Bispecific antibodies and uses thereof

Abstract

The invention relates to a bispecific antibody and application thereof, and belongs to the technical field of biological medicine. Wherein the bispecific antibody comprises a first antigen binding region, and the first antigen binding region comprises an anti-CD3 single-chain antibody; and a second antigen binding region, the second antigen binding region comprising an anti-GPC3 antibody or an antigen binding fragment thereof. The bispecific antibody can be combined with CD3 and GPC3, T cell activation and proliferation are promoted, cell factors are secreted, the killing function of PBMC on tumor cells is effectively improved, and the bispecific antibody has good anti-cancer activity; meanwhile, by reducing the risk of cytokine storm caused by the CD3 bispecific antibody, the treatment safety is effectively improved. In addition, the bispecific antibody has lower binding activity to T cells and higher binding activity to tumor cells, so that the specificity and targeting of treatment are improved.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically, relates to bispecific antibodies and applications thereof. Background Art

[0002] Cancer is a major global health threat, with approximately 19 million new cases and 10 million deaths each year, and the incidence and mortality rates continue to rise. Traditional treatments such as chemotherapy and radiotherapy have serious side effects and high recurrence rates. In recent years, immunotherapy, especially PD-1 / L1 therapy, has shown great anti-cancer potential, but its overall response rate is only about 30%, and it is not effective against "cold tumors". This type of tumor has few gene mutations and is difficult for T cells to recognize, which limits the effectiveness of PD-1 / L1 therapy.

[0003] CD3-based bispecific antibodies (hereinafter referred to as "CD3 bispecific antibodies") recruit T cells to reach the local tumor, bridge T cells and tumors, promote T cell activation, and kill tumors. This type of bispecific antibody does not require neoantigens and can guide T cells to kill "cold tumors." CD3 bispecific antibodies trigger strong activation signals after binding to T cells and tumor cells, so they can also "ignore" the inhibitory signals of immune checkpoint molecules to a certain extent; but there are safety risks, such as cytokine storms and excessive immune responses.

[0004] GPC3 is basically not expressed in normal human tissues, but highly expressed in liver cancer tissues, which makes GPC3 a potential therapeutic target. So far, there are no commercially available drugs targeting GPC3. Therefore, the development of CD3 dual antibodies targeting GPC3 has important clinical significance. Summary of the invention

[0005] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a highly safe bispecific antibody targeting GPC3 and CD3.

[0006] Specifically, this application provides the following technical solutions:

[0007] In the first aspect, the present application proposes a bispecific antibody. According to an embodiment of the present application, the bispecific antibody includes: a first antigen binding region, the first antigen binding region includes an anti-CD3 single-chain antibody; and a second antigen binding region, the second antigen binding region includes an anti-GPC3 antibody or an antigen binding fragment thereof. In some examples of the present application, the aforementioned bispecific antibody can bind to CD3 and GPC3, promote T cell activation, proliferation and secretion of cytokines, effectively improve the killing function of PBMC on tumor cells, and have good anti-cancer activity; at the same time, by reducing the risk of cytokine storm caused by CD3 bispecific antibodies, the safety of treatment is effectively improved. In addition, the bispecific antibody has a low binding activity to T cells, but a high binding activity to tumor cells, which improves the specificity and targeting of the treatment.

[0008] In a second aspect, 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 of the present application. In some examples of the present application, the aforementioned nucleic acid molecule may encode a bispecific antibody that can simultaneously target GPC3 and CD3.

[0009] In a third aspect, the present application proposes an expression vector. According to an embodiment of the present application, the expression vector carries the nucleic acid molecule described in the second aspect of the present application. In some examples of the present application, the aforementioned expression vector can express a large amount of bispecific antibodies that bind to GPC3 and CD3 with high specificity and high affinity in a suitable host cell.

[0010] In the fourth aspect, the present application proposes a method for preparing the bispecific antibody described in the first aspect. According to an embodiment of the present application, the method comprises: introducing the expression vector described in the third aspect of the present application into a cell; culturing the cell under conditions suitable for protein expression and secretion to obtain the bispecific antibody. In some examples of the present application, the bispecific antibody prepared based on the present method can bind to GPC3 and CD3 with high specificity and high affinity, promote T cell activation, proliferation and secretion of cytokines, effectively promote PBMC to kill tumor cells, and has good anti-cancer activity; and the prepared bispecific antibody has low binding activity with T cells and high binding activity with tumor cells.

[0011] In a fifth aspect, the present application proposes a recombinant cell. According to an embodiment of the present application, the recombinant cell carries the bispecific antibody described in the first aspect of the present application, the nucleic acid molecule described in the second aspect, or the expression vector described in the third aspect. Wherein, the aforementioned recombinant cell is obtained by transfecting or transforming the expression vector. In some examples of the present application, the aforementioned recombinant cell can efficiently express the aforementioned bispecific antibody under appropriate conditions.

[0012] In the sixth aspect, the present application proposes a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition includes: the bispecific antibody described in the first aspect of the present application, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect. In some examples of the present application, the pharmaceutical composition comprising the aforementioned bispecific antibody can promote T cell activation, proliferation and secretion of cytokines, and effectively improve the killing function of PBMC on tumor cells. The aforementioned pharmaceutical composition exhibits excellent anti-tumor activity, and at the same time effectively improves the safety of treatment by reducing the risk of cytokine storm caused by CD3 bispecific antibodies. In addition, the bispecific antibody has low binding activity to T cells, but high binding activity to tumor cells, which improves the specificity and targeting of the treatment. The pharmaceutical composition based on the bispecific antibody can be further used to prevent or treat diseases related to GPC3 and / or CD3.

[0013] In the seventh aspect, 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 fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a drug, and the drug is used to treat or prevent tumors. In some examples of the present application, the drug containing the aforementioned bispecific antibody can promote T cell activation, proliferation and secretion of cytokines, effectively improve the killing function of PBMC on tumor cells, and has good anti-cancer activity. The drug containing the aforementioned bispecific antibody is safer and effectively reduces the risk of cytokine storm caused by CD3 bispecific antibodies.

[0014] In an eighth aspect, 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 fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a kit for detecting GPC3 and / or CD3. It can be understood by those skilled in the art that the above features and advantages of the bispecific antibody are also applicable to this use and will not be repeated here.

[0015] In the ninth aspect, the present application proposes a kit. According to an embodiment of the present application, the kit comprises the bispecific antibody described in the first aspect. In some examples of the present application, the aforementioned bispecific antibody is capable of binding to GPC3 and CD3 proteins with high specificity and high affinity. Therefore, the kit comprising the bispecific antibody can be used to detect GPC3 and / or CD3 proteins. The aforementioned kit can be used for scientific research, such as qualitatively or quantitatively detecting GPC3 and CD3 proteins in biological samples, and can also be used to judge the status of an individual, such as after obtaining the GPC3 and CD3 protein levels of the individual, judging whether the GPC3 and CD3 protein levels are higher or lower than normal levels.

[0016] 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

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 It is a schematic diagram of the configuration of the CD3×GPC3 bispecific antibody according to a specific embodiment of the present application;

[0019] Figure 2 It is a schematic diagram of flow cytometry results of the binding of CD3×GPC3 bispecific antibody to Huh7 human liver cancer cells according to a specific embodiment of the present application;

[0020] Figure 3 This is a schematic diagram of the results of CD3×GPC3 bispecific antibody promoting PBMC to kill Huh7 human liver cancer cells according to a specific embodiment of the present application;

[0021] Figure 4 This is a schematic diagram of the results of CD3×GPC3 bispecific antibody promoting PBMC to kill HepG2 human liver cancer cells according to a specific embodiment of the present application;

[0022] Figure 5 It is a schematic diagram of flow cytometry results of the binding of CD3×GPC3 bispecific antibody to T cells according to a specific embodiment of the present application;

[0023] Figure 6 It is a schematic diagram of flow cytometry results of the binding of CD3×GPC3 bispecific antibody to HepG2 human liver cancer cells according to a specific embodiment of the present application;

[0024] Figure 7 It is a schematic diagram of flow cytometry results of the binding of CD3×GPC3 bispecific antibody to HepG2 human liver cancer cells according to a specific embodiment of the present application;

[0025] Figure 8 This is a schematic diagram of the results of CD3×GPC3 bispecific antibody promoting PBMC to secrete cytokines according to a specific embodiment of the present application;

[0026] Fig. 9 This is a schematic diagram of the results of CD3×GPC3 bispecific antibody promoting CD4 T cell proliferation according to a specific embodiment of the present application;

[0027] Fig.10 This is a schematic diagram of the results of CD3×GPC3 bispecific antibody promoting CD8 T cell proliferation according to a specific embodiment of the present application;

[0028] Fig.11 This is a schematic diagram of the results of CD3×GPC3 bispecific antibody promoting PBMC to kill Huh7 human liver cancer cells according to a specific embodiment of the present application;

[0029] Fig.12 This is a schematic diagram of the results of CD3×GPC3 bispecific antibody promoting PBMC to kill HepG2 human liver cancer cells according to a specific embodiment of the present application. DETAILED DESCRIPTION

[0030] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, but cannot be understood as limiting the present application.

[0031] In the present application, 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 technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present application, "plurality" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0032] In the present application, the term "antibody" refers to an antibody that can recognize one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain antibodies only, triabodies, single chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, as long as the desired biological activity is exhibited. 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 immunologically active portions of any of these polypeptides, i.e., molecules or portions thereof that contain 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. In the present application, "bispecific antibodies" refer to antibodies that can recognize CD3 and GPC3 antigenic epitopes.

[0033] The aforementioned "human antibody or humanized antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace all the amino acid sequences 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 sequences 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 sequences of non-CDR amino acids in the constant region and variable region are all humanized, they are called humanized antibodies. The method of humanization can be carried out with reference to conventional antibody engineering technology, which will not be repeated here.

[0034] 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).

[0035] In the present application, the amino acid sequences of the listed CDRs are shown in accordance with the IMGT definition rules. However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as the Kabat rule, the Chothia rule, etc. It should be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or a region thereof (such as a variable region) should be understood to cover the complementary determining region defined by any of the aforementioned known schemes described in the present invention. Although the scope of protection requested in the present application is based on the sequences shown in the IMGT definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also be included in the scope of protection of the present application.

[0036] In the present application, the term "amino acid" is represented by a single-letter or three-letter code and has the following meaning: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0037] With respect to nucleotides, the terms "homology", "identity" or "similarity" are used to describe or compare the degree of nucleotide similarity of two or more nucleotide sequences. The percentage of "sequence homology" between a first sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical to the nucleotides at corresponding positions in the second sequence] minus [the total number of nucleotides in the first sequence] and then multiplying by [100%], where each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - relative to the first nucleotide sequence - is considered to be a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings. Some other techniques, computer algorithms and arrangements for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768-A.

[0038] For polypeptides, the terms "(substantial) homology", "identity" or "similarity" are used to describe or compare the degree of amino acid similarity between two or more polypeptides or their designated sequences when optimally aligned and compared (where nucleotides are appropriately inserted or deleted). The % homology between two sequences varies with the number of identical positions shared by these sequences when the sequences are optimally aligned (i.e., % homology = number of identical positions / total number of positions × 100), wherein the optimal alignment is determined by taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences and the length of each gap. Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0039] In the present application, under the premise of not substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art may replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present application to obtain a variant of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The variant sequence described in the present application may have at least 80% identity (or homology) with the reference sequence, which means at least 80% with each reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity. The sequence consistency described in the present application can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, with default parameters is used. The amino acid sequences described in the present invention are all shown in the manner of N-terminus to C-terminus.

[0040] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred into and / or between host cells. The 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 vector also includes a vector with a variety of the aforementioned functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0041] As used herein, the term "pharmaceutical composition" generally refers to a 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.

[0042] 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.

[0043] 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. Except for any conventional excipients incompatible with the compound of the present invention, such as any adverse biological effect produced or any other component of the pharmaceutically acceptable composition that interacts in a harmful manner, their use is also within the scope of the present invention.

[0044] In this article, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by a suitable method. The antibody or antigen-binding fragment, recombinant protein, multispecific antibody, conjugate or pharmaceutical composition of the present invention 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 injection, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.

[0045] As used herein, the term "treatment" refers to the use of the term to refer to obtaining a desired pharmacological and / or physiological effect. 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 compound to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0046] 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.

[0047] In the present application, the amino acid sequences involved are shown in Table 1.

[0048] Aiming at the low safety problem of current CD3 bispecific antibodies due to overimmunization or cytokine storm, the inventors obtained a new configuration of CD3 and GPC3 bispecific antibodies through a large number of screening and experimental verifications. The developed anti-CD3 and GPC3 bispecific antibodies have been experimentally verified to have weak binding to T cells, strong binding to tumor cells, high promoting killing activity, better anti-cancer activity and higher safety.

[0049] The present application relates to bispecific antibodies, nucleic acid molecules, expression vectors, preparation methods, recombinant cells, pharmaceutical compositions, pharmaceutical uses, uses of preparation kits, kits, disease treatment methods, and disease treatment uses, which are described in detail below:

[0050] Bispecific Antibodies

[0051] In the first aspect of the present application, the present application proposes a bispecific antibody. According to an embodiment of the present application, the aforementioned bispecific antibody comprises: a first antigen binding region, the first antigen binding region comprises an anti-CD3 single-chain antibody; and a second antigen binding region, the second antigen binding region comprises an anti-GPC3 antibody or an antigen binding fragment thereof. Through a large number of experimental verifications and screenings, the inventors found that the bispecific antibody with the aforementioned configuration can specifically target CD3 and GPC3, promote T cell activation, proliferation and secretion of cytokines (such as IL-2 and INF-γ), effectively promote PBMC to kill tumor cells (such as human liver cancer cells), and has good anti-cancer activity. The antibody has weak binding activity with T cells, so that it is more distributed in the local tumor, increasing the local antibody concentration of the tumor, reducing the peripheral antibody concentration, reducing off-target toxicity, and improving safety.

[0052] In some examples of the present application, the aforementioned first antigen binding region further comprises: a first anti-GPC3 antibody or an antigen binding fragment thereof, wherein the anti-GPC3 antibody or the antigen binding fragment thereof is a first Fab fragment of anti-GPC3 ( Figure 1 C). The first Fab fragment is connected to the anti-CD3 single-chain antibody; wherein the first Fab fragment comprises: a first light chain variable region VL, a first heavy chain variable region VH, a first light chain constant region CL and a first heavy chain constant region 1CH1, and the anti-CD3 single-chain antibody comprises: a light chain variable region VL and a heavy chain variable region VH.

[0053] The aforementioned connection includes: the C-terminus of the CH1 region of the first Fab fragment is connected to the N-terminus of the heavy chain variable region of the anti-CD3 single-chain antibody; or the C-terminus of the CH1 region of the first Fab fragment is connected to the N-terminus of the light chain variable region of the anti-CD3 single-chain antibody.

[0054] In some examples of the present application, the second antigen binding region of the anti-GPC3 antibody or antigen binding fragment thereof includes: an anti-GPC3 antibody second Fab fragment ( Figure 1 A. Figure 1 C) or anti-GPC3 nanobody ( Figure 1 B).

[0055] In some exemplary configurations of the present application, the first antigen-binding region and the second antigen-binding region of the aforementioned bispecific antibody have the following compositions:

[0056] like Figure 1As shown in A, the first antigen binding region includes: an anti-CD3 single-chain antibody, and the second antigen binding region includes: an anti-GPC3 antibody second Fab fragment; the first antigen binding region further includes: a first Fc peptide segment; the second antigen binding region further includes: a second Fc peptide segment;

[0057] like Figure 1 As shown in B, the first antigen binding region includes: an anti-CD3 single-chain antibody, and the second antigen binding region includes: an anti-GPC3 nanobody; the first antigen binding region further includes: a first Fc peptide segment; the second antigen binding region further includes: a second Fc peptide segment;

[0058] like Figure 1 As shown in C, the first antigen binding region includes: an anti-CD3 single-chain antibody and a first Fab fragment of anti-GPC3, and the second antigen binding region includes: an anti-GPC3 antibody second Fab fragment; the first antigen binding region further includes: a first Fc peptide segment; the second antigen binding region further includes: a second Fc peptide segment.

[0059] The first Fc peptide segment of the above-mentioned bispecific antibody configuration can be selected from human Fc peptide segments. In some preferred examples of the present application, the first Fc peptide segment is a human IgG1 Fc peptide segment. The first Fc peptide segment can also have at least one of L234A and L235A mutations compared to the wild-type IgG1 Fc peptide segment.

[0060] The second Fc peptide segment of the above-mentioned bispecific antibody configuration can be selected from human Fc peptide segments. In some preferred examples of the present application, the second Fc peptide segment is a human IgG1 Fc peptide segment. The second Fc peptide segment can also have at least one of L234A and L235A mutations compared to the wild-type IgG1 Fc peptide segment.

[0061] By mutating the first peptide and the second peptide, the excessive immune response associated with Fc, such as cytokine storm, can be effectively reduced, and the safety of antibody use can be improved, especially in the treatment of "cold tumors", which can better control the inflammatory response.

[0062] In some examples of the present application, the aforementioned first Fc peptide segment and the second Fc peptide segment are connected via a knob-into-hole structure.

[0063] In some examples of this application, Figure 1The first antigen binding region of the bispecific antibody of configuration A has an amino acid sequence as shown in any one of SEQ ID NOs: 4-5 or an amino acid sequence having at least 80% identity therewith; the second antigen binding region has an amino acid sequence as shown in any two of SEQ ID NOs: 2-3, 7-14 or an amino acid sequence having at least 80% identity therewith. The bispecific antibody having the aforementioned sequence can promote T cell activation and proliferation, secrete immune activation factors IL-2 and INF-γ, and promote PBMC to kill human liver cancer cells.

[0064] In a specific example of the present application, having Figure 1 The first antigen binding region of the bispecific antibody of configuration A has an amino acid sequence as shown in SEQ ID NO:5; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO:2 and SEQ ID NO:3 (antibody name: UCHT1×AB1 1:1). Experimental verification shows that the bispecific antibody with the aforementioned sequence can promote T cell activation and proliferation, secrete immune activation factors IL-2 and INF-γ, and promote PBMC to kill human liver cancer cells. Compared with other bispecific antibodies of the present application, the bispecific antibody of this sequence has stronger binding activity with Huh7 human liver cancer cells and T cells.

[0065] In a specific example of the present application, having Figure 1 The first antigen binding region of the bispecific antibody of configuration A has an amino acid sequence as shown in SEQ ID NO:4; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO:2 and SEQ ID NO:3 (antibody name: Cross3×AB1 1:1). Experimental verification shows that the bispecific antibody having the aforementioned sequence can promote T cell activation and proliferation, secrete immune activation factors IL-2 and INF-γ, and promote PBMC to kill human liver cancer cells. Compared with other bispecific antibodies of the present application, the bispecific antibody of this sequence has the strongest binding activity with Huh7 human liver cancer cells, the strongest killing function for Huh7 human liver cancer cells, the strongest promotion of PBMC secretion of immune activation cytokines, and the strongest ability to promote T cell (CD4 T cells and CD8 T cells) proliferation.

[0066] In a specific example of the present application, having Figure 1 The first antigen binding region of the bispecific antibody of configuration A has an amino acid sequence as shown in SEQ ID NO: 4; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO: 7 and SEQ ID NO: 8 (antibody name: Cross3×AB2). Experimental verification shows that the bispecific antibody with the aforementioned sequence can promote PBMC to kill human liver cancer cells.

[0067] In a specific example of the present application, having Figure 1 The first antigen binding region of the bispecific antibody of configuration A has an amino acid sequence as shown in SEQ ID NO: 4; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO: 9 and SEQ ID NO: 10 (antibody name: Cross3×AB3). Experimental verification shows that the bispecific antibody with the aforementioned sequence can promote PBMC to kill human liver cancer cells.

[0068] In a specific example of the present application, having Figure 1 The first antigen binding region of the bispecific antibody of configuration A has an amino acid sequence as shown in SEQ ID NO: 4; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO: 11 and SEQ ID NO: 12 (antibody name: Cross3×AB4). Experimental verification shows that the bispecific antibody with the aforementioned sequence can promote PBMC to kill human liver cancer cells.

[0069] In a specific example of the present application, having Figure 1 The first antigen binding region of the bispecific antibody of configuration A has an amino acid sequence as shown in SEQ ID NO: 4; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO: 13 and SEQ ID NO: 14 (antibody name: Cross3×AB5). Experimental verification shows that the bispecific antibody with the aforementioned sequence can promote PBMC to kill human liver cancer cells.

[0070] In some examples of this application, Figure 1 The first antigen-binding region of the bispecific antibody of configuration B has an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having at least 80% identity thereto; the second antigen-binding region has an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence having at least 80% identity thereto. The bispecific antibody having the aforementioned sequence can promote T cell activation and proliferation, secrete immune activation factors IL-2 and INF-γ, and promote PBMC to kill human liver cancer cells.

[0071] In a specific example of the present application, having Figure 1 The first antigen binding region of the bispecific antibody of configuration B has an amino acid sequence as shown in SEQ ID NO: 4; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO: 15 (antibody name: Cross3×AB6). Experimental verification shows that the bispecific antibody with the aforementioned sequence can promote PBMC to kill human liver cancer cells.

[0072] In some examples of this application, Figure 1The first antigen-binding region of the bispecific antibody of configuration C has an amino acid sequence as shown in any two of SEQ ID NOs: 1, 3, and 6, or an amino acid sequence having at least 80% identity therewith; the second antigen-binding region has an amino acid sequence as shown in SEQ ID NOs: 2 and 3, or an amino acid sequence having at least 80% identity therewith. The bispecific antibody having the aforementioned sequence can promote T cell activation and proliferation, secrete immune activation factors IL-2 and INF-γ, and promote PBMC to kill human liver cancer cells.

[0073] In some examples of this application, Figure 1 The first antigen binding region of the bispecific antibody of C configuration has the amino acid sequence shown in SEQ ID NO:3 and SEQ ID NO:6; the second antigen binding region has the amino acid sequence shown in SEQ ID NO:2 and SEQ ID NO:3 (antibody name: UCHT1×AB1 2:1). Experimental verification shows that the bispecific antibody with the aforementioned sequence can promote T cell activation and proliferation, secrete immune activation factors IL-2 and INF-γ, and promote PBMC to kill human liver cancer cells.

[0074] In some examples of this application, Figure 1 The first antigen binding region of the bispecific antibody of C configuration has the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3; the second antigen binding region has the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3 (antibody name: Cross3×AB1 2: 1). Experimental verification shows that the bispecific antibody with the aforementioned sequence can promote T cell activation and proliferation, secrete immune activation factors IL-2 and INF-γ, and promote PBMC to kill human liver cancer cells.

[0075] Nucleic acid molecules

[0076] In another aspect of the present application, the present application provides a nucleic acid molecule, wherein the aforementioned nucleic acid molecule encodes the aforementioned bispecific antibody.

[0077] In some examples of the present application, the aforementioned nucleic acid molecule is DNA.

[0078] It should be noted that, for the nucleic acid molecules mentioned in the specification and claims of this application, those skilled in the art should 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 DNA form or RNA form, and disclosing one of them means that the other is also disclosed.

[0079] The nucleic acid molecule obtained based on the above method can encode a bispecific antibody targeting GPC3 and CD3.

[0080] Expression vector

[0081] In another aspect of the present application, the present application proposes an expression vector. According to an embodiment of the present application, the aforementioned expression vector carries the aforementioned nucleic acid molecule. The aforementioned expression vector includes an optional controlled amino acid sequence, such as the aforementioned controlled amino acid sequence is operably connected to the aforementioned nucleic acid molecule. Among them, the aforementioned controlled amino acid sequence can guide one or more controlled amino acid sequences of the aforementioned nucleic acid molecule to be expressed in the host. The vector thus constructed can effectively express the aforementioned bispecific antibody.

[0082] It should be noted that when the nucleic acid molecule is connected to the vector, the nucleic acid molecule and the control element on the vector can be directly or indirectly connected, 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 vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control element can be operably connected.

[0083] According to an embodiment of the present invention, the aforementioned vector may refer to a cloning vector or an expression vector, which can be obtained by operably connecting the aforementioned nucleic acid to a commercially available vector (such as a plasmid or a viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0084] In this article, the term "operably linked" refers to connecting the exogenous gene to the vector so that the control elements in the vector, such as the amino acid sequence of transcription control such as and the amino acid sequence of translation control such as, can play their intended function of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, viral vectors, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, 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 aforementioned nucleic acid molecules.

[0085] In some examples of the present application, the aforementioned vector is a eukaryotic vector or a prokaryotic vector.

[0086] In some examples of the present application, the aforementioned vector includes at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector, and an adeno-associated virus vector.

[0087] Method for preparing bispecific antibodies

[0088] In another aspect of the present application, the present application proposes a method for preparing the aforementioned bispecific antibody, comprising: introducing the aforementioned expression vector into cells; culturing the aforementioned cells under conditions suitable for protein expression and secretion, so as to obtain the aforementioned bispecific antibody. Based on this method, the aforementioned bispecific antibody can be expressed in large quantities in vitro.

[0089] In some examples of the present application, the aforementioned cells are eukaryotic cells.

[0090] Recombinant cells

[0091] In another aspect of the present application, the present application proposes a recombinant cell carrying the aforementioned nucleic acid molecule or expression vector. In some examples of the present application, the aforementioned recombinant cell is obtained by transfecting or transforming the aforementioned expression vector, and the aforementioned recombinant cell can efficiently express the aforementioned bispecific antibody under appropriate conditions.

[0092] In some examples of the present application, the aforementioned recombinant cells are prokaryotic cells, eukaryotic cells or bacteriophages. It should be noted that the aforementioned eukaryotic cells do not include animal reproductive cells, fertilized eggs or embryonic stem cells.

[0093] In some examples of the present application, the aforementioned prokaryotic cell is Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0094] In some examples of the present application, the aforementioned eukaryotic cell is a fungus, an insect cell, a plant cell or a mammalian cell.

[0095] In some examples of the present application, the aforementioned fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Trichoderma.

[0096] In some examples of the present application, the aforementioned insect cells are fall armyworm cells; in some examples of the present application, the aforementioned plant cells are tobacco plant cells; in some examples of the present application, the aforementioned mammalian cells are BHK cells, CHO cells, COS cells, myeloma cells or human embryonic kidney 293 cells; and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0097] In some examples of the present application, the aforementioned recombinant cell is a mammalian cell. When the cell is a mammalian cell, the expression efficiency of the aforementioned antibody or antigen-binding fragment thereof is higher.

[0098] In some examples of the present application, the aforementioned recombinant cell is a BHK cell, a CHO cell, a COS cell or a NSO cell.

[0099] It should be noted that the aforementioned "suitable conditions" in the specification of this application refer to conditions suitable for the expression of the bispecific antibodies of this application. It is easy for those skilled in the art to understand that conditions suitable for the expression of bispecific antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell states, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of bispecific antibodies according to the specific environment of the laboratory.

[0100] Pharmaceutical composition

[0101] In another aspect of the present application, the present application proposes a pharmaceutical composition, comprising: the aforementioned bispecific antibody, nucleic acid molecule, expression vector or recombinant cell. The aforementioned pharmaceutical composition can effectively promote PBMC to kill tumor cells, has good anti-cancer activity and higher safety; and can also reduce the risk of cytokine storm caused by CD3 bispecific antibody.

[0102] In some examples of the present application, a pharmaceutically acceptable excipient is further included.

[0103] In some examples of the present application, the aforementioned excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0104] In some examples of the present application, the aforementioned pharmaceutical composition is an injection.

[0105] It should be noted that the aforementioned pharmaceutical composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the components contained in the aforementioned pharmaceutical composition can be administered to the subject as a whole, or separately. When the components contained in the aforementioned pharmaceutical composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0106] The drug of the present application contains a safe and effective amount of the active ingredient (bispecific antibody) 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. Generally, the drug preparation should match the mode of administration. The dosage form of the drug of the present application is injection, oral preparation (tablet, capsule, oral solution), 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 aforementioned drug is preferably manufactured under sterile conditions.

[0107] The effective amount of the aforementioned active ingredient in the present application may vary with 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 aforementioned factors include, but are not limited to: pharmacokinetic parameters of the aforementioned 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.

[0108] The pharmaceutically acceptable excipients mentioned above in this 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.

[0109] Use in the preparation of medicines

[0110] In another aspect of the present application, the present application proposes the use of the aforementioned bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells or pharmaceutical compositions in the preparation of drugs, and the aforementioned drugs are used to treat or prevent tumors. Based on the aforementioned bispecific antibodies and corresponding nucleic acid molecules, vectors, recombinant cells or pharmaceutical compositions, drugs can be further prepared into drugs, which can be used clinically to prevent or treat diseases related to GPC3 and / or CD3-mediated diseases.

[0111] In some examples of the present application, the CD3-mediated related diseases include autoimmune diseases.

[0112] In some examples of the present application, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0113] In some examples of the present application, the GPC3-mediated related diseases are cancer, diseases caused by transplant rejection, autoimmune diseases, and infectious diseases.

[0114] In some examples of the present application, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0115] Use in the preparation of kits

[0116] In another aspect of the present application, the present application proposes the use of the aforementioned bispecific antibody, nucleic acid molecule, expression vector, recombinant cell or pharmaceutical composition in the preparation of a kit, wherein the aforementioned kit is used to detect GPC3 and / or CD3.

[0117] As mentioned above, the bispecific antibodies of the embodiments of the present application can specifically bind to GPC3 and CD3, and therefore, the aforementioned bispecific antibodies can be used to detect GPC3 and / or CD3. Further, it can be used to prepare GPC3 and / or CD3 related kits and used in scientific research, such as qualitatively or quantitatively detecting GPC3 and / or CD3 protein molecules in biological samples. More specifically, it can be used for immunoblotting, immunoprecipitation, and other kits involving the use of GPC3 and / or CD3 and antibody specific binding properties for detection. These kits may contain any one or more of the following: antagonists, bispecific antibodies of the present invention or drug reference materials; 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 test GPC3 and / or CD3-mediated related diseases by testing the serum or blood of the subject.

[0118] Reagent test kit

[0119] In another aspect of the present application, the present application provides a kit comprising the aforementioned bispecific antibody. The aforementioned kit can be portable and accurately detect GPC3 and CD3 proteins in a sample.

[0120] In some examples of the present application, the aforementioned kit is used to detect at least one of GPC3 and CD3.

[0121] Disease treatment methods

[0122] In another aspect of the present application, the present application provides a method for preventing and / or treating a disease mediated by GPC3 and / or CD3. According to an embodiment of the present application, the aforementioned method comprises: administering a pharmaceutically acceptable amount of a bispecific antibody, a nucleic acid molecule, a vector or a transformant, a recombinant cell or a pharmaceutical composition to a subject.

[0123] 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.

[0124] The effective amount of the aforementioned bispecific antibody, nucleic acid molecule, vector or transformant, recombinant cell or pharmaceutical composition of the present application may vary with 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 aforementioned factors include, but are not limited to: pharmacokinetic parameters of the aforementioned active ingredients 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, due to the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0125] The above diseases include CD3 and / or GPC3-mediated related diseases.

[0126] In some examples of the present application, the CD3-mediated related diseases include autoimmune diseases.

[0127] In some examples of the present application, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0128] In some examples of the present application, the GPC3-mediated related diseases are cancer, diseases caused by transplant rejection, autoimmune diseases, and infectious diseases.

[0129] In some examples of the present application, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0130] Disease treatment uses

[0131] In another aspect of the present application, the present application proposes the use of the aforementioned bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells or pharmaceutical compositions in treating or preventing diseases. In some examples of the present application, administering an effective dose of a bispecific antibody, nucleic acid molecule, vector or transformant, recombinant cell or pharmaceutical composition to a subject can effectively treat and / or prevent a disease.

[0132] The above diseases include CD3 and / or GPC3-mediated related diseases.

[0133] In some examples of the present application, the CD3-mediated related diseases include autoimmune diseases.

[0134] In some examples of the present application, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0135] In some examples of the present application, the GPC3-mediated related diseases are cancer, diseases caused by transplant rejection, autoimmune diseases, and infectious diseases.

[0136] In some examples of the present application, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0137] Table 1 Amino acid sequences and corresponding nucleotide sequences involved in this application

[0138]

[0139]

[0140]

[0141]

[0142] 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.

[0143] Example 1: Preparation of CD3 and GPC3 bispecific antibodies

[0144] The steps for preparing the antibody are as follows:

[0145] (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×10 6 / mL to obtain ExpiCHO cell solution;

[0146] (2) When the bispecific antibody is of A configuration ( Figure 1 ), add the pcDNA3.4 vector containing CD3 antibody, GPC3 antibody heavy chain, and GPC3 antibody light chain (commissioned by Nanjing GenScript for synthesis) into 2 mL OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a; or, when the dual antibody is in the B configuration ( Figure 1 ), add the pcDNA3.4 vector containing CD3 antibody and GPC3 antibody (commissioned by Nanjing GenScript for synthesis) into 2 mL OptiSFM medium (purchased from ThermoFisher) at a ratio of 1:1 to obtain solution a; when the double antibody is C configuration ( Figure 1 ), pcDNA3.4 vector containing GPC3 heavy chain-CD3 antibody, GPC3 antibody heavy chain, and GPC3 antibody light chain (commissioned by Nanjing GenScript for synthesis) was added to 2 mL OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a;

[0147] (3) Add 160 μL ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) into 2 mL OptiSFM medium (purchased from Thermo Fisher) to obtain solution b;

[0148] (4) Then, solution a and solution b were mixed to obtain a transfection mixture, and the entire transfection mixture was added to 50 mL of the ExpiCHO cell solution within 5 minutes;

[0149] (5) At 37°C and 5% CO 2 After culturing for 1 day under the same conditions, 8 mL of Feed and 300 μL of Enhancer (purchased from Thermo Fisher) were added and the cells were transferred to 32°C and 5% CO 2 The culture supernatant was harvested after 9 days of culture under the same conditions, and 8 mL of feed was added on the 5th day;

[0150] (6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from Nano-Micro).

[0151] In this example, a total of 9 CD3 and GPC3 bispecific antibodies were prepared to investigate the various properties of the bispecific antibodies of the present invention. Figure 1 , Table 1 and Table 2.

[0152] Table 2 Bispecific antibody structure and corresponding amino acid sequence in Example 1

[0153]

[0154]

[0155] Note: For antibody configuration, see Figure 1 .

[0156] Example 2: Bispecific antibody flow cytometry binding experiment

[0157] Flow cytometry experiments are used to detect the binding properties of bispecific antibodies. Antibodies are added to cells, and the strength of the signal after the addition of antibodies is used to determine the binding properties of the antibodies and cells.

[0158] (1) Dilute Huh7 and HepG2 liver cancer cells to 2×10 6 / ml, added to a 1.5ml EP tube at a volume of 100μl / tube, 10μl / tube of goat serum was added thereto, and blocked at 4℃ for 30min. Serial dilutions of Cross3×AB11:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), Cross3×AB2 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:7, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:8), Cross3×AB3 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:9, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:10), Cross3×AB4 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:11, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:12) were added. NO:12), Cross3×AB5 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:13, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:14), Cross3×AB6 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:4, and the amino acid sequence of the GPC3 antibody is shown in SEQ ID NO:15), UCHT1×AB1 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:5, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), UCHT1×AB1 2:1 (the amino acid sequence of the GPC3 tumor-CD3 single chain antibody is shown in SEQ ID NO:6, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), Cross3×AB2 2:1 (the amino acid sequence of the GPC3 tumor-CD3 single-chain antibody is shown in SEQ ID NO:1, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), control hIgG1LALA (purchased from Bio-Innovation), and incubated at 4°C for 30 min.Add 1ml PBS to the EP tube, centrifuge at 4℃3500rpm×5min, discard the supernatant, and wash again with PBS. After centrifugation, discard the supernatant, resuspend the cells with 100μl / tube PBS, add 1μl / tube Alexa-647 labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson lab), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, centrifuge and discard the supernatant. Resuspend the cells with 200μl / tube PBS and detect with flow cytometer.

[0159] The results are as follows Figure 2 As shown, Cross3×AB1 1:1, Cross3×AB2, Cross3×AB3, Cross3×AB4, Cross3×AB5, and Cross3×AB6 can all bind to Huh7 human liver cancer cells, and Cross3×AB1 1:1 antibody has the strongest binding.

[0160] The results are as follows Figure 6 As shown, Cross3×AB1 1:1, Cross3×AB1 2:1, UCHT1×AB1 1:1, and UCHT1×AB12:1 antibodies can all bind to HepG2 human liver cancer cells with similar binding abilities.

[0161] The results are as follows Figure 7 As shown, Cross3×AB1 1:1, Cross3×AB1 2:1, UCHT1×AB1 1:1, and UCHT1×AB12:1 antibodies can all bind to Huh7 human liver cancer cells, and the 1:1 configuration of the bispecific antibodies Cross3×AB1 1:1 and UCHT1×AB1 1:1 have stronger binding.

[0162] (2) Dilute PBMC to 2×10 6 / ml, added to a 1.5ml EP tube at a volume of 100μl / tube, 10μl / tube of goat serum was added thereto, and blocked at 4℃ for 30min. Serial dilutions of the bispecific antibodies Cross3×AB1 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), Cross3×AB1 2:1 (the amino acid sequence of the GPC3 tumor-CD3 single chain antibody is shown in SEQ ID NO:1, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), UCHT1×AB1 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:5, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), UCHT1×AB1 2:1 (the amino acid sequence of the GPC3 tumor-CD3 single chain antibody is shown in SEQ ID NO:6, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:7), NO:2, and the amino acid sequence of the light chain of the GPC3 antibody is shown in SEQ ID NO:3), control hIgG1LALA (purchased from Baiying Bio), and incubated at 4°C for 30min. Add 1ml PBS to the EP tube, centrifuge at 4°C 3500rpm×5min, discard the supernatant, and wash again with PBS. After centrifugation, discard the supernatant, resuspend the cells with 100μl / tube PBS, add 1μl / tube Alexa-647-labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson lab) and 0.5μl / tube PerCP-Cy5.5-labeled anti-human CD8 antibody, and incubate at 4°C in the dark for 30min. Wash twice with PBS, discard the supernatant after centrifugation. Resuspend the cells with 200μl / tube PBS and detect by flow cytometry.

[0163] The results are as follows Figure 5 As shown, Cross3×AB1 1:1, Cross3×AB1 2:1, UCHT1×AB1 1:1, and UCHT1×AB12:1 were all able to bind to T cells, and the T cell binding activities of Cross3×AB1 1:1 and Cross3×AB1 2:1 were similar, both weaker than those of UCHT1×AB1 1:1 and UCHT1×AB1 2:1.

[0164] Example 3: Experiment on Bispecific Antibodies Promoting PBMC to Kill Tumor Cells

[0165] Detect the ability of bispecific antibodies to promote PBMC to kill tumor cells.

[0166] (a) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate on the machine;

[0167] (b) HepG2 and Huh7 human liver cancer cells were diluted to 2×10 5 / mL, and added to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then incubated at 37°C, 5% CO 2 The xCELLigence RTCA MP instrument was used to detect the cell coefficient for 24 h under the conditions;

[0168] (c) using complete RPMI-1640 medium, serially diluted bispecific antibodies Cross3×AB1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), Cross3×AB2 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:7, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:8), Cross3×AB3 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:9, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:10), Cross3×AB4 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:11, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:12), and Cross3×AB5 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:13, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:14). NO:11, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:12), Cross3×AB5 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:13, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:14), Cross3×AB6 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:4, and the amino acid sequence of the GPC3 antibody is shown in SEQ ID NO:15), UCHT1×AB1 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:5, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), UCHT1×AB1 2:1 (the amino acid sequence of the GPC3 tumor-CD3 single chain antibody is shown in SEQ ID NO:6, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:7). NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), Cross3×AB2 2:1 (the amino acid sequence of the GPC3 tumor-CD3 single-chain antibody is shown in SEQ ID NO:1, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), and control hIgG1LALA (purchased from Bio-Bio), are added to the RTCA plate obtained in step (2), and the added volume is 20 μl / well;

[0169] (4) PBMC (purchased from Sai Li Biotechnology) were diluted to 1.25×10 6 pcs / ml, added to the RTCA plate obtained in step (3), with the added volume being 80 μl / well;

[0170] (5) The reaction system obtained in step (4) was heated at 37°C and 5% CO 2 The cell proliferation was measured using the xCELLigence RTCA MP instrument for 24 h.

[0171] The results are as follows Figure 3 As shown, Cross3×AB1 1:1, Cross3×AB2, Cross3×AB3, Cross3×AB4, Cross3×AB5, and Cross3×AB6 can all promote PBMC to kill Huh7 human liver cancer cells, and Cross3×AB1 1:1 has the strongest killing function.

[0172] The results are as follows Figure 4 As shown, Cross3×AB1 1:1, Cross3×AB2, Cross3×AB3, Cross3×AB4, Cross3×AB5, and Cross3×AB6 can all promote PBMC to kill HepG2 human liver cancer cells, and Cross3×AB1 1:1 has the strongest killing function.

[0173] The results are as follows Fig.11 As shown, Cross3×AB1 1:1, Cross3×AB1 2:1, UCHT1×AB1 1:1, and UCHT1×AB12:1 can all promote PBMC to kill Huh7 human liver cancer cells, and Cross3×AB1 1:1 has the strongest killing function.

[0174] The results are as follows Fig.12 As shown, Cross3×AB1 1:1, Cross3×AB1 2:1, UCHT1×AB1 1:1, and UCHT1×AB12:1 can all promote PBMC to kill HepG2 human liver cancer cells, and Cross3×AB1 1:1 has the strongest killing function.

[0175] Example 4: Experiment on Bispecific Antibodies Promoting Cytokine Secretion by PBMC

[0176] The bispecific antibody was added to the co-incubation system of PBMC and HepG2 human liver cancer cells. The culture supernatant was collected after 48 hours of culture, and the cytokine content in the supernatant was detected to determine the characteristics of the bispecific antibody in inducing cytokine release.

[0177] (a) HepG2 cells were diluted to 1×10 5 / ml, added to 96-well plate, 37°C, 5% CO 2 Culture in an incubator for 48 h;

[0178] (b) The bispecific antibodies Cross3×AB1 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), Cross3×AB1 2:1 (the amino acid sequence of the GPC3 tumor-CD3 single chain antibody is shown in SEQ ID NO:1, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), UCHT1×AB1 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:5, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), UCHT1×AB1 2:1 (the amino acid sequence of the GPC3 tumor-CD3 single chain antibody is shown in SEQ ID NO:5, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), and UCHT1×AB1 2:1 (the amino acid sequence of the GPC3 tumor-CD3 single chain antibody is shown in SEQ ID NO: NO:6, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), and the control hIgG1LALA (purchased from Bio-Bio) were serially diluted and added to a 96-well plate at 20 μl / well;

[0179] (c) PBMC (purchased from Sai Li Biotechnology) were diluted to 1.25×10 6 / ml, added into 96-well plate, 80 μl / well;

[0180] (d) Incubate the 96-well plate at 37°C with 5% CO 2 Culture in an incubator for 48 h;

[0181] (e) Centrifugation at 300 g for 10 min at room temperature to collect the cell culture supernatant;

[0182] (f) The cytokine content in the supernatant was detected using a CBA kit (purchased from BD).

[0183] The results are as follows Figure 8As shown, Cross3×AB1 1:1, Cross3×AB1 2:1, UCHT1×AB1 1:1, and UCHT1×AB12:1 can all promote PBMC to secrete immune-activating cytokines IL-2 and IFN-γ, among which Cross3×AB1 1:1 has the strongest promoting ability.

[0184] Example 5: Experiment on promoting T cell proliferation by bispecific antibodies

[0185] PBMC cells were labeled with CFSE fluorescence, and bispecific antibodies were added to the co-incubation system of PBMC and HepG2 human liver cancer cells. After 72 hours of culture, the CFSE fluorescence intensity in CD4 T cells and CD8 T cells was detected by flow cytometry to determine the characteristics of bispecific antibodies in inducing T cell proliferation.

[0186] (1) Dilute AGS cells to 1×10 using complete RPMI 1640 medium. 5 / ml, added into 96-well plate;

[0187] (2) The bispecific antibodies Cross3×AB1 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:4, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), Cross3×AB1 2:1 (the amino acid sequence of the GPC3 tumor-CD3 single chain antibody is shown in SEQ ID NO:1, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), UCHT1×AB1 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO:5, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), UCHT1×AB1 2:1 (the amino acid sequence of the GPC3 tumor-CD3 single chain antibody is shown in SEQ ID NO:5, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), and UCHT1×AB1 2:1 (the amino acid sequence of the GPC3 tumor-CD3 single chain antibody is shown in SEQ ID NO: NO:6, the amino acid sequence of the GPC3 antibody heavy chain is shown in SEQ ID NO:2, and the amino acid sequence of the GPC3 antibody light chain is shown in SEQ ID NO:3), and the control hIgG1LALA (purchased from Bio-Bio) were serially diluted and added to a 96-well plate at 20 μl / well;

[0188] (3) PBMCs were labeled with 5 μM CTV. After labeling, PBMCs (purchased from Sai Li Biotechnology) were diluted to 1.25 × 106 / ml using complete RPMI 1640 medium and added to a 96-well plate at 80 μl / well.

[0189] (4) Incubate the 96-well plate at 37°C and 5% CO 2 Culture in an incubator for 72 h;

[0190] (5) Add CD8 antibody labeled with PerCP-Cy5.5 and CD4 antibody labeled with BV605 and incubate at 4°C in the dark for 30 min.

[0191] (6) Wash twice with PBS and discard the supernatant after centrifugation.

[0192] (7) Resuspend the cells in 200 μl / tube of PBS and analyze them using a flow cytometer.

[0193] The results are as follows Fig. 9 As shown, Cross3×AB1 1:1, Cross3×AB1 2:1, UCHT1×AB1 1:1, and UCHT1×AB12:1 can all promote CD4 T cell proliferation, and Cross3×AB1 1:1 has the strongest ability to promote proliferation;

[0194] The results are as follows Fig.10 As shown, Cross3×AB1 1:1, Cross3×AB1 2:1, UCHT1×AB1 1:1, and UCHT1×AB12:1 can all promote the proliferation of CD8 T cells, and Cross3×AB1 1:1 has the strongest proliferation-promoting ability.

[0195] The above experimental results show that the bispecific antibody of the present application can bind to CD3 and GPC3, thereby promoting T cell activation, proliferation, and cytokine secretion, effectively promoting PBMC to kill tumor cells, and has good anti-cancer activity; the bispecific antibody of the present application can achieve high tumor binding activity, low T cell binding activity, and higher anti-cancer activity. In summary, the bispecific antibody of the present application can promote immune cells to fight cancer, has good anti-cancer activity, and has good clinical application value and drug development value.

[0196] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, the technical solution of the present application can be subjected to a variety of simple modifications, including combining various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed in the present application and belong to the protection scope of the present application.

[0197] 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.

[0198] 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 the 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 antigen binding region, the first antigen binding region comprising an anti-CD3 single-chain antibody; and The second antigen binding region comprises an anti-GPC3 antibody or an antigen binding fragment thereof.

2. The bispecific antibody according to claim 1, characterized in that The first antigen binding region further comprises: a first anti-GPC3 antibody or an antigen binding fragment thereof, wherein the anti-GPC3 antibody or the antigen binding fragment thereof is a first Fab fragment of anti-GPC3; Optionally, the first Fab fragment is linked to the anti-CD3 single-chain antibody; wherein the first Fab fragment comprises: a first light chain variable region VL, a first heavy chain variable region VH, a first light chain constant region CL and a first heavy chain constant region 1CH1, and the anti-CD3 single-chain antibody comprises: a light chain variable region VL and a heavy chain variable region VH; Optionally, the C-terminus of the CH1 region of the first Fab fragment is connected to the N-terminus of the heavy chain variable region of the anti-CD3 single-chain antibody; or The C-terminus of the CH1 region of the first Fab fragment is connected to the N-terminus of the light chain variable region of the anti-CD3 single-chain antibody; Optionally, the anti-GPC3 antibody or antigen-binding fragment thereof comprises: an anti-GPC3 antibody second Fab fragment or an anti-GPC3 nanobody.

3. The bispecific antibody according to claim 1 or 2, characterized in that: The first antigen binding region comprises: an anti-CD3 single-chain antibody, and the second antigen binding region comprises: an anti-GPC3 antibody second Fab fragment; Optionally, the first antigen binding region comprises: an anti-CD3 single-chain antibody, and the second antigen binding region comprises: an anti-GPC3 nanobody; Optionally, the first antigen-binding region comprises: an anti-CD3 single-chain antibody and a first Fab fragment of an anti-GPC3 antibody, and the second antigen-binding region comprises: a second Fab fragment of an anti-GPC3 antibody.

4. The bispecific antibody according to claim 3, characterized in that The first antigen binding region further comprises: a first Fc peptide segment; the second antigen binding region further comprises: a second Fc peptide segment; Optionally, the first Fc peptide segment is selected from a human Fc peptide segment, preferably a human IgG1 Fc peptide segment; Optionally, the first Fc peptide segment has at least one of L234A and L235A mutations compared to a wild-type IgG1 Fc peptide segment; Optionally, the second Fc peptide segment is selected from a human Fc peptide segment, preferably a human IgG1 Fc peptide segment; Optionally, the second Fc peptide segment has at least one of L234A and L235A mutations compared to the wild-type IgG1 Fc peptide segment; Optionally, the first Fc peptide segment and the second Fc peptide segment are connected via a knob-into-hole structure.

5. The bispecific antibody according to claim 4, characterized in that The first antigen-binding region has an amino acid sequence as shown in any one of SEQ ID NOs: 4-5, or an amino acid sequence having at least 80% identity thereto; the second antigen-binding region has an amino acid sequence as shown in any two of SEQ ID NOs: 2-3, 7-14, or an amino acid sequence having at least 80% identity thereto; Optionally, the first antigen-binding region has an amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having at least 80% identity thereto; the second antigen-binding region has an amino acid sequence as shown in SEQ ID NO: 15, or an amino acid sequence having at least 80% identity thereto; Optionally, the first antigen-binding region has an amino acid sequence as shown in any two of SEQ ID NOs: 1, 3, and 6, or an amino acid sequence that is at least 80% identical thereto; the second antigen-binding region has an amino acid sequence as shown in SEQ ID NOs: 2 and 3, or an amino acid sequence that is at least 80% identical thereto.

6. The bispecific antibody according to claim 4, characterized in that The first antigen binding region has an amino acid sequence as shown in SEQ ID NO: 5; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO: 2 and SEQ ID NO: 3; Optionally, the first antigen binding region has an amino acid sequence as shown in SEQ ID NO:4; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO:2 and SEQ ID NO:3; Optionally, the first antigen binding region has an amino acid sequence as shown in SEQ ID NO:4; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO:7 and SEQ ID NO:8; Optionally, the first antigen binding region has an amino acid sequence as shown in SEQ ID NO:4; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO:9 and SEQ ID NO:10; Optionally, the first antigen binding region has an amino acid sequence as shown in SEQ ID NO:4; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO:11 and SEQ ID NO:12; Optionally, the first antigen binding region has an amino acid sequence as shown in SEQ ID NO:4; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO:13 and SEQ ID NO:14; Optionally, the first antigen binding region has an amino acid sequence as shown in SEQ ID NO: 4; the second antigen binding region has an amino acid sequence as shown in SEQ ID NO: 15; Optionally, the first antigen binding region has the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 6; the second antigen binding region has the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3; Optionally, the first antigen-binding region has the amino acid sequence shown in SEQ ID NO:1 and SEQ ID NO:3; the second antigen-binding region has the amino acid sequence shown in SEQ ID NO:2 and SEQ ID NO:

3.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the bispecific antibody according to any one of claims 1 to 6; Optionally, the nucleic acid molecule is selected from DNA.

8. An expression vector, characterized in that Carrying the nucleic acid molecule according to claim 7.

9. A method for preparing the bispecific antibody according to any one of claims 1 to 6, characterized in that: include: Introducing the expression vector of claim 8 into a cell; culturing the cells under conditions suitable for protein expression and secretion to obtain the bispecific antibody; Optionally, the cell is a eukaryotic cell.

10. A recombinant cell, characterized in that The recombinant cell carries the bispecific antibody according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, or the expression vector according to claim 8.

11. A pharmaceutical composition, characterized in that include: The bispecific antibody according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8 or the recombinant cell according to claim 10; Optionally, further comprising a pharmaceutically acceptable excipient; Optionally, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers; Optionally, the pharmaceutical composition is an injection.

12. Use of the bispecific antibody according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the recombinant cell according to claim 10 or the pharmaceutical composition according to claim 11 in the preparation of a drug for treating or preventing GPC3 and / or CD3-mediated related diseases.

13. The use according to claim 12, characterized in that The CD3-mediated related diseases include autoimmune diseases; Optionally, the autoimmune disease comprises at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis; Optionally, the GPC3-mediated related diseases are cancer, diseases caused by transplant rejection, autoimmune diseases, infectious diseases; Optionally, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

14. Use of the bispecific antibody according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the recombinant cell according to claim 10 or the pharmaceutical composition according to claim 11 in the preparation of a kit for detecting GPC3 and / or CD3.

15. A kit, characterized in that: The kit comprises the bispecific antibody according to any one of claims 1 to 6.

16. The kit according to claim 15, characterized in that The kit is used to detect at least one of GPC3 and CD3.

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