific embodiments, the linker used is the one represented by SEQ ID NO: 18) to the N-terminal amino acid of the heavy chain variable region of the second antigen-binding portion. The C-terminal amino acid of the heavy chain variable region of the second antigen-binding portion is linked by a linker (according to specific embodiments, the linker used is the one represented by SEQ ID NO: 18) to the N-terminal amino acid of the light chain variable region of the second antigen-binding portion. According to specific embodiments, the provided bispecific antibody is BK-C-120, the first antigen-binding portion of which has the sequence represented by SEQ ID NO: 28, and the second antigen-binding portion has the heavy chain variable region sequence represented by SEQ ID NO: 41 and the light chain variable region sequence represented by SEQ ID NO: 42, and the Fc region sequence is the one represented by SEQ ID NO: 56. According to specific embodiments, the provided bispecific antibody is BK-C-120-hu1, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of antibody BK-C-120, and the second antigen-binding portion has the heavy chain variable region represented by SEQ ID NO: 44 and the light chain variable region sequence represented by SEQ ID NO: 51. According to specific embodiments, the provided bispecific antibody is BK-C-120-hu2, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of BK-C-120 antibody, and the second antigen-binding portion has the heavy chain variable region sequence represented by SEQ ID NO: 44 and the light chain variable region sequence represented by SEQ ID NO: 52. According to specific embodiments, the provided bispecific antibody is BK-C-120-hu3, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of BK-C-120 antibody, and the second antigen-binding portion has the heavy chain variable region represented by SEQ ID NO: 44 and the light chain variable region sequence represented by SEQ ID NO: 53. According to specific embodiments, the provided bispecific antibody is BK-C-120-hu4, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of antibody BK-C-120, and the second antigen-binding portion has the heavy chain variable region represented by SEQ ID NO: 45 and the light chain variable region sequence represented by SEQ ID NO: 51.According to a specific embodiment, the bispecific antibody provided is BK-C-120-hu5, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of antibody BK-C-120, and the second antigen-binding portion has a heavy-chain variable region represented by SEQ ID NO: 45 and a light-chain variable region sequence represented by SEQ ID NO: 52. According to a specific embodiment, the bispecific antibody provided is BK-C-120-hu6, which is a bispecific antibody obtained by humanizing the second antigen-binding portion of antibody BK-C-120, and the second antigen-binding portion has a heavy-chain variable region represented by SEQ ID NO: 45 and a light-chain variable region sequence represented by SEQ ID NO: 53.

[0129] Multispecific antibodies may 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. The tumor antigens mentioned include tumor-associated antigens (TAAs). Tumor-associated antigens are antigenic substances produced by tumor cells that can elicit a host immune response. Tumor-associated antigens may be derived from primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, ovarian cancer, breast cancer, etc., but are not limited thereto. TAAs may be patient-specific. For example, it includes, but is not limited to, p53, Ras, β-catenin, CDK4, α-actinin-4, CD20, PSMA, Her2, EGFR, etc. According to a specific embodiment, the tumor antigen includes at least one of AFP, BCMA, β-catenin, 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, WT1, but is not limited thereto. 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 the second cytokine or its receptor, and the second cytokine or its receptor referred to includes, but is 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. Note that the first cytokine or its receptor and the second cytokine or its receptor may be the same or different.

[0130] The multispecific antibodies provided may 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. According to specific embodiments, the polynucleotide encoding the multispecific antibody may be utilized for expressing the multispecific antibody in vivo or in vitro. The polynucleotides mentioned are separable and include, but are not limited to, DNA, RNA, cDNA, etc. Polynucleotide sequences separated using conventional methods in the art may be obtained and used to encode the multispecific antibody.

[0131] To produce the antibody mentioned, a polynucleotide sequence encoding the antibody may be inserted into a replicable expression vector and expressed in a host cell or a cell-free expression system. For this purpose, a construct containing the above polynucleotide is also provided. A "construct" refers to any recombinant polynucleotide molecule (such as a plasmid, virus, self-replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), which may be derived from any source that can integrate with or self-replicate in the host genome and complete the expression of the antibody in the host cell. Many methods commonly used in the art, such as in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology, may all be used to obtain the construct. For example, a polynucleotide may be inserted into the multiple cloning site of an expression vector to form a construct. The construct may optionally contain various regulatory elements such as a promoter, a terminator, and a marker gene, and these regulatory elements are operably linked to the polynucleotide. The promoter is generally used to provide a signal to initiate transcription, and as the promoter, optionally, the lactose promoter (Lac), the Trp promoter, the Tac promoter, the PL and PR promoters of phage may be selected. The terminator provides a signal to stop transcription during the transcription process, and the marker gene on the construct is often used for screening. Naturally, optionally, an enhancer that enhances protein expression may be contained. There is no particular limitation on the expression vector, and some commercially available expression vectors may be used. Optionally, an expression vector after artificial modification, such as a plasmid, phage, virus, etc., may be used. The virus may be a plant cell virus, a mammalian cell virus, etc. The construct may express the antibody or protein in vitro or may be introduced into cells to express the antibody or protein.

[0132] Also provided are host cells containing the above polynucleotide or the above construct for expressing or producing a multispecific antibody as a component of a therapeutic agent. Any cell suitable for expressing an antibody or a protein by the polynucleotide or construct may be used as a host cell. The host cell may be a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a yeast cell, a mammalian cell, etc.). Commonly used host cells include yeast cells, Chinese hamster ovary (CHO) cells (e.g., CHO-K1, CHO DG44, CHO-S, CHO-DXB11, CHO-M, etc.), HEK-293 cells, COS cells, insect cells such as Drosophila S2 or Sf9, NS0 cells, Hela cells, 3T3 cells, BHK cells, etc. Host cells containing the polynucleotide or construct can be obtained using methods commonly used in the art, such as microinjection, electroporation, chemical transfection, virus-mediated transformation, etc.

[0133] The present invention also provides a first container containing the first composition in the therapeutic agent described in the present invention, and a second container containing the second composition in the therapeutic agent described in the present invention or containing the multispecific antibody mentioned in the present invention, wherein the first container and the second container are independent, and a kit having a synergistic combination agent for treating tumors and / or cancers, comprising an instruction manual describing the timing and mode of administration.

[0134] The first composition, the second composition, and the multispecific antibody mentioned in the present specification may, in some cases, be formulated into different dosage forms to meet the needs regarding administration. For example, injection preparations, powders, liquid preparations, etc. When formulating into different dosage forms, the first active ingredient or the second active ingredient may be formulated with different pharmaceutically acceptable carriers.

[0135] The present invention also provides the use of the isolated nucleic acid having the labeled polypeptide coding sequence described in the present invention in the manufacture of a drug for treating and / or preventing tumors and / or cancers.

[0136] The present invention also provides the use of the kit in the manufacture of a drug for treating and / or preventing tumors and / or cancers.

[0137] The tumors and / or cancers include breast cancer, head and neck tumors, synovial sarcoma, kidney cancer, connective tissue-derived tumors, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain tumors, liver cancer, bone tumors, 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 tumors, osteochondroma, chondrosarcoma, Ewing sarcoma, cancer of unknown primary origin, carcinoid, fibrosarcoma, Paget's disease, cervical cancer, gallbladder cancer, eye tumors, Kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multiple myeloma, ovarian cancer, pancreatic endocrine tumors, glucagonoma, pancreatic cancer, penile cancer, pituitary tumors, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymic cancer, gestational trophoblast tumor, teratoma, endometrial cancer, vaginal cancer, vulvar cancer, fungating polyposis, insulinoma, heart tumors, meningeal cancer, peritoneal cancer, pleural tumors, blood cancers.

[0138] The present invention also provides a method for treating tumors and / or cancers, which includes administering the first composition in the therapeutic agent according to the present invention to a subject, and administering the second composition in the therapeutic agent according to the present invention to the subject, or administering the multispecific antibody referred to in the present invention. "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" referred to means that it can bring about a reduction in the severity of the symptoms of the disease, an increase in the frequency and duration of the asymptomatic period of the disease, or a reduction or elimination of the pain caused by the disease. After treatment, the inhibition rate of tumor cells or cancer cells in the subject's body reaches 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, and thus 90% or more or 95% or more compared to a subject who has not received treatment.

[0139] The first composition, the second composition or the multispecific antibody in the therapeutic agent may be administered simultaneously (for example, as a mixture), separately and simultaneously (for example, each administered by intratumoral or intravenous injection), or sequentially (for example, first administering the first composition and then administering the second composition or the multispecific antibody).

[0140] Preferably, as steps to be performed sequentially, 1) First, a step of administering the first composition to the subject; 2) After administration of the first composition, a step of administering the second composition in the therapeutic agent to the subject or administering the multispecific antibody.

[0141] Preferably, on the 1st to 60th day after first administering the first composition, the second composition or the multispecific antibody in the therapeutic agent is administered to the subject.

[0142] Taking the administration of the first composition and the second composition as an example, "administering the second composition in the therapeutic agent to the subject patient on the 1st to 60th day after first administering 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 to 60 days (for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days... 60 days), or the time interval between the first administration of the second composition and the immediately preceding administration of the first composition is 1 to 60 days (for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days... 60 days). Preferably, the time interval between the first administration of the second composition and the immediately preceding administration of the first composition is 1 to 30 days (for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days... 30 days). More preferably, the time interval between the first administration of the second composition and the immediately preceding administration of the first composition is 5 to 20 days (for example, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days... 20 days).

[0143] In addition, after administering the first composition to the subject, the second composition may be administered to the subject one or more times. For example, the second composition may be administered multiple times, such as 2 times, 3 times, 4 times, etc. The number of administrations of the second composition may be adjusted based on the condition of the subject. Similarly, before administering the second composition to the subject, the first composition may be administered one or more times. For example, the first composition may be administered multiple times, such as 2 times, 3 times, 4 times, etc. The number of administrations of the first composition may be adjusted based on the condition of the subject. Also, the first composition may be administered to the subject multiple times and the second composition may be administered to the subject multiple times. The number of administrations of both the first composition and the second composition may be adjusted based on the condition of the subject.

[0144] In a preferred embodiment of the present invention, the dosage of the nucleic acid for administration is 0.01 to 10 mg / day, administered 1 to 3 times a day, and continuously administered for 1 to 7 days. The nucleic acid can encode a labeled polypeptide in the body of the subject.

[0145] In a preferred embodiment of the present invention, the dosage of the recombinant oncolytic poxvirus is such that the total dosage range for each course is about 5×10 7 ~ about 5×10 12 virus particles (either a single administration or multiple administrations may be used), or any value within the above range, or the dosage of the recombinant oncolytic poxvirus is such that the total dosage range for each course is about 5×10 7 ~ about 5×10 12 PFU (either a single administration or multiple administrations may be used), or any value within the above range. The exact dosage also depends on the judgment of those skilled in the art and individual circumstances. In the case of multiple administrations, it is administered 1 to 3 times a day for 1 to 7 consecutive days.

[0146] In a preferred embodiment of the present invention, the dosage of the multispecific antibody is 0.1 to 50 mg / kg body weight, for example, 0.5 to 50 mg / kg body weight, 1 to 50 mg / kg body weight, 2 to 49 mg / kg body weight, 3 to 48 mg / kg body weight, 4 to 47 mg / kg body weight, 5 to 46 mg / kg body weight, 6 to 45 mg / kg body weight, 7 to 44 mg / kg body weight, 8 to 43 mg / kg body weight, 9 to 42 mg / kg body weight, 10 to 41 mg / kg body weight, 11 to 40 mg / kg body weight, 12 to 39 mg / kg body weight, 13 to 38 mg / kg body weight, 14 to 37 mg / kg body weight, 15 to 36 mg / kg body weight, 16 to 35 mg / kg body weight, 17 to 34 mg / kg body weight, 18 to 33 mg / kg body weight, 19 to 32 mg / kg body weight, 20 to 31 mg / kg, 21 to 30 mg / kg body weight, 22 to 29 mg / kg body weight, 23 to 28 mg / kg body weight, 24 to 27 mg / kg body weight, 25 to 26 mg / kg body weight, 10 to 20 mg / kg body weight, 15 to 25 mg / kg body weight.

[0147] In some embodiments, the method for treating the tumor and / or cancer further includes administering to the patient other drugs for treating the tumor and / or cancer, and / or drugs for regulating the patient's immune system, in order to improve the effect of tumor treatment. The other drugs for treating the tumor and / or cancer include, but are not limited to, chemotherapeutic drugs (e.g., cyclophosphamide, fludarabine), radiotherapeutic drugs, immunosuppressive drugs (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate, FK50), antibodies (e.g., antibodies against CD3, IL-2, IL-6, IL-17, TNFa).

[0148] The DNA may be formulated for intratumoral injection or intravenous administration, and the mRNA may be formulated for intratumoral injection or intravenous administration. For example, it may be directly injected into the tumor in the form of a plasmid, or may be injected into the tumor after being packaged with liposomes, or may be ligated to nanoparticles (e.g., polymers such as polylysine, polyamino acid, polyethyleneimine, chitosan) and then injected into the tumor, and the transfection rate may be improved by electrotransformation after intratumoral injection.

[0149] The recombinant virus may be formulated for different administration methods including, but not limited to, intratumoral injection, intravenous injection, intracranial injection, intraperitoneal injection, etc. Any administration method of injecting the recombinant virus into the body of the subject to exert a therapeutic effect is acceptable. The administration method may be adjusted according to the needs of the subject, or multiple administration methods may be combined for administration. The multispecific antibody may be administered in various ways such as, for example, intratumoral injection, intravenous injection, intraperitoneal injection, etc. The administration method of the multispecific antibody may be adjusted according to the needs of the subject, or multiple administration methods may be combined for administration.

[0150] The present invention also provides a novel immunotherapy, which comprises administering a therapeutically effective amount of an oncolytic virus to a subject, wherein the genome of the oncolytic virus contains a labeled polypeptide coding sequence, the labeled polypeptide has an extracellular antigen determinant, a spacer portion, and a transmembrane portion that are operably linked, the amino acid sequence of the extracellular antigen determinant contains the amino acid sequence of one or more epitope polypeptides, and in the natural state, the amino acid sequence of the cell membrane protein or secreted protein of the subject does not contain the amino acid sequence of the 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 antigen determinant of the labeled polypeptide, and 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 optionally further comprise 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. According to a specific embodiment, the second antigen-binding portion is capable of specifically recognizing and binding to an immune cell surface antigen.

[0151] According to a specific embodiment, as sequential steps, 1) administering a therapeutically effective amount of an oncolytic virus to the subject; and 2) after administration of the oncolytic virus, administering a therapeutically effective amount of the multispecific antibody to the subject.

[0152] The present invention also provides anti-CD3 antibodies that can specifically target the CD3 antigen and exhibit higher affinity for human and cynomolgus monkey CD3 antigens through sequence optimization and humanization modification of mouse SP34. According to specific embodiments, the anti-CD3 antibody has a heavy chain variable region sequence represented by SEQ ID NO: 43, 44, or 45. According to specific embodiments, the anti-CD3 antibody has a light chain variable region sequence represented by 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 represented by SEQ ID NO: 44 and a light chain variable region sequence represented by SEQ ID NO: 51. According to specific embodiments, the anti-CD3 antibody has a heavy chain variable region represented by SEQ ID NO: 44 and a light chain variable region sequence represented by SEQ ID NO: 52. According to specific embodiments, the anti-CD3 antibody has a heavy chain variable region represented by SEQ ID NO: 44 and a light chain variable region sequence represented by SEQ ID NO: 53. According to specific embodiments, the anti-CD3 antibody has a heavy chain variable region represented by SEQ ID NO: 45 and a light chain variable region sequence represented by SEQ ID NO: 51. According to specific embodiments, the anti-CD3 antibody has a heavy chain variable region represented by SEQ ID NO: 45 and a light chain variable region sequence represented by SEQ ID NO: 52. According to specific embodiments, the anti-CD3 antibody has a heavy chain variable region represented by SEQ ID NO: 45 and a light chain variable region sequence represented by SEQ ID NO: 53. The heavy chain constant domain sequence in the antibody is the one represented by SEQ ID NO: 57, and the light chain constant domain sequence is the one represented by SEQ ID NO: 55. According to specific embodiments, the provided anti-CD3 antibody binds to human CD3 or monkey (e.g., cynomolgus) CD3 at an EC 50 of 10 nM or less, and for example, the EC 50 value for affinity to 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.

[0153] The technical solution of the present invention will be described in detail below with reference to examples. These examples are merely provided to facilitate understanding by those skilled in the art and should not be regarded as a limitation on the protection scope of the present invention. The examples of the present invention will be described in detail below, and the described examples are illustrative and provided for interpreting the present invention and should not be understood as a limitation on the present invention.

Example

[0154] (Example) Information on the cell lines used in the examples is as follows. The human ovarian cancer cell line SKOV3, the human colorectal cancer cell line HCT116, and the human pancreatic cancer cell line PANC-1 were all purchased from ATCC.

[0155] The human liver cancer cell line SK-HEP-1, the human glioma cell line U251, and the human melanoma cell line A375 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

[0156] The MC38 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0157] The human fetal kidney cells 293T were provided by Shanghai Genechem Co., Ltd.

[0158] The human peripheral blood mononuclear cells (PBMC) were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd.

[0159] The Jurkat cells were purchased from Shanghai Ruizhi Chemical Research Co., Ltd.

[0160] SKOV3 and HCT116 were cultured in McCoy 5A (a commercial product purchased from Gibco) + 10% FBS (a commercial product purchased from PAN), SK-HEP-1 and U251 were cultured in MEM (a commercial product purchased from Gibco) + 10% FBS, and PANC-1, A375, and 293T were cultured in DMEM (a commercial product purchased from Gibco) + 10% FBS.

[0161] The buffer for flow cytometry was 1×DPBS + 2% FBS. 1×DPBS was a commercial product purchased from Anhui White Shark Biotechnology Co., Ltd., and FBS was a commercial product purchased from PAN.

[0162] (Packaging of LV-TT3-GFP lentivirus) After digesting 293T cells with 0.25% trypsin (a commercial product purchased from Beyotime Biotech Inc) to obtain single cells, 2×10 6Resuspended in the packaging medium at / mL. The composition of the packaging medium was Opti-MEM (commercially available product purchased from Gibco) + 5% FBS (commercially available product purchased from Gibco) + sodium pyruvate (commercially available product purchased from Gibco, final concentration 200 mM). Inoculated into a 6-well plate at 1 mL / well. A lentiviral plasmid mixture was prepared based on the Lipo3000 instruction manual. That is, (1) 250 μL of Opti-MEM + 7 μL of Lipo3000, (2) 250 μL of Opti-MEM + 6 μL of p3000 + lentiviral 4 plasmids (molar mass ratio = 1:1:1:1) were prepared. Next, after uniformly mixing (1) and (2), incubated at room temperature for 15 minutes, added 500 μL of the plasmid mixture to each well of the 6-well plate, then uniformly mixed and placed in an incubator for continuous culture. After 48 hours of transfection, the culture solution was obtained, centrifuged at 3000 rpm for 10 minutes, then the viral supernatant was obtained, aliquoted, and stored frozen at -80 °C.

[0163] (Construction of HCT116 cell lines (HCT116-TT3) and SK-HEP-1 (SK-HEP-1-TT3) stably expressing TT3) After digesting wild-type HCT116 with 0.25% trypsin to obtain single cells, 1×10 5Inoculate into a 48-well plate at 500 μL / well, add 300 μL of lentivirus LV-TT3-GFP and polybrene (a commercial product purchased from Shanghai Yisheng Biotechnology Co., Ltd., final concentration 8 μg / mL), place in an incubator for culture, passage and amplify. After one week, digest the infected cells and inoculate into a 96-well plate at 2 cells / well, with 100 μL per well, and place in an incubator for continuous culture. Continuously culture for about 10 days until monoclonal cells appear, then select wells under a microscope where all cells are GFP-positive and there is only one cluster of cell clones, passage and amplify. Measure the expression of GFP and TT3 by flow cytometry, screen based on the expression rate and intensity of GFP and TT3, and finally obtain the optimal monoclonal clone to obtain a stable transfected cell line (HCT116-TT3) expressing the TT3 antigen.

[0164] In the same way, a stable transfected cell line SK-HEP-1-TT3 expressing the TT3 antigen was constructed.

[0165] (Preparation of T cells) After resuscitating PBMC cells, add complete medium, incubate in an incubator for 2 hours. When the incubation is completed, inoculate into a 24-well plate at 5×10 5 / 500 μL / well, add 10 μL of Dynabeads® Human T-Activator CD3 / CD28 (a commercial product purchased from Gibco) to activate. Subsequently, replenish the complete medium every other day according to the cell growth state, continuously culture until the 10th day, remove the magnetic beads and cryopreserve the cells. The complete medium is AIM-V + 5% FBS + hIL-2 (final concentration 100 IU / mL), AIM-V is a commercial product purchased from Gibco, and human recombinant interleukin 2 (hIL-2) is a commercial product purchased from Jiangsu Kingsley Pharmaceutical Co., Ltd.

[0166] The poxvirus (DDVV-TT3) was constructed. The poxvirus DDVV-TT3 may be obtained by referring to the method described in the PCT patent with the international publication number WO2020038490A (international application number PCT / CN2019 / 102480). In the patent WO2020038490A, the poxvirus is denoted as vvDD-TT3. Both represent the same recombinant poxvirus.

[0167] The humanized anti-CD3 monoclonal antibody and the results of its activity analysis mentioned were specifically as follows. A humanized sequence template was searched in the database using the SP34 antibody sequence, and the importance of different amino acids in the 3D structure was analyzed. If necessary, reverse mutations of important amino acids that affect the structure were performed to determine the corresponding humanized antibody. The humanized antibody was fused with a human-derived constant region sequence and constructed into a pcDNA3.1 mammalian expression vector to construct a chimeric antibody. Next, the expression and purification of different antibodies obtained using a mammalian cell line were carried out to obtain different antibodies with a purity of at least 90% or more. The expressed antibody sequences are CD3 Ab01 to CD3 Ab15, respectively. The expressed antibodies are IgG-type antibodies. The heavy-chain constant domain of the antibody is shown by SEQ ID NO: 57, and the light-chain constant domain of the antibody is shown by SEQ ID NO: 55. The numbers of the sequences of the heavy-chain variable region and the light-chain variable region of each antibody in this specification are as follows. The heavy-chain variable region of antibody CD3 Ab01 is shown by SEQ ID NO: 43, and the light-chain variable region is shown by SEQ ID NO: 46. The heavy-chain variable region of antibody CD3 Ab02 is shown by SEQ ID NO: 43, and the light-chain variable region is shown by SEQ ID NO: 47. The heavy-chain variable region of antibody CD3 Ab03 is shown by SEQ ID NO: 43, and the light-chain variable region is shown by SEQ ID NO: 48. The heavy-chain variable region of antibody CD3 Ab04 is shown by SEQ ID NO: 43, and the light-chain variable region is shown by SEQ ID NO: 49. The heavy-chain variable region of antibody CD3 Ab05 is shown by SEQ ID NO: 43, and the light-chain variable region is shown by SEQ ID NO: 50. The heavy-chain variable region of antibody CD3 Ab06 is shown by SEQ ID NO: 44, and the light-chain variable region is shown by SEQ ID NO: 46. The heavy-chain variable region of antibody CD3 Ab07 is shown by SEQ ID NO: 44, and the light-chain variable region is shown by SEQ ID NO: 47. The heavy-chain variable region of antibody CD3 Ab08 is shown by SEQ ID NO: 44, and the light-chain variable region is shown by SEQ ID NO: 48. The heavy-chain variable region of antibody CD3 Ab09 is shown by SEQ ID NO: 44, and the light-chain variable region is shown by SEQ ID NO: 49. The heavy-chain variable region of anti-CD3 Ab10 is shown by SEQ ID NO: 44, and the light-chain variable region is shown by SEQ ID NO: 50.The heavy chain variable region of antibody CD3 Ab11 is shown by SEQ ID NO: 45, and the light chain variable region is shown by SEQ ID NO: 46. The heavy chain variable region of antibody CD3 Ab12 is shown by SEQ ID NO: 45, and the light chain variable region is shown by SEQ ID NO: 47. The heavy chain variable region of antibody CD3 Ab13 is shown by SEQ ID NO: 45, and the light chain variable region is shown by SEQ ID NO: 48. The heavy chain variable region of antibody CD3 Ab14 is shown by SEQ ID NO: 45, and the light chain variable region is shown by SEQ ID NO: 49. The heavy chain variable region of antibody CD3 Ab15 is shown by SEQ ID NO: 45, and the light chain variable region is shown by SEQ ID NO: 50.

[0168] Using the ELISA method, the activity of the prepared humanized antibody was measured. The experimental procedure was as follows. hCD3 (human CD3 antigen) was diluted to 0.5 μg / mL using the coating solution, and then added to a 96-well plate at 100 μL / well and incubated overnight at 4°C. It was washed with PBST, and then 200 μL of the blocking solution was added to each well and blocked at 37°C for 2 hours. It was washed with PBST, and then the diluted antibody was added at 100 μL / well (initial concentration 40 nM, 1:6 serial dilution) and incubated at 37°C for 2 hours. It was washed with PBST, and then HRP-labeled goat anti-human IgG was added at 100 μL / well and incubated at 37°C for 1 hour. It was washed with PBST, 100 μL / well of TMB was added, and color development was carried out at 37°C for 10 - 20 minutes. 50 μL of 2M H 2 SO 4 was added to stop the reaction. The numerical value was read at 450 nm. The EC 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, CD3 Ab15 50The measurement results were 0.0096 nM, 0.0117 nM, 0.0042 nM, 0.0068 nM, 0.0099 nM, 0.0126 nM, 0.0129 nM, 0.0116 nM, 0.0080 nM, 0.0020 nM, 0.0118 nM, 0.0095 nM, 0.0099 nM, 0.0087 nM, and 0.0099 nM respectively.

[0169] The binding activity of the humanized antibody produced was measured using FACS technology. The experimental procedure was as follows. Jurkat cells or 293T-CD3 cells were seeded, washed with FACS buffer, centrifuged at 1200 rpm for 3 - 5 minutes, then different concentrations of diluted antibody were added, incubated at 4°C for 30 minutes, centrifuged at 1200 rpm for 3 - 5 minutes, washed with FACS buffer, then goat anti-human IgG secondary antibody (manufactured by Abcam, ab98596) was added at 100 μL / well, incubated at 4°C for 30 minutes, centrifuged at 1200 rpm for 3 - 5 minutes, washed with FACS buffer, 50 μL of FACS buffer was added to resuspend the cells, and the measurement was performed using a flow cytometer. In addition, the EC 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, CD3 Ab15 against Jurkat cells by FACS 50 The measurement results were 3.299 nM, 2.642 nM, 2.284 nM, 1.182 nM, 1.024 nM, 3.309 nM, 2.415 nM, 1.823 nM, 0.862 nM, 0.784 nM, 4.451 nM, 3.518 nM, 3.194 nM, 1.142 nM, and 1.108 nM respectively, and the EC of the antibodies against 293T-CD3 cells by FACS 50 The measurement results were 22.600 nM, 12.300 nM, 10.060 nM, 5.234 nM, 4.267 nM, 12.990 nM, 9.020 nM, 4.238 nM, 4.401 nM, 2.438 nM, 21.550 nM, 15.770 nM, 12.360 nM, 5.728 nM, and 3.479 nM respectively.

[0170] The humanized anti-TT3 monoclonal antibody and the results of its activity analysis mentioned above were specifically as follows. Humanization modification was performed on the antibody (the number is Ab-TT3, its heavy chain variable region is SEQ ID NO: 26, and its light chain variable region is SEQ ID NO: 27). Specifically, a humanized sequence template was searched in the database with the Ab-TT3 antibody sequence. To maintain the three-dimensional structure of the CDR region, attention should be paid to three types of residues: residues located at the binding interface of VL and VH, residues near the CDR region and buried inside the protein, and residues that directly interact with the CDR region. The interactions include hydrophobic interactions, hydrogen bonds, and salt bridges. The importance of different amino acids in the 3D structure was analyzed, and if necessary, reverse mutations of important amino acids that affect the structure were performed to determine the corresponding humanized antibody.

[0171] The humanized antibody was fused with the human-derived constant region sequence and constructed into the pcDNA3.1 mammalian expression vector to construct a chimeric antibody. Next, the expression and purification of different antibodies obtained using the mammalian cell line were performed to obtain different antibodies with a purity of at least 90% or more. The expressed antibodies are TT3 Ab-1, TT3 Ab-2, and TT3 Ab-3 respectively. The heavy chain constant domain of the antibody is shown by SEQ ID NO: 57, and the light chain constant domain of the antibody is shown by SEQ ID NO: 55. TT3 Ab-1 has a heavy chain variable region shown by SEQ ID NO: 29 and a light chain variable region shown by SEQ ID NO: 32. TT3 Ab-2 has a heavy chain variable region shown by SEQ ID NO: 30 and a light chain variable region shown by SEQ ID NO: 33. TT3 Ab-3 has a heavy chain variable region shown by SEQ ID NO: 31 and a light chain variable region shown by SEQ ID NO: 34. In addition, the Fc region of the TT3 Ab-3 antibody was mutated, and the antibody number after mutation is TT3 Ab-3mu. Its heavy chain constant domain is shown by SEQ ID NO: 54, and its light chain constant domain is shown by SEQ ID NO: 55.

[0172] The activity of the humanized antibody produced was measured using the ELISA method. The experimental procedure was as follows. The TT3 antigen was diluted to 0.5 μg / mL using the coating solution and added to a 96-well plate at 100 μL / well, and incubated overnight at 4°C. It was washed with PBST, and then 200 μL of the antibody blocking solution was added to each well and blocked at 37°C for 2 hours. It was washed with PBST, different antibodies were added at 100 μL / well (initial concentration 100 nM, 1:6 gradient dilution), and incubated at 37°C for 2 hours. It was washed with PBST, HRP-conjugated goat anti-human / mouse IgG antibody was added at 100 μL / well, and incubated at 37°C for 1 hour. It was washed with PBST, TMB was added at 100 μL / well, developed color at 37°C for 10 - 20 minutes, and 50 μL of 2M sulfuric acid was added to each well to stop the reaction. The OD value was read at a wavelength of 450 nm. The EC 50 values of the 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.

[0173] The activity of the humanized antibody produced was measured using the FACS method, and the experimental procedure was as follows. Different cells (HCT116-TT3, SK-HEP-1-TT3, MC38-TT3) were seeded, then washed with FACS buffer and centrifuged at 1200 rpm for 3 - 5 minutes. Diluted antibodies at different concentrations were added (initial concentration 100 nM, 1:5 gradient dilution), incubated at 4°C for 30 minutes, and centrifuged at 1200 rpm for 3 - 5 minutes. It was washed with FACS buffer, then the goat anti-human IgG secondary antibody (Abcam, ab98596) was diluted 1:250 and added to the plate at 100 μL / well, incubated at 4°C for 30 minutes, centrifuged at 1200 rpm for 3 - 5 minutes, washed with FACS buffer, then 50 μL of FACS buffer was added to resuspend the cells, and measured with a flow cytometer. Here, the measurement results of the affinity of the antibodies TT3 Ab-1, TT3 Ab-2, and TT3 Ab-3 for HCT116-TT3 cells by FACS EC 50They were 0.568 nM, 0.958 nM, and 0.692 nM respectively, which were the EC results of the affinity measurement for SK-HEP-1-TT3 cells by FACS. 50 They were 0.546 nM, 1.527 nM, and 0.738 nM respectively, which were the EC results of the affinity measurement for MC38-TT3 cells by FACS. 50 They were 1.201 nM, 2.327 nM, and 0.588 nM respectively.

[0174] When the antibody after Fc mutation (TT3 Ab-3mu) was measured by the same method, both before and after Fc mutation, it could bind to the TT3 antigen, had biological activity, and there was not much difference in the binding activity between the two.

[0175] (Example 1) The bispecific antibody BK-A-101 was prepared by genetic engineering methods. Specifically, after synthesizing the nucleotide sequences encoding the heavy chain part and the light chain part of the bispecific antibody, they were cloned into the pcDNA3.1 expression vector (purchased from GenScript) by ordinary techniques in the art. Next, the bispecific antibody protein was expressed by transient transfection of CHO-S cells by the electrotransformation method, the supernatant was collected, purified by a protein A affinity column, and the purity of the purified antibody was measured by HPLC and SDS-PAGE. The measurement showed that the purity of the bispecific antibody BK-A-101 was 90% or more. Next, the binding activity of the bispecific antibody BK-A-101 with the target antigen was measured by flow cytometry. 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, BK-A-101-hu6) were obtained by the same method.

[0176] Using a similar method, transient transfection of 293F cells by electroporation yielded 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, BK-B-110-hu6), bispecific antibodies with BK-C structure (BK-C-120, BK-C-120-hu1, BK-C-120-hu2, BK-C-120-hu3, BK-C-120-hu4, BK-C-120-hu5, BK-C-120-hu6), and bispecific antibodies with BK-D structure (including 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, BK-D-13).

[0177] The binding activity of the bispecific antibody to T cells was measured by flow cytometry (FACS), and the steps were as follows. T cells were resuspended in a buffer for flow cytometry and dispensed into a 96-well plate. Next, different antibodies were prepared and incubated. Group 1 was a blank control group and no antibody was added for staining. Group 2 was added with an isotype control antibody (negative control, a commercial product purchased from Biolegend, human IgG1), and its final concentration was 50 nM. Group 3 was added with an anti-CD3 monoclonal antibody (L2K antibody, a commercial product purchased from KYinno Biotechnology Co., Ltd, the same anti-CD3 monoclonal antibody used below), and its final concentration was 50 nM. Group 4 was added with the BK-A-101 bispecific antibody, and its final concentration was 50 nM each. After each group was incubated at 2-8 °C for 30 minutes, it was diluted, washed, and resuspended with a buffer for flow cytometry. Each group was added with a PE-labeled anti-human IgG secondary antibody (a commercial product purchased from ABCAM, diluted 1:200) and incubated at 2-8 °C for 30 minutes. After dilution and washing with a buffer for flow cytometry, it was resuspended with 200 μL of a buffer for flow cytometry and measured with a flow cytometer (Novocyte). The results are shown in Figure 5. The measurement results by flow cytometry of the blank control and negative control were negative. The positive rate measured by flow cytometry for the positive control was 99.45%, and the positive rate measured from the bispecific antibody was 99.44%. This result reveals that the bispecific antibody BK-A-101 has high T cell binding activity.

[0178] The binding activity of the bispecific antibody BK-A-101 to HCT116 cells or HCT116-TT3 cells was measured by flow cytometry (FACS), and the steps were as follows. HCT116 and HCT116-TT3 cells were digested with 0.25% trypsin respectively to obtain single cells, which were then resuspended in the buffer for flow cytometry. The BK-A-101 bispecific antibody was added respectively, and its final concentration was set to 0.1 nM, 1 nM, 10 nM, and 100 nM respectively. After uniform mixing, it was incubated at 2-8°C for 30 minutes. After dilution and washing with the buffer for flow cytometry, it was resuspended. Each group was added with a PE-labeled anti-human IgG secondary antibody (1:200 dilution) and incubated at 2-8°C for 30 minutes. After dilution and washing with the buffer for flow cytometry, it was resuspended with 200 μL of the buffer for flow cytometry and measured with a flow cytometer (Novocyte). The results are shown in Figures 6 and 7.

[0179] Among them, Figure 6 shows the measurement results of the binding activity of the bispecific antibody to the HCT116 cell line. The results reveal that the bispecific antibody does not show binding activity to the HCT116 cell line even when its concentration increases. Figure 7 shows the binding activity of the bispecific antibody BK-A-101 to HCT116-TT3 cells. The results indicate that as the concentration of the bispecific antibody increases, the positive rate measured by flow cytometry shows a tendency to gradually increase. The results of Figures 6 and 7 demonstrate that the bispecific antibody has specificity in binding to TT3.

[0180] In the same way, the binding activities of the bispecific antibody to the SK-HEP-1 cell line and the SK-HEP-1-TT3 cell line were measured. As is obvious from the experimental results, the bispecific antibody does not show binding activity to the SK-HEP-1 cell line, but shows binding activity to SK-HEP-1-TT3. Moreover, as the concentration of the bispecific antibody increases, the binding rate shows a tendency to gradually increase. Therefore, it is further proved that the bispecific antibody has specificity in binding to TT3.

[0181] By flow cytometry, the affinity (i.e., avidity) 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, BK-D-12) with the CD3 terminus and the TT3 terminus was measured. The measurement method and results were as follows respectively. Jurkat cells were seeded, washed with FACS buffer, centrifuged at 1200 rpm for 3 - 5 minutes, then different concentrations of diluted antibodies were added at 100 μL / well, incubated at 4°C for 30 minutes, centrifuged at 1200 rpm for 3 - 5 minutes, washed with FACS buffer, then diluted goat anti-human IgG (FITC, Abcam, ab97224) antibody was added, incubated at 4°C for 30 minutes, centrifuged at 1200 rpm for 3 - 5 minutes, washed with FACS buffer, 50 μL of FACS buffer was added to resuspend the cells, and the cells were measured with a flow cytometer.

[0182] Similarly, the affinity of the bispecific antibodies with the TT3 terminus was measured, and the experimental steps were as follows. HCT116-TT3 cells were seeded, washed with FACS buffer, centrifuged at 1200 rpm for 3 - 5 minutes, then different concentrations of diluted antibodies were added, incubated at 4°C for 30 minutes, centrifuged at 1200 rpm for 3 - 5 minutes, washed with FACS buffer, diluted goat anti-human IgG Fc (Dylight 650, Abcam, ab98622) antibody was added, incubated at 4°C for 30 minutes, centrifuged at 1200 rpm for 3 - 5 minutes, washed with FACS buffer, 50 μL of FACS buffer was added to resuspend the cells, and the cells were measured with a flow cytometer.

[0183] The respective measurement results are shown in the following table and Figure 8.

Table 5

[0184] As is clear from the experimental results, the prepared bispecific antibody showed high affinity with the TT3 terminus and the CD3 terminus. The affinity of the bispecific antibody with the TT3 terminus was measured using SKHEP1-TT3 cells or MC38-TT3 cells in the same way, and the results revealed that the bispecific antibody showed high affinity with the TT3 terminus.

[0185] Other bispecific antibodies with BK-B and BK-D structures also showed similar affinities with the CD3 terminus and the TT3 terminus.

[0186] Furthermore, the non-specific binding of the bispecific antibody with wild-type tumor cell lines was measured by FACS. The steps were as follows. HCT116 and SK-HEP-1 cells were digested with 0.25% trypsin respectively to obtain single cells, and then resuspended in the buffer for flow cytometry. BK-A-101 and BK-D-01-09 bispecific antibodies were added respectively, with their final concentrations being 10 nM and 100 nM. After uniform mixing, they were incubated at 2-8 °C for 30 minutes. After dilution and washing with the buffer for flow cytometry, they were resuspended. Each group was added with an APC-labeled anti-human IgG secondary antibody (1:200 dilution) and incubated at 2-8 °C for 30 minutes. After dilution and washing with the buffer for flow cytometry, they were resuspended in 200 μL of the buffer for flow cytometry and measured with a flow cytometer (Novocyte). The results are shown in Figure 9. The bispecific antibody BK-A-101 and BK-D-01-09 groups had a low non-specific binding rate under the normal concentration of 10 nM. The bispecific antibody BK-D-01, 02, 03, 04, 05, 06, 08 groups also had a low non-specific binding rate even under the very high concentration of 100 nM. This reveals that when there is no specific target, the bispecific antibody of the present invention shows low non-specific binding characteristics and has good safety.

[0187] (Example 2) Example 2 showed the measurement by an RTCA real-time killing analyzer of the killing effect on HCT116 and HCT116-TT3 cell lines of the combined use of a bispecific antibody and pre-activated and amplified T cells.

[0188] First, 96 E-Plates (commercial products purchased from Agilent Technologies Co., Ltd.) were equilibrated with 50 μL of McCoy 5A + 10% FBS culture medium. Next, wild-type HCT116 and HCT116-TT3 were digested with 0.25% trypsin respectively to obtain single cells, resuspended in McCoy 5A + 10% FBS culture medium, and 5×10 3 cells / 50 μL / well of tumor cells were seeded in 96 E-Plates. Five hours after seeding, T cells were added to the corresponding wells of the 96 E-Plates at a cell ratio of E:T = 2:1 (E:T is the effector / target ratio, representing the ratio of effector cells to target cells), at 50 μL / well. At the same time, after gradient dilution of the bispecific antibody (BK-A-101) or anti-CD3 monoclonal antibody (CD3 Ab), they were added to the corresponding wells of the 96 E-Plates. 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 of each well was 200 μL, and it was set to collect data with one scan every 15 minutes.

[0189] The results are shown in Figure 10. The single action of T cells and the combined use of CD3 Ab or BK-A-101 at each concentration with T cells did not show a significant killing effect on wild-type HCT116. The single action of T cells and the combined use of CD3 Ab at each concentration with T cells also did not show a significant killing effect on HCT116-TT3. However, the combined use of BK-A-101 at each concentration with T cells showed a very significant killing effect on HCT116-TT3 and showed a dose-correlation with the concentration of BK-A-101. Specifically, the combined use of 1 nM and 1 nM of BK-A-101 with T cells could almost completely remove HCT116-TT3, showing good continuity. The combined use of 0.1 nM of BK-A-101 with T cells could almost completely remove HCT116-TT3 by 40 hours.

[0190] The measurement results of the bispecific antibody with the BK-D structure were obtained in the same way and were different in the following points. After seeding the tumor cell line and incubating overnight, effector cells and bispecific antibody were added, and the ratio of effector cells to target cells was 5:1. The results are shown in Figure 11. Among them, A and B in Figure 11 show the killing effects of the HCT116-TT3 cell line by the combined use of different concentrations (0.1 nM or 1 nM) of the bispecific antibody and pre-activated and amplified T cells, respectively. Among them, numbers 1 to 9 represent the HCT116-TT3 + T cell + bispecific antibody BK-D-01 to BK-D-09 groups, number 10 represents the HCT116-TT3 + T cell + BK-A-101 group, number 11 represents only the HCT116-TT3 cells, indicating that bispecific antibody and T cells were not added for treatment, and number 12 represents that only T cells were added to treat the HCT116-TT3 cells and no bispecific antibody was added. As can be seen from the results, the combined use of the bispecific antibody of the present invention with T cells can almost completely remove specific cancer cells at each concentration, showing excellent continuity and excellent tumor cell killing effect.

[0191] The present inventors tested the killing of wild-type tumor cell lines HCT116 (A in Fig. 12) and SK-HEP-1 (B in Fig. 12) at a high concentration of bispecific antibody in a similar manner. Among them, numbers 1 to 6 represent wild-type tumor cell line + T cell + bispecific antibody BK-D-01, 04, 05, 06, 07, 08 groups, number 7 represents wild-type tumor cell line + T cell + TT3 monoclonal antibody-1 group, number 8 represents wild-type tumor cell line + T cell + CD3 monoclonal antibody SP34 group, number 9 represents only wild-type tumor cell line cells without adding antibodies or T cells for treatment, and number 10 represents adding only T cells to wild-type tumor cell line cells for treatment without adding antibodies. In A of Fig. 12, the concentration of the bispecific antibody is 100 nM, and in B of Fig. 12, the concentration of the bispecific antibody is 10 nM. The results are shown in Fig. 12. Even at a very high concentration, the bispecific antibodies BK-D-04, 05, 06, 07, 08 of the present invention did not show a significant killing effect on wild-type cell lines, indicating that these bispecific antibodies have good killing specificity.

[0192] (Example 3) Example 3 showed the measurement of the killing effect of the combination of bispecific antibody and PBMC cells on the HCT116 cell line by an RTCA real-time killing analyzer.

[0193] After resuscitating PBMC, it was resuspended in complete culture medium and recovered overnight in an incubator. The next day, first, 96 E-Plates (Agilent) were equilibrated with 50 μL of McCoy 5A + 10% FBS culture medium. Next, for HCT116-TT3, it was digested with 0.25% trypsin to obtain single cells, resuspended in McCoy 5A + 10% FBS culture medium, and 5×10 3Tumor cells were inoculated into 96 E-Plates at 1 cell / 50 μL / well. Five hours after seeding, PBMCs were added to the corresponding wells of the 96 E-Plates at a cell ratio of E:T = 2:1, with a volume of 50 μL / well. At the same time, BK-A-101 was serially diluted and then added to the corresponding wells of the 96 E-Plates. The final concentrations of BK-A-101 were 10 nM and 1 nM, respectively. The final volume of each well was 200 μL, and data was set to be collected by scanning once every 15 minutes.

[0194] The results are shown in Figure 13. The single action of PBMC cells showed no significant killing effect on HCT116-TT3. However, the combined use of BK-A-101 at each concentration and PBMC cells showed a very significant killing effect on HCT116-TT3. The combined use of 10 nM and 1 nM BK-A-101 and PBMC cells could almost completely remove HCT116-TT3, and there was good continuity.

[0195] (Example 4) In Example 4, the binding of BK-A-101 after the infection of human tumor cell lines of DDvv-TT3 was measured by flow cytometry (FACS). The steps were as follows. SKOV3, PANC1, A375, and U251 were digested with 0.25% trypsin to obtain single cells, respectively, and then resuspended in their respective complete media at 1×10 5Cells were seeded in a 6-well plate at 2 mL / well. The next day, the 6-well plate was taken out, the culture medium was discarded, and the DDvv-TT3 recombinant oncolytic poxvirus was serially diluted with the corresponding medium supplemented with 5% FBS and then added at 500 μL / well. The MOIs were 2, 0.2, and 0.02. After gently shaking to make it uniform, it was placed in an incubator at 37 °C and incubated for 2 hours. Then, the 6-well plate was taken out, and each well was replenished with 1.5 mL of medium containing 5% FBS to make it 2 mL. After gently shaking to make it uniform, it was placed in the incubator and cultured continuously. After infecting the cells with the virus for 48 hours, the cells were obtained and resuspended in the buffer for flow cytometry. Then, the following antibodies were added respectively and incubated at 2 - 8 °C for 30 minutes.

[0196] Group 1: Isotype control antibody (isotype, human IgG1, negative control, final concentration 100 nM) Group 2: Bispecific antibody BK-A-101 (final concentration 100 nM) Group 3: Anti-Strep tag II antibody (positive control, anti-TT3 monoclonal antibody, a commercial product purchased from GenScript, final concentration 10 μg / mL)

[0197] After washing and resuspending with the buffer for flow cytometry, here, for Group 1 and Group 2, a PE-labeled anti-human IgG secondary antibody (a commercial product purchased from ABCAM, diluted 1:200) was added respectively, and for Group 3, a PE-labeled anti-mouse IgG antibody (a commercial product purchased from ABCAM, diluted 1:200) was added and incubated at 2 - 8 °C for 30 minutes. After diluting and washing with the buffer, it was resuspended with 200 μL of the buffer and measured with a flow cytometer (Novocyte).

[0198] The results are shown in Fig. 14. As the MOI value increased, all the measurement results by flow cytometry of the isotype control group were negative, while the measurement results by flow cytometry of the positive control and bispecific antibody staining group showed a tendency to gradually increase. As is clear from the experimental results, after infecting human tumor cell lines with DDvv-TT3 and adding bispecific antibodies, they could specifically bind to the tumor cells infected with oncolytic virus, and moreover, as the MOI value increased, the binding percentage increased.

[0199] (Example 5: Experiment on Stimulating Proliferation and Activation of T Cells) In Example 5, flow cytometry (FACS) was used to measure the activation of T cells after 3-day co-incubation of bispecific antibodies with T cells (A in Fig. 15) or PBMC (B in Fig. 15) cultured in vitro. The steps were as follows. Pre-activated and amplified T cells or PBMC were resuspended in complete medium at 1×10 5Cells were inoculated into a 48-well plate at 150 μL / well. After inoculation, 150 μL of bispecific antibody diluted to different concentrations was added to the corresponding wells. The final concentrations of the bispecific antibody were 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, and 250 nM. The blank group was added with no bispecific antibody but only an equal volume of blank medium. After culturing for 3 days, the 48-well plate was taken out, and the cells of each group were obtained. After dilution and washing with buffer for flow cytometry and then resuspension, FITC anti-human CD3, PE anti-human CD4, APC-Cy7 anti-human CD25, and APC anti-human CD127 were added to each group. After uniform mixing, the mixture was incubated at 2-8 °C for 30 minutes. After dilution and washing with buffer for flow cytometry, it was resuspended with 200 μL of buffer for flow cytometry and measured with a flow cytometer (Novocyte). The results are shown in Figure 15. In the bispecific antibody BK-D-01~09 groups, the expression rates of the activation marker CD25 on the T cell surface were all low under normal concentrations such as 0.01 nM, 0.1 nM, 1 nM, and 10 nM. In the bispecific antibody BK-D-04~09 groups, the expression rates of the activation marker CD25 on the T cell surface were also low even under very high concentrations such as 100 nM and 250 nM. These results demonstrate that in the absence of specific targets, the bispecific antibody of the present invention exhibits low T cell activation ability and has good safety.

[0200] In Example 5, the release of cytokine IFN-γ in the culture supernatant after 3-day co-incubation of the bispecific antibody with PBMC and SK-HEP1 cell line was also measured by ELISA (Figure 16), and the final concentrations of the bispecific antibody were 0.1 nM, 1 nM, 10 nM, 100 nM, and 250 nM. In Figure 16, A shows the results of 3-day co-culture of PBMC and the bispecific antibody, B shows the results of 3-day co-culture of PBMC, the bispecific antibody, and the wild-type SK-HEP-1 cell group, and C shows the results of 3-day co-culture of PBMC, the bispecific antibody, and SK-HEP-1-TT3. As is clear from the results, when the bispecific antibody was co-cultured with PBMC or the bispecific antibody was co-cultured with PBMC and SK-HEP-1, in the case of the bispecific antibody BK-D-01, 04-08 groups, the secretion amounts of IFN-γ were all low under normal concentrations such as 0.1 nM, 1 nM, and 10 nM. When the bispecific antibody was co-cultured with PBMC, in the case of the bispecific antibody BK-D-04, 05, 06 groups, the secretion amounts of IFN-γ were also low even under very high concentrations such as 100 nM and 250 nM. These results reveal that in the case of no specific target, the bispecific antibody of the present invention has low T cell activation ability and good safety. When the bispecific antibody was co-cultured with PBMC and SK-HEP-1-TT3, since all bispecific antibody groups secreted a large amount of IFN-γ, it is revealed that in the case of having a specific target, the bispecific antibody of the present invention can highly activate T cells.

[0201] (Example 6) Example 6 showed the measurement by RTCA real-time killing analyzer of the killing effect on HCT116 cell line by in vitro combined use of the bispecific antibody with recombinant oncolytic poxvirus DDvv-TT3 and pre-activated and amplified T cells (see Figure 17).

[0202] First, a 96 E-Plate (a commercial product purchased from Agilent Technologies Co., Ltd.) was equilibrated with 50 μL of McCoy 5A + 10% FBS culture medium. Next, for wild-type HCT116, it was digested with 0.25% trypsin to obtain single cells, resuspended in McCoy 5A + 10% FBS culture medium, and 3 tumor cells were seeded into the 96 E-Plate at 5×10 3 cells / 50 μL / well. After seeding and incubating overnight, DDvv-TT3 was seeded into the 96 E-Plate at 50 μL / well (MOI = 1). After adding the oncolytic poxvirus and incubating overnight, T cells were added to the corresponding wells of the 96 E-Plate at a cell ratio of E:T = 10:1, with a volume of 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 the 96 E-Plate at 50 μL / well. The final concentration of the bispecific antibody was 1 nM, and the final volume of each well was 250 μL. Data was set to be collected by scanning once every 15 minutes. In Figure 17A, number 1 represents the HCT116 group without adding T cells, poxvirus, and bispecific antibody; number 2 represents the HCT116 + T cell group; numbers 3 - 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 adding T cells, poxvirus, and bispecific antibody; number 2 represents the HCT116 + DDvv-TT3 group; number 3 represents the HCT116 + DDvv-TT3 + T cell group; numbers 4 - 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 to the tumor growth inhibition rate (IR, %), and Figure 17B was obtained. The cell index at 80 hours in Figure 17C was converted to the tumor growth inhibition rate, and Figure 17D was obtained.

[0203] As is clear from the results, when the recombinant oncolytic poxvirus DDvv-TT3 was not added, the tumor cells continued to proliferate (see A in Fig. 17). When the recombinant oncolytic poxvirus DDvv-TT3 was added, the combined use of the bispecific antibodies BK-D-04, 06, 07 or BK-A-101 and T cells all showed significant killing effects (see C in Fig. 17). Referring to the results shown in B and D of Fig. 17 together, in the bispecific antibody BK-D-04, 06, 07 + T cell group, the growth inhibition rate of HCT116 cells was less than 10%. In the DDvv-TT3 + T cell group, the growth inhibition rate of HCT116 cells was about 40%. In the combined group of bispecific antibodies BK-D-04, 06, 07, DDvv-TT3 and T cells, the growth inhibition rate of HCT116 cells exceeded 80%. Therefore, it was revealed that the combined use of bispecific antibodies BK-D-04, 06, 07, DDvv-TT3 and T cells showed a synergistic killing effect.

[0204] In the description of the present invention, unless otherwise specifically limited, the meaning of the term "plurality" is at least two, for example, two, three, etc.

[0205] In the description of this specification, descriptions using terms such as "one embodiment", "several embodiments", "example", "specific example" or "several examples" mean that the specific features, structures, materials or characteristics described in the said embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the exemplary descriptions using the above terms are not necessarily directed to the same embodiment or example. Also, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. Also, unless there is a contradiction, those skilled in the art may combine and combine the different embodiments or examples and the features of different embodiments or examples described in this specification.

[0206] The embodiments of the present invention have been shown and described above. However, the above embodiments are exemplary and should not be construed as limitations on the present invention. It should be understood that those skilled in the art may make changes, corrections, replacements, and modifications to the above embodiments within the scope of the present invention.

Claims

1. (a) A first composition comprising a recombinant oncolytic virus, wherein the genome of the recombinant oncolytic virus has a labeled polypeptide coding sequence for introduction into tumor cells and / or cancer cells, the labeled polypeptide has an operably linked extracellular antigenic determinant, a spacer portion and a transmembrane portion, and the amino acid sequence of the extracellular antigenic determinant comprises the amino acid sequence of Strep Tag II, (b) A second composition comprising a multispecific antibody, wherein the multispecific antibody comprises 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 Strep tag II, and further comprising, The first antigen-binding region includes a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having sequences indicated by SEQ ID NOs. 20, 21, and 22, respectively, based on the IMGT scheme, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having sequences indicated by SEQ ID NOs. 23, 24, and 25, respectively; The second antigen-binding portion is capable of specifically recognizing and binding to CD3, and the second antigen-binding portion comprises a heavy chain variable region containing HCDR1, HCDR2, and HCDR3 having sequences shown in SEQ ID NOs. 35, 36, and 37, respectively, based on the IMGT scheme, and a light chain variable region containing LCDR1, LCDR2, and LCDR3 having sequences shown in SEQ ID NOs. 38, GTN, and 40, respectively, the second antigen-binding portion is humanized, and the bispecific antibody has a reduced affinity for CD3 compared to a humanized anti-CD3 monoclonal antibody having the same HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; and A therapeutic agent characterized in that the heavy chain variable region and light chain variable region of the first antigen-binding region 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 second antigen-binding region, and the carboxyl-terminal amino acid of the light chain variable region of the second antigen-binding region is linked to the amino-terminal amino acid of the heavy chain variable region of the second antigen-binding region.

2. The therapeutic agent according to claim 1, characterized in that the second antigen-binding portion comprises a heavy chain variable region sequence represented by SEQ ID NO: 43, 44, or 45 and a light chain variable region sequence represented by SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, or 53.

3. The second antigen-binding portion is (c-1) has a heavy chain variable region sequence shown in sequence number 43 and a light chain variable region sequence shown in sequence number 46, or (c-2) has a heavy chain variable region sequence shown in Sequence ID 43 and a light chain variable region sequence shown in Sequence ID 47, or (c-3) has a heavy chain variable region sequence shown in Sequence ID 43 and a light chain variable region sequence shown in Sequence ID 48, or (c-4) has a heavy chain variable region sequence shown in Sequence ID 43 and a light chain variable region sequence shown in Sequence ID 49, or (c-5) has a heavy chain variable region sequence shown in Sequence ID 43 and a light chain variable region sequence shown in Sequence ID 50, or (c-6) has a heavy chain variable region sequence shown in Sequence ID 43 and a light chain variable region sequence shown in Sequence ID 51, or (c-7) has a heavy chain variable region sequence shown in Sequence ID 43 and a light chain variable region sequence shown in Sequence ID 52, or (c-8) has a heavy chain variable region sequence indicated by Sequence ID 43 and a light chain variable region sequence indicated by Sequence ID 53, or (d-1) has a heavy chain variable region sequence shown in SEQ ID NO: 44 and a light chain variable region sequence shown in SEQ ID NO: 46, or (d-2) has a heavy chain variable region sequence shown in Sequence ID 44 and a light chain variable region sequence shown in Sequence ID 47, or (d-3) has a heavy chain variable region sequence shown in Sequence ID 44 and a light chain variable region sequence shown in Sequence ID 48, or (d-4) has a heavy chain variable region sequence shown in Sequence ID 44 and a light chain variable region sequence shown in Sequence ID 49, or (d-5) has a heavy chain variable region sequence indicated by Sequence ID 44 and a light chain variable region sequence indicated by Sequence ID 50, or Having a heavy chain variable region sequence indicated by Sequence ID 44 and a light chain variable region sequence indicated by Sequence ID 51 (d-6), or (d-7) has a heavy chain variable region sequence shown in Sequence ID 44 and a light chain variable region sequence shown in Sequence ID 52, or (d-8) has a heavy chain variable region sequence indicated by Sequence ID 44 and a light chain variable region sequence indicated by Sequence ID 53, or (e-1) has a heavy chain variable region sequence indicated by sequence number 45 and a light chain variable region sequence indicated by sequence number 46, or (e-2) has a heavy chain variable region sequence indicated by Sequence ID 45 and a light chain variable region sequence indicated by Sequence ID 47, or (e-3) has a heavy chain variable region sequence indicated by sequence number 45 and a light chain variable region sequence indicated by sequence number 48, or (e-4) has a heavy chain variable region sequence indicated by sequence number 45 and a light chain variable region sequence indicated by sequence number 49, or (e-5) has a heavy chain variable region sequence indicated by Sequence ID 45 and a light chain variable region sequence indicated by Sequence ID 50, or Having a heavy chain variable region sequence indicated by Sequence ID 45 and a light chain variable region sequence indicated by Sequence ID 51 (e-6), or (e-7) has a heavy chain variable region sequence indicated by Sequence ID 45 and a light chain variable region sequence indicated by Sequence ID 52, or The therapeutic agent according to claim 1, characterized in that it is selected from (e-8) having a heavy chain variable region sequence shown in SEQ ID NO: 45 and a light chain variable region sequence shown in SEQ ID NO:

53.

4. (i) The amino acid sequence of the extracellular antigenic determinant is shown in SEQ ID NO: 3; and / or (ii) The spacer portion originates from the hinge region of CD8α, the hinge region of IgG, or the hinge region of IgD, and The therapeutic agent according to claim 1, characterized in that the transmembrane portion originates from the transmembrane region of CD8, CD3ζ, CD4, or CD28.

5. (i) The labeled polypeptide has the amino acid sequence shown in SEQ ID NO: 13; and / or (ii) The spacer portion has the amino acid sequence shown in Sequence ID No. 6; and / or (iii) The transmembrane portion is characterized in that it has the amino acid sequence shown in Sequence ID No. 7, The therapeutic agent according to claim 1.

6. The therapeutic agent according to claim 1, characterized in that the first antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 29, 30, or 31 and a light chain variable region sequence represented by SEQ ID NO: 32, 33, or 34.

7. The first antigen-binding portion is Having a heavy chain variable region sequence shown in Sequence ID No. 29 and a light chain variable region sequence shown in Sequence ID No. 32 (1-1), or Having a heavy chain variable region sequence indicated by SEQ ID NO: 29 and a light chain variable region sequence indicated by SEQ ID NO: 33 (1-2), or (1-3) Having a heavy chain variable region sequence indicated by SEQ ID NO: 29 and a light chain variable region sequence indicated by SEQ ID NO: 34, or (1-4) having a heavy chain variable region sequence indicated by SEQ ID NO: 30 and a light chain variable region sequence indicated by SEQ ID NO: 32, or Having a heavy chain variable region sequence indicated by SEQ ID NO: 30 and a light chain variable region sequence indicated by SEQ ID NO: 33 (1-5), or Having a heavy chain variable region sequence indicated by SEQ ID NO: 30 and a light chain variable region sequence indicated by SEQ ID NO: 34 (1-6), or Having a heavy chain variable region sequence indicated by SEQ ID NO: 31 and a light chain variable region sequence indicated by SEQ ID NO: 32 (1-7), or Having a heavy chain variable region sequence indicated by SEQ ID NO: 31 and a light chain variable region sequence indicated by SEQ ID NO: 33 (1-8), or The therapeutic agent according to claim 1, characterized in that it is selected from (1-9) having a heavy chain variable region sequence shown in SEQ ID NO: 31 and a light chain variable region sequence shown in SEQ ID NO:

34.

8. The therapeutic agent according to claim 1, characterized in that the recombinant oncolytic virus is derived from adenovirus, poxvirus, herpes simplex virus, measles virus, Semryki forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, Seneca Valley virus, echovirus, coxsackievirus, Newcastle disease virus, and Maraba virus, which have oncolytic activity.

9. The therapeutic agent according to claim 1, characterized in that the recombinant oncolytic virus is formulated for intratumoral injection, intravenous injection, intraperitoneal injection, or intracranial injection.

10. 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 and are not mixed with each other.

11. The therapeutic agent according to claim 1, characterized in that the therapeutic agent comprises the first composition and the second composition.

12. The therapeutic agent according to claim 1, characterized in that the Fc region is a humanized sequence or a mutated humanized sequence.

13. 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 II, The second antigen-binding portion includes one or more domains capable of specifically recognizing and binding to CD3, the second antigen-binding portion includes a heavy chain variable region and a light chain variable region, and based on the IMGT scheme, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 having sequences shown in SEQ ID NOs. 35, 36, and 37, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 having sequences shown in SEQ ID NOs. 38, GTN, and SEQ ID NOs. 40, respectively, the second antigen-binding portion is humanized, and the bispecific antibody has a reduced affinity for CD3 compared to a humanized anti-CD3 monoclonal antibody having the same HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; and The first antigen-binding region comprises a heavy chain variable region and a light chain variable region, and according to the IMGT scheme, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having sequences shown in SEQ ID NOs. 20, 21, and 22, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having sequences shown in SEQ ID NOs. 23, 24, and 25, respectively; and The heavy chain variable region and light chain variable region of the first antigen-binding region 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 second antigen-binding region, and the carboxyl-terminal amino acid of the light chain variable region of the second antigen-binding region is linked to the amino-terminal amino acid of the heavy chain variable region of the second antigen-binding region. Multispecific antibodies.

14. The multispecific antibody according to claim 13, characterized in that the second antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 43, 44, or 45 and a light chain variable region sequence represented by SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, or 53.

15. The multispecific antibody according to claim 13, characterized in that the first antigen-binding portion has a heavy chain variable region sequence represented by SEQ ID NO: 29, 30, or 31 and a light chain variable region sequence represented by SEQ ID NO: 32, 33, or 34.

16. The second antigen-binding portion is (c-1) has a heavy chain variable region sequence shown in sequence number 43 and a light chain variable region sequence shown in sequence number 46, or (c-2) has a heavy chain variable region sequence shown in Sequence ID 43 and a light chain variable region sequence shown in Sequence ID 47, or (c-3) has a heavy chain variable region sequence shown in Sequence ID 43 and a light chain variable region sequence shown in Sequence ID 48, or (c-4) has a heavy chain variable region sequence shown in Sequence ID 43 and a light chain variable region sequence shown in Sequence ID 49, or (c-5) has a heavy chain variable region sequence shown in Sequence ID 43 and a light chain variable region sequence shown in Sequence ID 50, or (c-6) has a heavy chain variable region sequence shown in Sequence ID 43 and a light chain variable region sequence shown in Sequence ID 51, or (c-7) has a heavy chain variable region sequence shown in Sequence ID 43 and a light chain variable region sequence shown in Sequence ID 52, or (c-8) has a heavy chain variable region sequence indicated by Sequence ID 43 and a light chain variable region sequence indicated by Sequence ID 53, or (d-1) has a heavy chain variable region sequence shown in SEQ ID NO: 44 and a light chain variable region sequence shown in SEQ ID NO: 46, or (d-2) has a heavy chain variable region sequence shown in Sequence ID 44 and a light chain variable region sequence shown in Sequence ID 47, or (d-3) has a heavy chain variable region sequence shown in Sequence ID 44 and a light chain variable region sequence shown in Sequence ID 48, or (d-4) has a heavy chain variable region sequence shown in Sequence ID 44 and a light chain variable region sequence shown in Sequence ID 49, or (d-5) has a heavy chain variable region sequence indicated by Sequence ID 44 and a light chain variable region sequence indicated by Sequence ID 50, or Having a heavy chain variable region sequence indicated by Sequence ID 44 and a light chain variable region sequence indicated by Sequence ID 51 (d-6), or (d-7) has a heavy chain variable region sequence shown in Sequence ID 44 and a light chain variable region sequence shown in Sequence ID 52, or (d-8) has a heavy chain variable region sequence indicated by Sequence ID 44 and a light chain variable region sequence indicated by Sequence ID 53, or (e-1) has a heavy chain variable region sequence indicated by sequence number 45 and a light chain variable region sequence indicated by sequence number 46, or (e-2) has a heavy chain variable region sequence indicated by Sequence ID 45 and a light chain variable region sequence indicated by Sequence ID 47, or (e-3) has a heavy chain variable region sequence indicated by sequence number 45 and a light chain variable region sequence indicated by sequence number 48, or (e-4) has a heavy chain variable region sequence indicated by sequence number 45 and a light chain variable region sequence indicated by sequence number 49, or (e-5) has a heavy chain variable region sequence indicated by Sequence ID 45 and a light chain variable region sequence indicated by Sequence ID 50, or Having a heavy chain variable region sequence indicated by Sequence ID 45 and a light chain variable region sequence indicated by Sequence ID 51 (e-6), or (e-7) has a heavy chain variable region sequence indicated by Sequence ID 45 and a light chain variable region sequence indicated by Sequence ID 52, or The multispecific antibody according to claim 13, characterized in that it is selected from (e-8) having a heavy chain variable region sequence shown in SEQ ID NO: 45 and a light chain variable region sequence shown in SEQ ID NO:

53.

17. The first antigen-binding portion is Having a heavy chain variable region sequence shown in Sequence ID No. 29 and a light chain variable region sequence shown in Sequence ID No. 32 (1-1), or Having a heavy chain variable region sequence indicated by SEQ ID NO: 29 and a light chain variable region sequence indicated by SEQ ID NO: 33 (1-2), or (1-3) Having a heavy chain variable region sequence indicated by SEQ ID NO: 29 and a light chain variable region sequence indicated by SEQ ID NO: 34, or (1-4) having a heavy chain variable region sequence indicated by SEQ ID NO: 30 and a light chain variable region sequence indicated by SEQ ID NO: 32, or Having a heavy chain variable region sequence indicated by SEQ ID NO: 30 and a light chain variable region sequence indicated by SEQ ID NO: 33 (1-5), or Having a heavy chain variable region sequence indicated by SEQ ID NO: 30 and a light chain variable region sequence indicated by SEQ ID NO: 34 (1-6), or Having a heavy chain variable region sequence indicated by SEQ ID NO: 31 and a light chain variable region sequence indicated by SEQ ID NO: 32 (1-7), or Having a heavy chain variable region sequence indicated by SEQ ID NO: 31 and a light chain variable region sequence indicated by SEQ ID NO: 33 (1-8), or The multispecific antibody according to claim 13, characterized in that it is selected from (1-9) having a heavy chain variable region sequence shown in SEQ ID NO: 31 and a light chain variable region sequence shown in SEQ ID NO:

34.

18. The multispecific antibody according to claim 13, characterized in that the Fc region is a humanized sequence or a mutated humanized sequence.

19. A polynucleotide characterized by encoding a multispecific antibody according to any one of claims 13 to 18.

20. A construct comprising the polynucleotide described in claim 19.

21. A host cell comprising the construct according to claim 20.

22. Use of a therapeutic agent according to any one of claims 1 to 12 in the manufacture of a drug for treating tumors and / or cancer.

23. The aforementioned tumors and / or cancers include breast cancer, head and neck tumors, synovial sarcoma, kidney cancer, connective tissue tumors, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain tumors, liver cancer, bone tumors, 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, cancer of unknown primary origin, carcinoid, fibrosarcoma, Paget's disease, cervical cancer, gallbladder cancer, eye tumors, Kaposi's sarcoma, prostate cancer, testicular cancer, and squamous epithelium of the skin. The use according to claim 22, comprising at least one of the following: mesothelioma, multiple myeloma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, penile cancer, pituitary tumor, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymic cancer, trophoblastic tumor, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, cardiac tumor, meningeal cancer, peritoneal cancer, pleural tumor, and hematological cancer.

24. The use according to claim 22, wherein the second composition is administered after the first composition.

25. A kit of synergistic combination agents for treating tumors and / or cancer, comprising the therapeutic agent according to any one of claims 1 to 12, the kit comprises, A first container containing the first composition of the therapeutic agent, A second container containing the second composition of the therapeutic agent, wherein the first container and the second container are independent of each other. A kit including instructions describing the timing and method of administration.

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