Bispecific antibodies targeting PD-1 and LAG3 and uses thereof

Abstract

The invention discloses a bispecific antibody. According to the embodiment of the invention, the bispecific antibody comprises a first binding region, and the first binding region comprises a first nano antibody and a second nano antibody which are used for binding LAG3; the first binding region is used for binding PD-1, the second binding region is used for binding PD-1, and the first binding region is connected with the second binding region. According to the bispecific antibody, T cells can be more comprehensively activated by jointly blocking PD-1 and LAG-3, T cell activation is promoted, anti-tumor immune response is enhanced, and immune cells can be effectively promoted to kill tumor cells. Therefore, the bispecific antibody provided by the invention can overcome the limitation of a single inhibitor, provides more effective treatment selection for cancer patients, and has remarkable clinical application potential and important research value.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to a bispecific antibody targeting PD-1 and LAG3 and its use, and more specifically to a bispecific antibody, nucleic acid molecule, expression vector, recombinant cell, product and its pharmaceutical application. Background Art

[0002] Cancer immunotherapy, as a new generation of anti-tumor treatment, aims to identify and kill cancer cells by activating the human immune system, and has become a hot topic in current research. Its core concept is to relieve immunosuppression in the tumor microenvironment, improve the antigen presentation function of dendritic cells, and promote the production of protective T cells. PD-1 (Programmed Cell Death Protein 1) is one of the most concerned immune checkpoints in recent years and has important physiological functions.

[0003] PD-1 is a cell surface receptor that belongs to the immune checkpoint family and is mainly found on the surface of T cells. Its function is to regulate the activity of T cells by binding to its ligand PD-L1 (Programmed Death-Ligand 1) or PD-L2, thereby preventing T cells from attacking normal tissues, maintaining the balance of the immune system, and preventing excessive activation of autoimmune responses. When PD-1 binds to its ligand, it inhibits the activity of T cells, reduces their ability to attack tumor cells, and helps tumor cells escape the surveillance of the immune system.

[0004] Currently, multiple PD-1 inhibitors such as Nivolumab (Opdivo) and Pembrolizumab (Keytruda) have achieved remarkable success in the clinic and are widely used in the treatment of various cancers such as melanoma and non-small cell lung cancer. However, the treatment response rate of PD-1 inhibitors is still limited, and data show that only 4% (such as pancreatic cancer) to 70% (such as melanoma) of patients have a significant response to them. This limitation is mainly due to the fact that tumor cells may escape the attack of the immune system through other immune checkpoint molecules.

[0005] Lymphocyte activation gene 3 (LAG-3) is another important immune checkpoint molecule that belongs to the CD4 superfamily and is mainly expressed on the surface of activated immune cells such as T cells, B cells, natural killer cells (NK cells) and dendritic cells. LAG-3 inhibits the activity of T cells by binding to its ligand, maintaining the balance of the immune system and preventing excessive immune response.

[0006] Single PD-1 or LAG-3 inhibitors have limited efficacy in some patients. Therefore, the development of bispecific antibodies for PD-1 and LAG-3 has become an important research direction in current cancer immunotherapy, which has higher therapeutic potential and broader application prospects. Summary of the invention

[0007] The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present application provides a bispecific antibody targeting PD-1 and LAG3.

[0008] This application is completed based on the following findings of the inventors:

[0009] Bispecific antibodies (BsAbs) are a class of antibodies that have the ability to recognize two or more different antigens or epitopes and are widely used in cancer treatment, immunotherapy, and other disease areas. Compared with monoclonal antibodies, BsAbs can more comprehensively activate the immune system, increase the attack power of immune cells, and improve resistance to cancer cells by simultaneously targeting multiple antigens or epitopes. This makes BsAbs more advantageous for patients who do not respond well to single inhibitory point therapy.

[0010] The choice of bispecific antibody structure depends on the specific application requirements, such as therapeutic goals, half-life, tissue penetration ability and production difficulty. On the market, there are already several anti-PD1+LAG3 bispecific antibodies with different structures entering the clinical stage. These bispecific antibodies are mainly used for the treatment of solid tumors. The designed structures include DART-Fc (Tebotelimab) (Luke et al., 2023), Crossmab (RO-7247669) (Chocarro et al., 2022) and FIT-IgG (EMB-02) (Day et al., 2023). In addition, some anti-PD1+LAG3 bispecific antibodies (such as AK-129 and INCA32459) have entered the clinical research stage, but their specific structural types have not yet been disclosed. However, the effectiveness of current PD-1 and LAG-3 bispecific antibody molecules in cancer treatment needs to be improved.

[0011] The inventors of this application designed 7 different anti-PD1+LAG3 dual antibodies for the anti-PD1+LAG3 target combination ( Figure 1 ). The structures of these bispecific antibodies are different from the current anti-PD1+LAG3 bispecific antibodies and can be roughly divided into the following three types:

[0012] Monovalent IgG bispecific antibody: This type of bispecific antibody does not contain an Fc terminal and is composed of one Fab and two VHHs in series, with a size of approximately 75 kD. Due to its smaller diameter, monovalent IgG has good penetration into solid tumors in vivo.

[0013] ART-Ig (Asymmetric Re-engineering Technology-Immunoglobulin) bispecific antibodies: This type of bispecific antibody introduces different mutations into the CH3 region of the antibody Fc, so that the specific regions of the CH3 segments of the A and B chains of the mutated antibody carry positive and negative charges, thereby enabling heterologous fitting of the AB heavy chains.

[0014] IgG+VHH type bispecific antibody: This type of bispecific antibody is a bispecific antibody that connects VHH to the C-terminus of the heavy chain or light chain of the IgG antibody. The 25VHH and 27VHH of anti-LAG3 can produce a good interference effect after being connected in series, so 25VHH and 27VHH are connected in series to the C-terminus of the anti-PD1 IgG antibody.

[0015] Based on this, the above-mentioned bispecific antibodies can not only block the binding of PD-1 to its ligand, but also inhibit the binding of LAG-3 to its ligand, thus achieving dual immune regulation of tumor cells. The above-mentioned bispecific antibodies can more comprehensively activate T cells and enhance anti-tumor immune response by jointly blocking PD-1 and LAG-3, thereby improving the therapeutic effect. By inhibiting PD-1 and LAG-3 at the same time, drug resistance can be overcome, the overall remission rate of patients can be improved, and the immune system's ability to recognize and eliminate tumors can be enhanced.

[0016] Therefore, in the first aspect of the present application, the present application proposes a bispecific antibody. According to an embodiment of the present application, the bispecific antibody comprises: a first binding region, the first binding region comprises a first nanobody and a second nanobody for binding to LAG3; a second binding region, the second binding region is used to bind to PD-1, and the first binding region and the second binding region are connected. The bispecific antibody of the present application can more comprehensively activate T cells, promote T cell activation, enhance anti-tumor immune response, and effectively promote immune cells to kill tumor cells by jointly blocking PD-1 and LAG-3. As a result, the bispecific antibody of the present application has good anti-cancer activity.

[0017] In the second aspect of the present application, the present application proposes a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the bispecific antibody described in the first aspect. According to an embodiment of the present application, the nucleic acid molecule can encode a bispecific antibody that can simultaneously target PD-1 and LAG3.

[0018] In the third aspect of the present application, the present application proposes an expression vector. According to an embodiment of the present application, the expression vector comprises the nucleic acid molecule described in the second aspect. The nucleic acid molecule according to the embodiment of the present application can express a bispecific antibody targeting PD-1 and LAG3.

[0019] In the fourth aspect of the present application, the present application proposes a recombinant cell. According to an embodiment of the present application, the recombinant cell carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect; or expresses the bispecific antibody described in the first aspect. According to an embodiment of the present application, the recombinant cell can be obtained by transfecting or transforming the expression vector described in the third aspect, and can efficiently express the bispecific antibody described in the first aspect under appropriate conditions.

[0020] In the fifth aspect of the present application, the present application proposes a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition comprises: the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression described in the third aspect, or the recombinant cell described in the fourth aspect. As can be seen from the foregoing, the bispecific antibody described in the first aspect or the bispecific antibody prepared by the nucleic acid molecule described in the second aspect, the expression described in the third aspect, or the recombinant cell described in the fourth aspect can simultaneously target PD-1 and LAG3, thereby promoting T cell activation, effectively promoting immune cells to kill tumor cells, and having good anti-cancer activity. Thus, the obtained pharmaceutical composition can effectively prevent and / or treat cancer.

[0021] In the sixth aspect of the present application, the present application proposes a kit. According to an embodiment of the present application, the kit includes the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect. According to an embodiment of the present application, the kit can be combined with PD-1 protein and / or LAG3 protein, and can effectively identify PD-1 protein and / or LAG3 protein.

[0022] In the seventh aspect of the present application, the invention proposes the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of a kit for detecting PD-1 and / or LAG3. As can be seen from the foregoing, the bispecific antibody described in the first aspect or the bispecific antibody prepared by the nucleic acid molecule described in the second aspect, the expression described in the third aspect, or the recombinant cell described in the fourth aspect can simultaneously target PD-1 and LAG3. Thus, the method of the present application can effectively identify PD-1 protein and / or LAG3 protein.

[0023] In the eighth aspect of the present application, the present application proposes the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a drug, and the drug is used to prevent and / or treat LAG3-mediated related diseases. As can be seen from the foregoing, the bispecific antibody described in the first aspect or the bispecific antibody prepared using the nucleic acid molecule described in the second aspect, the expression described in the third aspect, or the recombinant cell described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect can effectively prevent and / or treat cancer. Thus, the bispecific antibody and the corresponding nucleic acid, vector or transformant or pharmaceutical composition of the present application can be further prepared into a drug, which can be used clinically to prevent or treat cancer.

[0024] The advantages of the present invention are:

[0025] 1. The bispecific antibody of the present application can simultaneously target PD-1 and LAG-3, regulate T cell activity through different signaling pathways, and its combined use can give full play to their complementary effects and completely relieve the inhibition of T cells.

[0026] 2. The problem of drug resistance caused by single PD-1 or LAG-3 inhibitors. The bispecific antibody of the present application can simultaneously target PD-1 and LAG-3, and their combined use can overcome drug resistance and ensure sustained and effective therapeutic effects.

[0027] 3. The bispecific antibody of the present application can target PD-1 and LAG-3 simultaneously, which can improve the treatment response rate and benefit more patients.

[0028] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0030] Figure 1 Schematic diagram of the structure of each bispecific antibody in Example 1 of the present application;

[0031] Figure 2 The SDS-PAGE results of each bispecific antibody in Example 1 of the present application;

[0032] Figure 3 SEC 250 results of each bispecific antibody in Example 1 of the present application;

[0033] Figure 4LC-MS results of Bs003 and Bs004 in Example 1 of the present application;

[0034] Figure 5 These are the results of two ELISAs of each bispecific antibody and 293T-LAG3 cells in Example 2 of the present application. Figure 5 BMS vs.Bs004(***), BMS vs.Bs012(*), NC-Fc vs.Bs004(****), NC-his vs.Bs010(*), NC-his vs.Bs012(**) in A, Figure 5 NC-Fc vs. Bs003 (**), NC-his vs. Bs011 (*), NC-his vs. Bs012 (*) in B, where *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0035] Figure 6 The FACS results of each bispecific antibody and 293T-LAG3 cells in Example 3 of the present application, BMS vs. Bs003 (*), IgG4 vs. Bs003 (***), IgG4 vs. Bs004 (*), where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0036] Figure 7 The results of the effects of each bispecific antibody in Example 4 of the present application on T cell activation and IL-2 secretion in human PBMCs are shown in FIG. Figure 7 **P<0.01 in A: aLAG3 mAb vs.Bs003, aLAG3 mAb vs.Bs004, aLAG3 mAb vs.Bs021, ***P<0.001: aLAG3 mAb vs.Bs020, aLAG3 mAb vs.Bs010, aLAG3 mAb vs.Bs011, aLAG3 mAb vs.Bs012; Figure 7 *P<0.05 in B: aLAG3 mAb vs.Bs003, aLAG3 mAb vs.Bs004, aLAG3mAb vs.Bs021, **P<0.01: aLAG3 mAbvs.Bs020, aLAG3 mAb vs.Bs010, aLAG3 mAbvs.Bs011, ***P<0.001: aLAG3 mAb vs.Bs012;

[0037] Figure 8 This is a schematic diagram of the design of the mouse efficacy experiment in Example 5 of the present application;

[0038] Fig. 9 The results of the mouse efficacy experiments of the bispecific antibodies in Example 5 of the present application are shown in FIG. Fig. 9 A is the weight change of mice after antibody injection. Fig. 9 B is the tumor volume in mice after antibody injection. DETAILED DESCRIPTION

[0039] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0040] This application details

[0041] Definitions and general terms

[0042] The articles "a", "an", and "the" herein include plural references unless the context clearly indicates otherwise. For example, "an antibody" refers to one antibody or more than one antibody.

[0043] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0044] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0045] In order to make the present application more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present application belongs. The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0046] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0047] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0048] As used herein, the term "antibody" includes any immunoglobulin that can bind to a specific antigen. The term "antibody" is used in the broadest sense, covering various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), triabodies, single-chain Fv (scFv), nanobodies, chimeric antibodies, etc., or dimers or polymers, and also includes antibody fragments, as long as they exhibit the desired biological activity. Antibodies can be murine, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy chains, full-length light chains, complete immunoglobulin molecules; or an immunologically active portion of any of these polypeptides, i.e., a molecule or portion thereof that contains an antigen binding site that immunospecifically binds to a target antigen of interest, such targets include but are not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. Typically, a natural complete antibody contains two heavy (H) chains and two light (L) chains. Antibodies can be divided into five major classifications or isotypes: IgA, IgD, IgE, IgG, and IgM, depending on whether they contain α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classifications can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). Each heavy chain consists of a variable region (VH) and the first, second, third, and fourth (optional) constant regions (CH1, CH2, CH3, CH4, respectively). The variable region of the heavy chain determines the binding of the antigen. The variable region of each chain usually contains three hypervariable regions, called "complementarity determining regions (CDRs)", of which the heavy chain CDRs include HCDR1, HCDR2, and HCDR3. The variable regions (VH and VL) are each composed of three complementarity determining regions connected by four framework regions (FRs). Typically, the variable region VH of the heavy chain can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0049] In this article, certain regions in the variable region have a higher degree of variation in amino acid composition and arrangement order, which are called "hypervariable regions (HVR)". The hypervariable region is the location where the antigen and antibody bind, so it is also called the complementarity-determining region (CDR). Among them, the heavy chain complementary determining region (heavy chain variable region CDR) is represented by "HCDRs" or "HCDR", which includes HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2) and HCDR3 (also known as CDR-H3); the light chain complementary determining region (light chain variable region CDR) is represented by "LCDRs" or "LCDR", which includes LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2) and LCDR3 (also known as CDR-L3). Commonly used CDR definition schemes in the art include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition.

[0050] Herein, the term "anti-PD-1 antibody" or "antibody that binds to PD-1" refers to an antibody that can bind to PD-1. The term "anti-LAG3 antibody" or "antibody that binds to LAG3" refers to an antibody that can bind to LAG3.

[0051] In this article, the term "bispecific antibodies (BsAbs)" refers to a class of antibodies that have the ability to recognize two or more different antigens or epitopes, and are widely used in cancer treatment, immunotherapy, and other disease areas. Compared with monoclonal antibodies, BsAbs can more comprehensively activate the immune system, increase the attack power of immune cells, and improve resistance to cancer cells by simultaneously targeting multiple antigens or epitopes.

[0052] As used herein, the term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of the constant region amino acids of a monoclonal antibody from one species (such as mouse) with the constant region of an antibody from another species (such as human).

[0053] Herein, the term "humanized antibody" belongs to chimeric antibodies, specifically refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of non-CDR (Fv framework region (FR)) amino acids in the constant region and variable region of a monoclonal antibody from one species (such as mouse) with the amino acid sequence of non-CDR amino acids in the constant region and variable region of an antibody from another species (such as human). That is, when the constant region of an antibody is humanized, it is called a chimeric antibody, and when the amino acid sequence of non-CDR amino acids in the constant region and variable region is fully humanized, it is called a humanized antibody. The method of humanization can be carried out with reference to conventional antibody engineering technology, which will not be repeated here.

[0054] As used herein, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 48:443); the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:24 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0055] As used herein, the term "conservatively modified amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the binding properties of the antibody comprising the amino acid sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present application by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0056] As used herein, the terms "variant Fc fragment", "Fc mutant", "Fc fragment carrying mutation", "mutant Fc fragment", "mutant Fc fragment", "Fc fragment variant", "Fc variant", "variant Fc fragment" and "mutant Fc fragment" and similar terms are used interchangeably to refer to an Fc region that comprises at least one amino acid modification and is different from a native sequence Fc region / wild-type Fc region.

[0057] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, the nucleic acid molecule comprises a nucleotide sequence that can express a target protein, and the nucleic acid molecule can be transferred into a host cell and / or between host cells. The expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with a variety of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0058] In this article, the term "recombinant cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique trait of stable inheritance. Wherein, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant expression vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as expression vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present application, and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used for the present application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid) and CoS cells.

[0059] As used herein, the term "pharmaceutical composition" generally refers to a composition in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0060] As used herein, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic response), ie, substances with a reasonable benefit / risk ratio.

[0061] In this article, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. In addition to any conventional excipients that are incompatible with the bispecific antibody of the present application, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of consideration of the present application.

[0062] In this article, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The multispecific antibody or pharmaceutical composition of the present application can be administered by any common route as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present application is not limited to these exemplified modes of administration. Preferably, the multispecific antibody or pharmaceutical composition of the present application is administered by intravenous or subcutaneous injection.

[0063] As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or bispecific antibody to an individual to treat, cure, alleviate, improve, reduce or inhibit an individual's disease, including but not limited to administering a drug containing the bispecific antibody described herein to an individual in need.

[0064] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0065] Detailed description of the bispecific antibody targeting PD-1 and LAG3 and its use in this application

[0066] The present application proposes a bispecific antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a product and pharmaceutical applications thereof, which will be described in detail below.

[0067] Bispecific Antibodies

[0068] In the first aspect of the present application, the present application proposes a bispecific antibody. According to an embodiment of the present application, the bispecific antibody comprises: a first binding region, the first binding region comprises a first nanobody and a second nanobody for binding to LAG3; a second binding region, the second binding region is used to bind to PD-1, and the first binding region and the second binding region are connected. The bispecific antibody of the present application can more comprehensively activate T cells, promote T cell activation, enhance anti-tumor immune response, and effectively promote immune cells to kill tumor cells by jointly blocking PD-1 and LAG-3. As a result, the bispecific antibody of the present application has good anti-cancer activity.

[0069] Herein, the terms "single domain antibody", "nanoantibody" and "VHH antibody" are used interchangeably, which were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to antigen proteins (e.g., LAG-3) through the heavy chain variable region.

[0070] According to an embodiment of the present application, the above-mentioned bispecific antibody may further include at least one of the following technical features:

[0071] According to an embodiment of the present application, the first Nanobody and the second Nanobody are the same or different.

[0072] According to an embodiment of the present application, the first Nanobody and the second Nanobody are different.

[0073] According to an embodiment of the present application, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the first Nanobody are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0074] According to an embodiment of the present application, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the second Nanobody are shown as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0075] According to an embodiment of the present application, the amino acid sequence of the first Nanobody is shown in SEQ ID NO:13.

[0076] According to an embodiment of the present application, the amino acid sequence of the second Nanobody is shown in SEQ ID NO:14.

[0077] In an optional embodiment of the present application, the HCDRs and LCDRs are determined by the Kabat, Chothia, AbM, Contact or IMGT definitions. Exemplarily, the HCDR1, HCDR2 and HCDR3 shown in the above SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 are obtained based on the amino acid sequence described in SEQ ID NO:13 according to the Kabat definition, and other definitions (such as Chothia, AbM, Contact or IMGT) can be converted with reference to conventional methods in the art, all within the scope of protection of the present application. Exemplarily, the HCDR1, HCDR2 and HCDR3 shown in the above SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 are obtained based on the amino acid sequence described in SEQ ID NO:14 according to the Kabat definition, and other definitions (such as Chothia, AbM, Contact or IMGT) can be converted with reference to conventional methods in the art, all within the scope of protection of the present application.

[0078] According to an embodiment of the present application, the first Nanobody and the second Nanobody are linked or not linked.

[0079] According to an embodiment of the present application, the first nanobody and the second nanobody are linked.

[0080] It should be noted that, in this article, unless otherwise specified, "the first Nanobody and the second Nanobody are connected" means directly connected or connected through any connecting peptide.

[0081] According to an embodiment of the present application, the first Nanobody and the second Nanobody are not linked.

[0082] It should be noted that, in this article, unless otherwise specified, "the first Nanobody and the second Nanobody are not connected" means that they are not directly connected or are not connected through any connecting peptide.

[0083] According to an embodiment of the present application, the first Nanobody and the second Nanobody are connected, the first binding region further includes a first connecting peptide, and the first Nanobody and the second Nanobody are connected through the first connecting peptide.

[0084] According to an embodiment of the present application, the C-terminus of the first Nanobody is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the second Nanobody; or

[0085] The C-terminus of the second nanobody is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the first nanobody.

[0086] According to an embodiment of the present application, the amino acid sequence of the first connecting peptide is shown in SEQ ID NO:15.

[0087] According to an embodiment of the present application, the first binding region has an amino acid sequence as shown in SEQ ID NO:16 and SEQ ID NO:17.

[0088] According to an embodiment of the present application, the second binding region includes a Fab antibody, or a F(ab) 2 Antibody.

[0089] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which is composed of the VH and CH1 of the heavy chain and a complete light chain (ie, VL and CL), with the light chain and the heavy chain connected by a disulfide bond.

[0090] As used herein, the term "F(ab') 2 Antibody" or "F(ab') 2 A "fragment" has two antigen-binding F(ab') portions linked together by a disulfide bond.

[0091] According to the embodiments of the present application, the Fab antibody and F(ab) 2 The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the antibody are shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively;

[0092] The Fab antibody, and F(ab) 2 The amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0093] According to the embodiments of the present application, the Fab antibody and F(ab) 2 The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 18;

[0094] The Fab antibody, and F(ab) 2 The amino acid sequence of the light chain variable region in the antibody is shown in SEQ ID NO:19.

[0095] According to the embodiments of the present application, the Fab antibody and F(ab) 2 The CH1 region and CL region in the antibody have the amino acid sequences shown in SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

[0096] According to an embodiment of the present application, the bispecific antibody further comprises one or more second connecting peptides, and the first binding region and the second binding region are connected by the second connecting peptide.

[0097] According to an embodiment of the present application, the second connecting peptide has an amino acid sequence as shown in SEQ ID NO:22.

[0098] According to an embodiment of the present application, the bispecific antibody has any of the following structures:

[0099] 1) the first Nanobody and the second Nanobody are not linked,

[0100] The second binding region is selected from a Fab antibody,

[0101] The C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the first nanobody,

[0102] The C-terminus of the CL region in the second binding region is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the second Nanobody;

[0103] 2) the first Nanobody and the second Nanobody are not linked,

[0104] The second binding region is selected from a Fab antibody,

[0105] The C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the second connecting peptide, the C-terminus of the second connecting peptide is connected to the N-terminus of the second Nanobody, and the C-terminus of the CL region in the second binding region is connected to the N-terminus of the first Nanobody;

[0106] 3) the first binding region is selected from the group consisting of a first nanobody-a first connecting peptide-a second nanobody, or a second nanobody-a first connecting peptide-a first nanobody,

[0107] The second binding region is selected from a Fab antibody,

[0108] The C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the first binding region;

[0109] 4) the first binding region is selected from the group consisting of a first nanobody-a first connecting peptide-a second nanobody, or a second nanobody-a first connecting peptide-a first nanobody,

[0110] The second binding region is selected from a Fab antibody,

[0111] The C-terminus of the CL region in the second binding region is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the first binding region.

[0112] It should be noted that if the first binding region contains two or more sequences, "the C-terminus of the second connecting peptide is connected to the N-terminus of the first binding region" includes two or more schemes.

[0113] Exemplarily, for the above 3), including a) or b):

[0114] a) the first binding region is selected from the group consisting of a first nanobody-a first connecting peptide-a second nanobody,

[0115] The second binding region is selected from a Fab antibody,

[0116] The C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the first Nanobody;

[0117] b) the first binding region is selected from the group consisting of a second Nanobody-a first connecting peptide-a first Nanobody,

[0118] The second binding region is selected from a Fab antibody,

[0119] The C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the second nanobody.

[0120] Exemplarily, for the above 4), including c) or d):

[0121] c) the first binding region is selected from the group consisting of a first nanobody-a first connecting peptide-a second nanobody,

[0122] The second binding region is selected from a Fab antibody,

[0123] The C-terminus of the CL region in the second binding region is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the first Nanobody;

[0124] d) the first binding region is selected from the group consisting of a second Nanobody-a first connecting peptide-a first Nanobody,

[0125] The second binding region is selected from a Fab antibody,

[0126] The C-terminus of the CL region in the second binding region is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the second nanobody.

[0127] In this article, without special instructions, the ABC fragment refers to A, B, and C from the N-terminus to the C-terminus. Exemplarily, "first nanobody-first connecting peptide-second nanobody" refers to a fragment consisting of the first nanobody, the first connecting peptide, and the second nanobody from the N-terminus to the C-terminus.

[0128] According to an embodiment of the present application, the bispecific antibody further comprises an Fc fragment, and the first binding region and / or the second binding region is connected to the Fc fragment.

[0129] In an optional embodiment of the present application, the second binding region is connected to the Fc fragment.

[0130] In an optional embodiment of the present application, the first binding region and the second binding region are connected to the Fc fragment.

[0131] According to an embodiment of the present application, the first binding region and / or the second binding region is connected to the N-terminus of the Fc fragment; or

[0132] The first binding region and / or the second binding region is connected to the C-terminus of the Fc fragment; or

[0133] The first binding region is connected to the N-terminus of the Fc fragment, and the second binding region is connected to the C-terminus of the Fc fragment; or

[0134] The first binding region is connected to the C-terminus of the Fc fragment, and the second binding region is connected to the N-terminus of the Fc fragment.

[0135] It should be noted that "the first binding region and / or the second binding region is connected to the C-terminus of the Fc fragment" and "the first binding region and / or the second binding region is connected to the N-terminus of the Fc fragment" can be directly connected or indirectly connected, preferably directly connected.

[0136] In an optional embodiment of the present application, the first binding region and / or the second binding region is connected to the N-terminus of the Fc fragment and is selected from:

[0137] The second binding region is connected to the N-terminus of the Fc fragment; or

[0138] The first binding region and the second binding region are respectively connected to the N-termini of the two chains in the Fc fragment.

[0139] In an optional embodiment of the present application, the first binding region and / or the second binding region is connected to the C-terminus of the Fc fragment and is selected from:

[0140] The second binding region is connected to the C-terminus of the Fc fragment; or

[0141] The first binding region and the second binding region are respectively connected to the C-termini of the two chains in the Fc fragment.

[0142] According to an embodiment of the present application, the species of the Fc fragment is derived from cattle, horses, pigs, sheep, mice, rats, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese or humans.

[0143] It should be noted that the heavy chain variable region, IgG CH1 region, IgG hinge region, IgM CH2-CH3-CH4 region, light chain variable region and CL region are of the same or different species origin.

[0144] According to an embodiment of the present application, the subtype of the Fc fragment is derived from IgG1, IgG2a, IgG2b, IgG3 or IgG4.

[0145] In some optional examples of the present application, the Fc fragment may be a wild-type Fc fragment or a mutant Fc fragment.

[0146] According to an embodiment of the present application, the Fc fragment is a human Fc peptide segment or a mutant thereof.

[0147] According to an embodiment of the present application, the human Fc peptide segment is a human IgG4 Fc peptide segment or a mutant thereof.

[0148] In some optional examples of the present application, the Fc fragment may be a human IgG4 Fc peptide segment. When the two Fc fragments are the same, no special instructions are required, and the two Fc fragments are connected by a disulfide bond.

[0149] According to an embodiment of the present application, the Fc fragment has an amino acid sequence as shown in SEQ ID NO:23.

[0150] In some optional examples of the present application, the Fc fragment may be a mutant of a human IgG4 Fc peptide segment. Exemplarily, two Fc fragments are connected via a knob into hole structure.

[0151] As used herein, the term "knob into hole structure" refers to a knob-into-hole mutation formed in the CH3 region of the heavy chain constant region of an antibody, which facilitates the binding of the heavy chains to form a heterodimer.

[0152] According to an embodiment of the present application, the first binding region is selected from the group consisting of a first nanobody-a first connecting peptide-a second nanobody, or a second nanobody-a first connecting peptide-a first nanobody.

[0153] The second binding region is selected from a Fab antibody,

[0154] The C-terminus of the first binding region is connected to the N-terminus of one chain in the Fc fragment, and the C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the other chain in the Fc fragment.

[0155] According to an embodiment of the present application, the first binding region has an amino acid sequence as shown in SEQ ID NO: 16; the second binding region is selected from a Fab antibody, the C-terminus of the first binding region is connected to the N-terminus of one chain in the Fc fragment, and the C-terminus of the CH1 region in the second binding region is connected to the N-terminus of another chain in the Fc fragment. The inventors found that the yield of the bispecific antibody is significantly higher than that of other bispecific antibodies. For example, in the present application, the yield of the Bs003 antibody is more than twice that of Bs010 and Bs021, and more than three times that of Bs004, Bs011, Bs012 and Bs020; and according to the LC-MS test results, the purity of the Bs003 bispecific antibody after expression is significantly improved, and it has fewer impurity peaks.

[0156] According to an embodiment of the present application, the first binding region is selected from the group consisting of a first nanobody-a first connecting peptide-a second nanobody, or a second nanobody-a first connecting peptide-a first nanobody.

[0157] The second binding region is selected from F(ab) 2 Antibody,

[0158] The C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the Fc fragment, and the N-terminus of the first binding region is connected to the C-terminus of the CL region in the second binding region; or, the C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the Fc fragment, and the N-terminus of the first binding region is connected to the C-terminus of the Fc fragment.

[0159] According to an embodiment of the present application, the C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the Fc fragment, and the N-terminus of the first binding region is connected to the C-terminus of the CL region in the second binding region.

[0160] According to an embodiment of the present application, the bispecific antibody further includes a third connecting peptide, and the first binding region is connected to the Fc fragment or the second binding region through the third connecting peptide.

[0161] According to an embodiment of the present application, the third connecting peptide has an amino acid sequence as shown in SEQ ID NO:24.

[0162] According to an embodiment of the present application, the C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the Fc fragment, the N-terminus of the first binding region is connected to the C-terminus of the third connecting peptide, and the N-terminus of the third connecting peptide is connected to the C-terminus of the CL region in the second binding region; or,

[0163] The C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the Fc fragment, the N-terminus of the first binding region is connected to the C-terminus of the third connecting peptide, and the N-terminus of the third connecting peptide is connected to the C-terminus of the Fc fragment.

[0164] According to an embodiment of the present application, the bispecific antibody comprises:

[0165] has a first peptide chain as shown in the amino acid sequence of SEQ ID NO: 25, has a second peptide chain as shown in the amino acid sequence of SEQ ID NO: 26, has a third peptide chain as shown in the amino acid sequence of SEQ ID NO: 27; or

[0166] has a first peptide chain as shown in the amino acid sequence of SEQ ID NO: 28, has a second peptide chain as shown in the amino acid sequence of SEQ ID NO: 26, has a third peptide chain as shown in the amino acid sequence of SEQ ID NO: 27; or

[0167] has a first peptide chain as shown in the amino acid sequence of SEQ ID NO: 29, and has a second peptide chain as shown in the amino acid sequence of SEQ ID NO: 30; or

[0168] has a first peptide chain as shown in the amino acid sequence of SEQ ID NO: 31, and has a second peptide chain as shown in the amino acid sequence of SEQ ID NO: 32; or

[0169] has a first peptide chain as shown in the amino acid sequence of SEQ ID NO: 33, and has a second peptide chain as shown in the amino acid sequence of SEQ ID NO: 34; or

[0170] has a first peptide chain as shown in the amino acid sequence of SEQ ID NO: 35, and has a second peptide chain as shown in the amino acid sequence of SEQ ID NO: 36; or

[0171] It has a first peptide chain as shown in the amino acid sequence of SEQ ID NO:37, and a second peptide chain as shown in the amino acid sequence of SEQ ID NO:38.

[0172] Therefore, the bispecific antibody of the present application can simultaneously target PD-1 and LAG-3, regulate T cell activity through different signaling pathways, and the combined use can give full play to their complementary effects, which can significantly improve the T cell activation effect and immunotherapy effect.

[0173] In particular, compared with the anti-LAG3 monoclonal antibody (2527-Fc) group, in the 44.45 and 133.34 nM dual antibody experimental groups, Bs003, Bs004, Bs020, Bs021, Bs010, Bs011 and Bs012 had higher T cell activation and secreted more IL-2 levels after stimulation with SEE (150 pg / ml) or CD3 monoclonal antibody + SEE (150 pg / ml), and this difference was significantly stronger than that in the monoclonal antibody group and the Combo group (a mixture of anti-PD1 and anti-LAG3 monoclonal antibodies).

[0174] Nucleic acid molecules, expression vectors and recombinant cells

[0175] In the second aspect of the present application, the present application proposes a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the bispecific antibody described in the first aspect. According to an embodiment of the present application, the nucleic acid molecule can encode a bispecific antibody that can simultaneously target PD-1 and LAG3.

[0176] According to an embodiment of the present application, the nucleic acid molecule is DNA.

[0177] It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, in this specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequence in this application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0178] Those skilled in the art will appreciate that the features and advantages described above for the bispecific antibody are also applicable to the nucleic acid molecule and will not be described in detail here.

[0179] In the third aspect of the present application, the present application proposes an expression vector. According to an embodiment of the present application, the expression vector includes the nucleic acid molecule described in the second aspect. When the above-mentioned nucleic acid molecule is connected to the expression vector, the nucleic acid molecule can be directly or indirectly connected to the control element on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the expression vector itself, or they can be exogenous, that is, not from the expression vector itself. Of course, the nucleic acid molecule can be operably connected to the control element.

[0180] Herein, "operably linked" means connecting the foreign gene to the expression vector so that the control elements in the expression vector, such as transcription control sequences and translation control sequences, can play their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors can be, for example, plasmids, bacteriophages, and the like.

[0181] According to some specific embodiments of the present application, after the expression vector is introduced into a suitable recipient cell, the expression of the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein or the aforementioned multi-specific antibody can be effectively achieved under the mediation of the regulatory system, thereby achieving large-scale in vitro acquisition of the antibody or antigen-binding fragment, recombinant protein or multi-specific antibody.

[0182] According to the embodiments of the present application, the expression vector may refer to a cloning vector, which can be obtained by operably connecting the nucleic acid molecule to a commercially available vector (such as a plasmid or a viral vector). The vector in the present application is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0183] In this article, the term "operably linked" refers to connecting an exogenous gene to an expression vector so that the control elements in the expression vector, such as transcription-controlled amino acid sequences such as and translation-controlled amino acid sequences such as, can play their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used expression vectors can be, for example, viral vectors, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of the present application are introduced into a suitable host cell, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of the regulatory system, thereby achieving the in vitro acquisition of a large amount of protein encoded by the nucleic acid molecules.

[0184] According to an embodiment of the present application, the vector is a eukaryotic vector or a prokaryotic vector.

[0185] According to an embodiment of the present application, the vector includes at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.

[0186] Those skilled in the art will appreciate that the features and advantages described above for the bispecific antibody and nucleic acid molecule are also applicable to the expression vector and will not be described in detail here.

[0187] In the fourth aspect of the present application, the present application proposes a recombinant cell. According to an embodiment of the present application, the recombinant cell carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect; or expresses the bispecific antibody described in the first aspect. According to an embodiment of the present application, the recombinant cell can be obtained by transfecting or transforming the expression vector described in the third aspect, and can efficiently express the bispecific antibody described in the first aspect under appropriate conditions.

[0188] According to some specific embodiments of the present application, the recombinant cells can efficiently and massively express bispecific antibodies under appropriate conditions. The bispecific antibodies have stronger specificity, longer half-life and higher efficacy, and can deliver antibody drugs to target cells at a lower dosage, thereby achieving effective treatment or prevention of cancer.

[0189] It should be noted that "suitable conditions" refer to conditions suitable for the expression of the multispecific antibodies described in the present application. It is easy for those skilled in the art to understand that conditions suitable for the expression of the multispecific antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell states, suitable host cell densities, suitable cell culture environments, and suitable cell culture times. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the above-mentioned multispecific antibodies according to the specific environment of the laboratory.

[0190] According to an embodiment of the present application, the recombinant cell is obtained by introducing the expression vector described in the third aspect into a host cell.

[0191] It should be noted that the recombinant cells described in the present application are not particularly limited and can be prokaryotic cells, eukaryotic cells or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as S. frugiperda, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments of the present application, the recombinant cells described in the present application are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0192] Those skilled in the art will appreciate that the features and advantages described above for the bispecific antibody, nucleic acid molecule, and expression vector are also applicable to the recombinant cell and will not be described in detail here.

[0193] Pharmaceutical compositions and kits

[0194] In the fifth aspect of the present application, the present application proposes a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition comprises: the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression described in the third aspect, or the recombinant cell described in the fourth aspect. As can be seen from the foregoing, the bispecific antibody described in the first aspect or the bispecific antibody prepared by the nucleic acid molecule described in the second aspect, the expression described in the third aspect, or the recombinant cell described in the fourth aspect can simultaneously target PD-1 and LAG3, thereby promoting T cell activation, effectively promoting immune cells to kill tumor cells, and having good anti-cancer activity. Thus, the obtained pharmaceutical composition can effectively prevent and / or treat cancer.

[0195] According to an embodiment of the present application, the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0196] According to an embodiment of the present application, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0197] According to an embodiment of the present application, the pharmaceutical composition is an injection.

[0198] It should be noted that the pharmaceutical composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present application. For example, the components contained in the composition can be administered to the subject as a whole, or separately. When the components contained in the composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0199] The pharmaceutical composition of the present application contains a safe and effective amount of the active ingredient of the present application and a pharmaceutically acceptable adjuvant. Such adjuvants include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should match the mode of administration. The dosage form of the pharmaceutical composition of the present application is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained release agent. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0200] The effective amount of the active ingredient described in the present application may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0201] The pharmaceutically acceptable excipients described in the present application include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide substances, cellulose, nanogels, or combinations thereof. The choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.

[0202] Those skilled in the art will appreciate that the features and advantages described above for the bispecific antibodies, nucleic acid molecules, expression vectors and recombinant cells are also applicable to the pharmaceutical composition and will not be described in detail here.

[0203] In the sixth aspect of the present application, the present application proposes a kit. According to an embodiment of the present application, the kit includes the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect. According to an embodiment of the present application, the kit can be combined with PD-1 protein and / or LAG3 protein, and can effectively identify PD-1 protein and / or LAG3 protein.

[0204] As mentioned above, the bispecific antibodies of the embodiments of the present application can specifically bind to PD-1 and LAG3. The PD-1 protein and / or LAG3 protein related kits developed using this property can be used for PD-1 protein and / or LAG3 protein related research, such as for detecting and / or enriching and / or separating and purifying PD-1 protein and / or LAG3 protein of humans or other mammals.

[0205] The kit can effectively detect, enrich or separate and purify PD-1 protein and / or LAG3 protein in biological samples, and further be used for scientific research, such as qualitatively or quantitatively detecting PD-1 protein and / or LAG3 protein molecules in biological samples. More specifically, it can be used for immunoblotting, immunoprecipitation, etc., which involve using the specific binding properties of PD-1 protein and / or LAG3 protein and bispecific antibodies for detection. In addition to the bispecific antibodies of the present application, these kits may also include any one or more of the following: protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents. The bispecific antibodies of the present application can be used for different types of diagnostic tests, for example, various diseases or the presence of drugs, toxins or other proteins can be detected in vitro or in vivo. For example, it can be used to diagnose cancer by testing the serum or blood of the subject.

[0206] Those skilled in the art will appreciate that the features and advantages described above for the bispecific antibodies, nucleic acid molecules, expression vectors, and recombinant cells are also applicable to the kit and will not be described in detail herein.

[0207] use

[0208] In the seventh aspect of the present application, the invention proposes the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of a kit for detecting PD-1 and / or LAG3. As can be seen from the foregoing, the bispecific antibody described in the first aspect or the bispecific antibody prepared by the nucleic acid molecule described in the second aspect, the expression described in the third aspect, or the recombinant cell described in the fourth aspect can simultaneously target PD-1 and LAG3. Thus, the method of the present application can effectively identify PD-1 protein and / or LAG3 protein.

[0209] As mentioned above, the bispecific antibodies of the embodiments of the present application can specifically bind to PD-1 and LAG3, and therefore, the bispecific antibodies can be used to detect PD-1 and / or LAG3. Further, it can be used to prepare PD-1 and / or LAG3 related kits and used for scientific research, such as qualitative or quantitative detection of PD-1 and / or LAG3 protein molecules in biological samples. More specifically, it can be used for immunoblotting, immunoprecipitation, and other kits that involve detection using the specific binding properties of PD-1 and / or LAG3 and antibodies. In addition to the bispecific antibodies of the present application, these kits may also include any one or more of the following: antagonists; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents. The bispecific antibodies of the present application can be used for different types of diagnostic tests, for example, the presence of various diseases or drugs, toxins or other proteins can be detected in vitro or in vivo.

[0210] Those skilled in the art will appreciate that the features and advantages described above for the bispecific antibodies, nucleic acid molecules, expression vectors and recombinant cells are also applicable to this use and will not be described in detail here.

[0211] In the eighth aspect of the present application, the present application proposes the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a drug, and the drug is used to prevent and / or treat LAG3-mediated related diseases. As can be seen from the foregoing, the bispecific antibody described in the first aspect or the bispecific antibody prepared using the nucleic acid molecule described in the second aspect, the expression described in the third aspect, or the recombinant cell described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect can effectively prevent and / or treat cancer. Thus, the bispecific antibody and the corresponding nucleic acid, vector or transformant or pharmaceutical composition of the present application can be further prepared into a drug, which can be used clinically to prevent or treat cancer.

[0212] According to an embodiment of the present application, the cancer is selected from colorectal cancer.

[0213] Those skilled in the art will appreciate that the features and advantages described above for the bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells and pharmaceutical compositions are also applicable to this use and will not be described in detail here.

[0214] method

[0215] In the ninth aspect of the present application, the present application provides a method for preventing and / or treating cancer. According to an embodiment of the present application, the method comprises: administering to a subject a pharmaceutically acceptable amount of the bispecific antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect.

[0216] It should be noted that the terms "subject", "individual" and "patient" are used interchangeably herein and refer to a mammal that is evaluated for treatment and / or treated. In one embodiment, the mammal is a human. The terms "subject", "individual" and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infection, etc. The subject can be a human, but also includes other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0217] The effective amount of the bispecific antibody, nucleic acid molecule, expression vector, recombinant cell or pharmaceutical composition described in the present application may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0218] According to an embodiment of the present application, the cancer is selected from colorectal cancer.

[0219] The amino acid sequences involved in this article are shown in Table A:

[0220] Table A: Amino acid sequences

[0221]

[0222]

[0223]

[0224]

[0225]

[0226] The scheme of the present application will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the document in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained by commercial purchase.

[0227] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0228] In the embodiments of the present invention, the nucleotide sequence used to prepare the expression vector can be obtained according to its amino acid sequence using conventional methods or conventional software (such as the online program Vectorbuilder (website: https: / / www.vectorbuilder.cn / tool / codon-optimization.html), GeneOptimizer online program, etc.).

[0229] Example 1: Expression and purification of anti-PD1+LAG3 bispecific antibodies

[0230] 1. Antibody Preparation

[0231] 1) Antibodies containing Fc:

[0232] In this example, bispecific antibodies Bs003, Bs004, Bs020 and Bs021 were prepared by gene synthesis. Figure 1 . Among them, Bs003 includes a first peptide chain p1 with an amino acid sequence as shown in SEQ ID NO:25, a second peptide chain p2 with an amino acid sequence as shown in SEQ ID NO:26, and a third peptide chain p3 with an amino acid sequence as shown in SEQ ID NO:27; Bs003 includes a first peptide chain p1 with an amino acid sequence as shown in SEQ ID NO:28, a second peptide chain p2 with an amino acid sequence as shown in SEQ ID NO:26, and a third peptide chain p3 with an amino acid sequence as shown in SEQ ID NO:27; Bs020 includes a first peptide chain p1 with an amino acid sequence as shown in SEQ ID NO:29, and a second peptide chain p2 with an amino acid sequence as shown in SEQ ID NO:30; Bs021 includes a first peptide chain p1 with an amino acid sequence as shown in SEQ ID NO:31, and a second peptide chain p2 with an amino acid sequence as shown in SEQ ID NO:32.

[0233] The above polypeptide sequences were constructed into pTT5 vectors by molecular cloning technology to obtain the plasmids (p1 plasmid, p2 plasmid, and optionally p3 plasmid) required for expressing each bispecific antibody. Among them, for Bs003 and Bs004 bispecific antibodies, p1 plasmid: p2 plasmid: p3 plasmid were mixed at 1:1:1; for Bs020 and Bs021 bispecific antibodies, p1 plasmid: p2 plasmid were mixed at 1:1. The mixed plasmids were then transfected into ExpiCHO cells for transfection expression, and the cells were cultured in a 32°C shaker for 12 days. The cell culture supernatant was harvested, centrifuged at 3000 rpm for 30 minutes, and then filtered with a 0.22 μm filter. The clarified supernatant was purified with AKTA and MabSelect Sure (1 ml) columns and eluted with 20 mM sodium acetate (PH 3.4) buffer. After concentration, the eluted antibody was further purified by Superdex 200 (GE).

[0234] 2) Antibody without Fc but with 6x His:

[0235] In this example, bispecific antibodies Bs010, Bs011 and Bs012 were prepared by gene synthesis. Figure 1 Among them, Bs010 includes a first peptide chain p1 having an amino acid sequence as shown in SEQ ID NO:33, and a second peptide chain p2 having an amino acid sequence as shown in SEQ ID NO:34; Bs011 includes a first peptide chain p1 having an amino acid sequence as shown in SEQ ID NO:35, and a second peptide chain p2 having an amino acid sequence as shown in SEQ ID NO:36; Bs012 includes a first peptide chain p1 having an amino acid sequence as shown in SEQ ID NO:37, and a second peptide chain p2 having an amino acid sequence as shown in SEQ ID NO:38.

[0236] The above polypeptide sequences were constructed into the pTT5 vector by molecular cloning technology to obtain the plasmids (p1 plasmid, p2 plasmid) required for expressing each bispecific antibody. Among them, the p1 plasmid and p2 plasmid required for each bispecific antibody of Bs010, Bs011, and Bs012 were mixed at a ratio of 1:1 and transfected into ExpiCHO cells for transfection expression. The cells were cultured in a shaker at 32°C for 12 days. The cell culture supernatant was harvested, centrifuged at 3000 rpm for 30 minutes, and then filtered with a 0.22 μm filter. The clarified supernatant was purified with AKTA and Ni Sepharose excel (1 ml) columns and eluted with 250 mM imidazole (PH 7.9). The eluted protein was further purified by Superdex 75 (GE) after concentration.

[0237] 2. Result analysis:

[0238] The 7 bispecific antibodies (referred to as bispecific antibodies) prepared above were subjected to SDS-PAGE, SEC, and LC-MS analysis. The results are shown in Figure 2 to Figure 4 and Table 1. The details are as follows:

[0239] Among the Fc-containing antibodies, there are Bs003 and Bs004 bispecific antibodies with ART-Fc structure (both with molecular weight of 125kD), and Bs020 and Bs021 antibodies targeting two PD1 and two LAG3 antigen epitopes (both with molecular weight of 202kD). The yields of all of them after transfection and expression in 50ml of cells are slightly different. Bs003 has the highest yield of 15mg; Bs021 is second at 6mg; Bs004 and Bs020 have the lowest yields, both at 2.4mg.

[0240] In the three types of bispecific antibodies without Fc, each antibody has a 6×his tail at the C-terminus, which is helpful for subsequent purification. The molecular weight of the three bispecific antibodies is about 75kD, and the final purified antibody amounts of Bs010 (6.4mg), Bs011 (4mg) and Bs012 (3.2mg) were obtained respectively. All antibodies were dissolved in PBS (pH7.4) buffer and stored at -80℃ for future use.

[0241] 1) SDS-PAGE and SEC analysis

[0242] SDS-PAGE analysis of 7 antibodies was performed under non-reducing and reducing conditions. The results are shown in Figure 2 and Figure 3 .

[0243] The results showed that all band sizes were consistent with the theoretical values ​​( Figure 2 ). Then, the SEC250 column was used for detection. All six antibodies showed a single peak, which corresponded to the theoretical size. Only Bs004 had a small miscellaneous peak in addition to the main peak ( Figure 3 , among which 2527-ART is Bs003 and 2725-ART is Bs004).

[0244] 2) LC-MS analysis

[0245] To further confirm, LC-MS tests were performed on Bs003 and Bs004 bispecific antibodies. LC-MS can accurately measure the specific molecular weight of antibodies in expression samples. The sample treatment conditions were non-reducing and non-deglycosylated. The results are as follows: Figure 4 shown.

[0246] from Figure 4It can be seen that the antibody size of Bs003 is mainly concentrated around 128kD, and some peaks of different sizes are distributed around it, which may be caused by different glycoforms. However, Bs004 has a main peak near 128kD and a secondary peak near 148kD. From the size of the secondary peak, it is estimated that it may be an anti-PD1 monoclonal antibody.

[0247] Table 1: Expression results of anti-PD1+LAG3 dual antibodies with different structures

[0248] Name lsotype MW(kDa) Exp volume (ml) Quantity(mg) Buffer / PH Theoretical PI Bs003 IgG4 125 50 15 PBS 7.4 6.65 Bs004 IgG4 125 50 2.4 PBS 7.4 6.65 Bs010 6xHis 77 50 6.4 PBS 7.4 8.28 Bs011 6xHis 80 50 4 PBS 7.4 8.28 Bs012 6xHis 77 50 3.2 PBS 7.4 8.33 Bs020 IgG4 202 50 2.4 PBS 7.4 7.96 Bs021 IgG4 202 50 6 PBS 7.4 7.98

[0249] Example 2: ELISA detection of anti-PD1+LAG3 dual antibodies and 293T-LAG3 cells

[0250] 1. Experimental steps:

[0251] 1) 293T-hLAG3 cells were digested and the cells were counted using trypan blue staining. The viable cell density was 3.92×10 6 / ml, volume 20ml, cell viability was 96.66%.

[0252] 2) Wash 293T-LAG3 cells three times with PBS and resuspend the cells to 5×10 6 cells / mL.

[0253] 3) Add 100 μl of cell suspension to each well of a 96-well plate.

[0254] 4) The antibodies of the experimental group (Bs003, Bs004, Bs010, Bs011, Bs012, Bs020 and Bs021 prepared in Example 1), the positive control (BMS (amino acid sequence as shown in SEQ ID NO: 39 and 40) and the negative control (NC-Fc (IgG4) and NC-his) were prepared into a concentration gradient (133.33, 44.44, 14.81, 4.94, 1.65, 0.55, 0.18 and 0.061 nM) and added sequentially to a 96-well plate.

[0255] 5) After resuspending the cells, incubate at 4°C for 1 hour.

[0256] 6) Wash the cells five times by centrifugation at 400 × g for 5 minutes.

[0257] 7) Use 3% BSA / PBS to dilute Mouse anti-human IgG4 Fc antibody HRP and 6x his tag monoclonal antibody HRP to the optimal concentration of 1:5000, add the diluted secondary antibodies to the corresponding cell resuspension, and incubate at 4°C for 1 hour.

[0258] 8) Wash the cells 5 times with PBS, add 100 μl TMB colorimetric solution to each well, and incubate at room temperature in the dark for 10 to 30 minutes. The solution will turn blue.

[0259] 9) Add 50-100 μl of stop solution to each well to stop the reaction.

[0260] 10) Centrifuge at 400 μg for 5 minutes.

[0261] 11) Take 100 μl of supernatant from each well and read using Envision at 450 nm. Figure 5 .

[0262] 2. Result analysis:

[0263] from Figure 5 The results showed that compared with the negative control group, Bs003( ** P<0.01) and Bs004( *** P<0.001) could bind to the LAG3 receptor on the surface of 293T-LAG3 cells, and the binding curves were obvious, showing significant differences between the two.

[0264] Compared with the negative control group, Bs010, Bs011, and Bs012 can also significantly bind to the LAG3 receptor on the surface of 293T-LAG3 cells, and the binding curves are clear and significantly different ( * P<0.05). Compared with the negative control group, there was no significant difference in the binding of positive control BMS.

[0265] Therefore, the results show that compared with the positive control antibody BMS, the bispecific antibody of the present application has a stronger binding affinity to the human LAG3 protein on the surface of 293T-LAG3 cells.

[0266] Example 3: FACS detection of anti-PD1+LAG3 dual antibodies and 293T-LAG3 cells

[0267] 1. Experimental steps:

[0268] 1) 293T-hLAG3 cells were digested and counted using trypan blue staining, and the viable cell density was 4.65×10 6 / ml, the volume was 20ml, and the cell viability was 94.21%.

[0269] 2) Wash 293T-LAG3 cells three times with PBS and resuspend the cells to 5×10 6 According to the platemap, add 100 μl of cell suspension to each well of a 96-well plate.

[0270] 3) Prepare antibody solution of corresponding concentration using 3% BSA / PBS.

[0271] 4) Add the prepared bispecific antibody prepared in Example 1 to a 96-well plate according to the platemap, 100 μl / well, mix well and incubate at 4° C. for 1 h. The specific concentrations are as follows:

[0272] Experimental groups Bs003, Bs004, and negative control IgG4 antibody: 133.33, 44.44, 14.81, 4.94, 1.65, 0.55, 0.18, and 0.061 nM.

[0273] Experimental groups Bs020, Bs021, positive control BMS (amino acid sequence as shown in SEQ ID NO: 39 and 40), negative control IgG1 antibody: 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09 and 0.030 nM.

[0274] The reason for dividing the antibodies into two groups of gradients is that the variable region domains of anti-LAG3 in the group with a high concentration gradient are monovalent, and the variable region domains of anti-LAG3 in the group with a low concentration gradient are bivalent or the molecular weight of the bispecific antibody molecule is halved, thereby doubling the molar concentration.

[0275] 5) Wash the cells five times by centrifugation at 400×g for 5 minutes.

[0276] 6) Use 3% BSA / PBS to dilute the secondary antibody Goat polyclonal Secondary Antibody to Human IgG-Fc (650) (Abcam, Ab98622) to the optimal concentration (1:100), then resuspend the corresponding cells and incubate at 4°C for 1 hour.

[0277] 7) Wash cells 5 times with PBS.

[0278] 8) Resuspend the cells in 3% BSA / PBS (200 μl) and perform flow cytometry (detected by Shengtang Biotechnology). Figure 6 .

[0279] 2. Result analysis:

[0280] from Figure 6 Judging from the curve trend, the binding of Bs020 and Bs021 antibodies to human LAG3 receptor is also better than that of anti-LAG3 BMS monoclonal antibody.

[0281] Example 4: Effects of anti-PD1+LAG3 dual antibodies on T cell activation and IL-2 secretion in human PBMCs

[0282] 1. Experimental steps:

[0283] 1) Prepare 0.5 μg / ml anti-CD3e (B6928) antibody solution using sterile PBS, add to the corresponding 96-well plate, and incubate at 4°C overnight.

[0284] 2) Aspirate the supernatant, slowly add 100 μl PBS to wash once, and remove the supernatant.

[0285] 3) PBMC cells were revived, resuspended in RPMI 1640 (Gibco, A10491-01) + 10% FBS complete medium, and the cells were counted.

[0286] 4) Dilute the cells to 2×10 6 / ml, add 100 μl of cell suspension into each well of a 96-well plate.

[0287] 5) The working concentration of each bispecific antibody prepared in Example 1 and the control group antibody (aLAG3 mAb (amino acid sequence as shown in SEQ ID NOs: 39 and 40), aPD-1 mAb (amino acid sequence as shown in SEQ ID NOs: 41 and 42), aLAG3 mAb + aPD-1 mAb) was 133.335 nM, and 50 μl was taken into each well and added to the corresponding 96-well plate.

[0288] 6) 100 μg / ml Staphylococcus aureus enterotoxin E (SEE) (Toxintechnology, ET404) solution was diluted to the corresponding concentration with culture medium, and 50 μl was added to the cells in the corresponding 96-well plate.

[0289] 7) The 96-well plate was placed in a 37°C incubator for 72 h for ELISA testing.

[0290] 8) Prepare a solution of Capture Antibody (Anti-Human IL-2 monoclonal antibody) with Coating buffer at a ratio of 1:250, add 100 μl to each well of a 96-well plate, and incubate overnight at 4°C.

[0291] 9) Prepare Wash buffer (20x) to 1x with distilled water, discard the antibody diluent in the 96-well plate, add 300 μl Wash buffer to each well and wash three times. After the last wash, tap the plate on paper.

[0292] 10) Add 200 μl of Assay diluent to each well and block at room temperature for 1 hour.

[0293] 11) Discard the Assay diluent in the 96-well plate, add 300 μl of Wash buffer to each well and wash three times. After the last wash, tap the plate on paper.

[0294] 12) Prepare standards with concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.81 and 0 pg / ml, and add 100 μl to each well of a 96-well plate.

[0295] 13) To prevent the sample OD value from being too high, dilute the sample 5 times with Assay diluent and add 100 μl to each well and incubate at room temperature for 2 h.

[0296] 14) Discard the liquid, add 300 μl of Wash buffer to each well and wash five times. After the last wash, tap the plate on paper.

[0297] 15) Prepare secondary antibody solution by mixing Detection Antibody and Enzyme Reagent at a ratio of 1:250, add 100 μl to each well, and incubate in a 96-well plate for 1 hour.

[0298] 16) Discard the secondary antibody, add 300 μl of Wash buffer to each well and wash seven times. After the last wash, tap the plate on paper.

[0299] 17) Prepare color developing solution (mix A and B solution in a ratio of 1:1), add 100 μl to each well of a 96-well plate, wait for the color to change, add stop solution, and read the OD 450 value.

[0300] 2. Result analysis:

[0301] Compared with the anti-LAG3 monoclonal antibody (2527-Fc) group, the 133.34nM dual antibody experimental groups Bs003, Bs004, Bs020, Bs021, Bs010, Bs011 and Bs012 had a higher degree of T cell activation and secreted more IL-2 levels after SEE (150pg / ml) stimulation or CD3 monoclonal antibody + SEE (150pg / ml) stimulation, and there were significant differences. This example exemplifies the results of some antibodies, see Figure 7 , among which, under the co-stimulation of CD3 monoclonal antibody + SEE (150pg / ml), the difference was more significant ( Figure 7 A).

[0302] Example 5: Establishment of MC380-hPD-L1 colon cancer animal model using B-hPD-1 / hPD-L1 / hLAG3 humanized mice and drug efficacy experiments

[0303] 1. Experimental steps:

[0304] MC38-hPD-L1 colon cancer cells were plated at 5×10 6 The concentration of 2×10 / mL was inoculated subcutaneously on the right side of female humanized B-hPD-1 / hPD-L1 / hLAG3 mice. 6 When the tumor grows to about 118 mm 3 The mice were randomly divided into groups according to the tumor volume, with 6 mice in each group, including a negative control group: human IgG (20 mg / kg), a positive control group: BMS (20 mg / kg, the amino acid sequence is shown in SEQ ID NO: 39 and 40), an anti-LAG3 monoclonal antibody group: 25-27-2 (14.14 mg / kg, the amino acid sequence is shown in SEQ ID NO: 43) and experimental groups (each bispecific antibody prepared in Example 1 (16.6 mg / kg) or Bisp6).

[0305] All groups were intraperitoneally injected with a volume of 133.4 nmol. Tumor volume and body weight were measured twice a week, and mouse body weight and tumor volume were recorded. See the experimental design diagram for details. Figure 8 At the end of the experiment, the animals were euthanized, the tumors were removed, weighed, photographed, and the relative tumor inhibition rate (TGI%) was calculated. This part of the experiment was completed by Biocytogen.

[0306] 2. Result analysis:

[0307] During the entire experiment, no obvious general clinical symptoms or mouse deaths were observed. All experimental animals were active and ate well during the administration period, indicating that the experimental animals tolerated the test product well. From the first administration to 26 days, it was found that the bispecific antibody in this experiment could significantly inhibit the growth of subcutaneous transplanted tumors of MC38-hPD-L1 colon cancer cells in B-hPD-1 / hPD-L1 / hLAG3 humanized mice, and its inhibitory effect was significantly better than that of the negative control, positive control group and anti-LAG3 monoclonal antibody group.

[0308] This example shows some test results. Fig. 9 , where G1 is the negative control group, G2 is the positive control group, G3 is the anti-LAG3 monoclonal antibody group, and G5 is Bisp6 (its structure is the same as that of Bs003 bispecific antibody, the only difference is that 2527 of the p1 chain has been humanized, and the amino acid sequence of the p1 chain is shown in SEQ ID NO: 44, the amino acid sequence of the p2 chain is shown in SEQ ID NO: 45, and the amino acid sequence of the p3 chain is shown in SEQ ID NO: 46). The specific analysis is as follows:

[0309] From the first administration to the 26th day, compared with the body weight of the G1 negative control group mice, the body weights of the G2, G3, and G5 groups showed no significant changes ( Fig. 9 A) At day 26, the mean tumor volume in the G1 group was 2402±437mm 3 The average tumor volume in the G2 group was 1396±393mm 3 The average tumor volume in the G3 group was 2223±343mm 3 The average tumor volume in the G5 group was 971±160mm 3 The TGI% of G2, G3 and G5 groups were 44.1%, 7.8% and 62.6% respectively ( Fig. 9 B).

[0310] At a dose level of 133.4 nmol, the bispecific antibody in this experiment (G5 bispecific antibody group) can significantly inhibit the growth of subcutaneously transplanted tumors of MC38-hPD-L1 colon cancer cells in B-hPD-1 / hPD-L1 / hLAG3 humanized mice, and its inhibitory effect is significantly better than that of the negative control groups G1, G2 and G3 ( Fig. 9 B).

[0311] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0312] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A bispecific antibody, characterized in that: include: A first binding region, comprising a first nanobody and a second nanobody for binding to LAG3; The second binding region is used to bind to PD-1, and the first binding region and the second binding region are connected.

2. The bispecific antibody according to claim 1, characterized in that The first Nanobody and the second Nanobody are the same or different; Preferably, the first Nanobody and the second Nanobody are different; Preferably, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the first Nanobody are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; Preferably, the amino acid sequence of the first Nanobody is as shown in SEQ ID NO: 13; Preferably, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the second Nanobody are shown as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; Preferably, the amino acid sequence of the second Nanobody is as shown in SEQ ID NO: 14; and / or, the first Nanobody and the second Nanobody are linked or not linked; And / or, the first Nanobody and the second Nanobody are connected, the first binding region further comprises a first connecting peptide, and the first Nanobody and the second Nanobody are connected via the first connecting peptide; Preferably, the C-terminus of the first Nanobody is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the second Nanobody; or The C-terminus of the second Nanobody is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the first Nanobody; Preferably, the amino acid sequence of the first connecting peptide is as shown in SEQ ID NO: 15; Preferably, the first binding region has an amino acid sequence as shown in SEQ ID NO:16 and SEQ ID NO:

17.

3. The bispecific antibody according to claim 2, characterized in that The second binding region includes a Fab antibody or a F(ab)2 antibody; Preferably, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region in the Fab antibody and the F(ab)2 antibody are shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region in the Fab antibody and the F(ab)2 antibody are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; Preferably, the amino acid sequence of the heavy chain variable region of the Fab antibody and the F(ab)2 antibody is as shown in SEQ ID NO: 18; The amino acid sequence of the light chain variable region in the Fab antibody and the F(ab)2 antibody is shown in SEQ ID NO: 19; Preferably, the CH1 region and CL region in the Fab antibody and F(ab)2 antibody have the amino acid sequences shown in SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

4. The bispecific antibody according to claim 3, characterized in that The bispecific antibody further comprises one or more second connecting peptides, and the first binding region and the second binding region are connected by the second connecting peptide; Preferably, the second connecting peptide has an amino acid sequence as shown in SEQ ID NO: 22; Preferably, the bispecific antibody has any of the following structures: 1) the first Nanobody and the second Nanobody are not linked, The second binding region is selected from a Fab antibody, The C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the first nanobody, The C-terminus of the CL region in the second binding region is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the second Nanobody; 2) the first Nanobody and the second Nanobody are not linked, The second binding region is selected from a Fab antibody, The C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the second connecting peptide, the C-terminus of the second connecting peptide is connected to the N-terminus of the second Nanobody, and the C-terminus of the CL region in the second binding region is connected to the N-terminus of the first Nanobody; 3) the first binding region is selected from the group consisting of a first nanobody-a first connecting peptide-a second nanobody, or a second nanobody-a first connecting peptide-a first nanobody, The second binding region is selected from a Fab antibody, The C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the first binding region; 4) the first binding region is selected from the group consisting of a first nanobody-a first connecting peptide-a second nanobody, or a second nanobody-a first connecting peptide-a first nanobody, The second binding region is selected from a Fab antibody, The C-terminus of the CL region in the second binding region is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the first binding region.

5. The bispecific antibody according to claim 3, characterized in that The bispecific antibody further comprises an Fc fragment, and the first binding region and / or the second binding region are connected to the Fc fragment; Preferably, the first binding region and / or the second binding region is connected to the N-terminus of the Fc fragment; or The first binding region and / or the second binding region is connected to the C-terminus of the Fc fragment; or The first binding region is connected to the N-terminus of the Fc fragment, and the second binding region is connected to the C-terminus of the Fc fragment; or The first binding region is connected to the C-terminus of the Fc fragment, and the second binding region is connected to the N-terminus of the Fc fragment; Preferably, the species of the Fc fragment is derived from cattle, horses, pigs, sheep, mice, rats, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese or humans; Preferably, the subtype of the Fc fragment is derived from IgG1, IgG2a, IgG2b, IgG3 or IgG4; Preferably, the Fc fragment is a human Fc peptide or a mutant thereof; Preferably, the human Fc peptide segment is a human IgG4 Fc peptide segment or a mutant thereof; Preferably, the Fc fragment has the amino acid sequence shown in SEQ ID NO:

23.

6. The bispecific antibody according to claim 5, characterized in that The first binding region is selected from the group consisting of a first nanobody-a first connecting peptide-a second nanobody, or a second nanobody-a first connecting peptide-a first nanobody, The second binding region is selected from a Fab antibody, The C-terminus of the first binding region is connected to the N-terminus of one chain in the Fc fragment, and the C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the other chain in the Fc fragment; Preferably, the first binding region has the amino acid sequence shown in SEQ ID NO:

16.

7. The bispecific antibody according to claim 5, characterized in that The first binding region is selected from the group consisting of a first nanobody-a first connecting peptide-a second nanobody, or a second nanobody-a first connecting peptide-a first nanobody, The second binding region is selected from F(ab)2 antibodies, The C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the Fc fragment, and the N-terminus of the first binding region is connected to the C-terminus of the CL region in the second binding region; or, the C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the Fc fragment, and the N-terminus of the first binding region is connected to the C-terminus of the Fc fragment; Preferably, the C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the Fc fragment, and the N-terminus of the first binding region is connected to the C-terminus of the CL region in the second binding region; And / or, the bispecific antibody further comprises a third connecting peptide, and the first binding region is connected to the Fc fragment or the second binding region via the third connecting peptide; Preferably, the third connecting peptide has an amino acid sequence as shown in SEQ ID NO: 24; Preferably, the C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the Fc fragment, the N-terminus of the first binding region is connected to the C-terminus of the third connecting peptide, and the N-terminus of the third connecting peptide is connected to the C-terminus of the CL region in the second binding region; or, The C-terminus of the CH1 region in the second binding region is connected to the N-terminus of the Fc fragment, the N-terminus of the first binding region is connected to the C-terminus of the third connecting peptide, and the N-terminus of the third connecting peptide is connected to the C-terminus of the Fc fragment.

8. The bispecific antibody according to claim 1, characterized in that The bispecific antibody comprises: has a first peptide chain as shown in the amino acid sequence of SEQ ID NO: 25, has a second peptide chain as shown in the amino acid sequence of SEQ ID NO: 26, has a third peptide chain as shown in the amino acid sequence of SEQ ID NO: 27; or has a first peptide chain as shown in the amino acid sequence of SEQ ID NO: 28, has a second peptide chain as shown in the amino acid sequence of SEQ ID NO: 26, has a third peptide chain as shown in the amino acid sequence of SEQ ID NO: 27; or has a first peptide chain as shown in the amino acid sequence of SEQ ID NO: 29, and has a second peptide chain as shown in the amino acid sequence of SEQ ID NO: 30; or has a first peptide chain as shown in the amino acid sequence of SEQ ID NO: 31, and has a second peptide chain as shown in the amino acid sequence of SEQ ID NO: 32; or has a first peptide chain as shown in the amino acid sequence of SEQ ID NO: 33, and has a second peptide chain as shown in the amino acid sequence of SEQ ID NO: 34; or has a first peptide chain as shown in the amino acid sequence of SEQ ID NO: 35, and has a second peptide chain as shown in the amino acid sequence of SEQ ID NO: 36; or It has a first peptide chain as shown in the amino acid sequence of SEQ ID NO:37, and a second peptide chain as shown in the amino acid sequence of SEQ ID NO:

38.

9. A nucleic acid molecule, characterized in that Encoding the bispecific antibody according to any one of claims 1 to 8.

10. An expression vector, characterized in that it comprises the nucleic acid molecule according to claim 9; Optionally, the expression vector is a eukaryotic vector or a prokaryotic vector; Optionally, the expression vector comprises at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.

11. A recombinant cell, characterized in that Carrying the nucleic acid molecule of claim 9 or the expression vector of claim 10; or expressing the bispecific antibody of any one of claims 1 to 8; Optionally, the recombinant cell is obtained by introducing the expression vector of claim 7 into a host cell; Optionally, the recombinant cell is a prokaryotic cell or a eukaryotic cell.

12. A pharmaceutical composition or kit, characterized in that: Comprising the multispecific antibody according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, the expression vector according to claim 10 or the recombinant cell according to claim 11; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

13. Use of the multispecific antibody according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, the expression vector according to claim 10 or the recombinant cell according to claim 11 in preparing a kit for detecting PD-1 and / or LAG3.

14. Use of the multispecific antibody according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, the expression vector according to claim 10, the recombinant cell according to claim 11 or the pharmaceutical composition according to claim 12 in the preparation of a drug for preventing and / or treating cancer; Optionally, the cancer is selected from colorectal cancer.

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