Recombinant antibodies and their use

Abstract

The application provides a bispecific antibody and application thereof, and the antibody is a double-chain antibody, which comprises a first peptide chain and a second peptide chain, wherein the first peptide chain comprises a CD3 single-chain antibody and a first Fc region, and the CD3 single-chain antibody comprises a heavy chain variable region and a light chain variable region; the second peptide chain comprises a TIGIT extracellular region and a second Fc region; the C terminal of the CD3 single-chain antibody is connected with the N terminal of the first Fc region; and the C terminal of the TIGIT extracellular region is connected with the N terminal of the second Fc region. The prepared bispecific antibody can simultaneously target CD3 and CD155, thereby mediating the killing of tumor cells by T cells, and has strong tumor inhibition capacity.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to recombinant antibodies and their applications, and more specifically, to the use of recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, the use of said recombinant antibodies or antigen-binding fragments or nucleic acid molecules or expression vectors or recombinant cells or compositions in the preparation of drugs, drugs, the use of said recombinant antibodies in the preparation of kits, and kits. Background Technology

[0002] In recent years, bispecific antibodies have become a research hotspot in immunotherapy. Bispecific antibodies are artificial antibodies containing two specific antigen-binding sites, which can bridge the gap between target cells (tumor cells) and effector cells (immune cells) to produce targeted tumor killing effector function. However, due to (1) the risk of immunogenicity of bispecific antibody drugs: the antibodies themselves are not native to humans, but are produced by animals such as mice through antigen immunization. Humanization can only partially reduce their immunogenicity. The human immune system will still produce antibodies against the antibody, i.e., anti-antibodies, which will lead to reduced efficacy or even failure of the drug. (2) If the antibody targeting tumor-associated antigens has a light and heavy chain dimer structure, it will be difficult to produce and purify; if the antibody targeting tumor-associated antigens is a ScFv fragment, its affinity for tumor-associated antigens will be greatly reduced, affecting the anti-cancer effect.

[0003] Therefore, further development of bispecific antibodies with low immunogenicity and high affinity is still needed. Summary of the Invention

[0004] This application is based on the inventor's discoveries and understanding of the following facts and problems:

[0005] In tumor tissues, CD155 is differentially highly expressed compared to normal tissues. Furthermore, CD155 binds to the immune checkpoint receptor TIGIT, thereby inhibiting the anti-cancer function of immune cells. The CD3 antibody single-chain variable fragment (ScFv) can bind to T cells. Based on this, the inventors designed a bispecific antibody targeting CD155, CD3×TIGIT extra. After extensive screening, a superior bispecific antibody was obtained. This antibody can simultaneously bind to CD155, which is highly expressed in tumor tissues, and T cells, effectively mediating the killing of tumor cells by T cells and exhibiting strong anti-cancer ability.

[0006] Therefore, in a first aspect, the present invention provides a recombinant antibody. According to an embodiment of the present invention, it comprises: a first peptide chain containing a CD3 single-chain antibody and a first Fc region, wherein the CD3 single-chain antibody includes a heavy chain variable region and a light chain variable region; and a second peptide chain containing a TIGIT extracellular region and a second Fc region; wherein the C-terminus of the CD3 single-chain antibody is linked to the N-terminus of the first Fc region, and the C-terminus of the TIGIT extracellular region is linked to the N-terminus of the second Fc region. The recombinant antibody according to an embodiment of the present invention can simultaneously bind to CD3 and CD155, effectively mediating the killing effect of T cells on tumor cells, and exhibiting strong tumor suppressor ability.

[0007] In a second aspect, the present invention provides a nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the recombinant antibody described in the first aspect. The recombinant antibody encoded by the nucleic acid according to an embodiment of the present invention can bind to both CD3 and CD155 simultaneously, effectively mediating the killing effect of T cells on tumor cells, and exhibiting strong tumor suppressor ability.

[0008] In a third aspect, the present invention provides an expression vector. According to embodiments of the invention, the vector carries the nucleic acid described in the second aspect. The expression vector may include an optional control sequence operatively linked to the nucleic acid molecule. The control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The expression vector proposed in the embodiments of the present invention can efficiently express the recombinant antibody in suitable host cells. The recombinant antibody can simultaneously bind to CD3 and CD155, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor capabilities.

[0009] In a fourth aspect, the present invention provides a method for preparing the recombinant antibody described in the first aspect. According to embodiments of the present invention, the method includes: introducing the expression vector described in the third aspect into cells; culturing the cells under conditions suitable for protein expression and secretion to obtain the recombinant antibody. The method according to embodiments of the present invention can effectively obtain the recombinant antibody, which can simultaneously bind to CD3 and CD155, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor ability.

[0010] In a fifth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, the recombinant cell carries the nucleic acid described in the second aspect or the expression vector described in the third aspect. The recombinant cell is obtained by transfection or transformation of the expression vector. According to some specific embodiments of the invention, the recombinant cell can efficiently express the aforementioned recombinant antibody under suitable conditions. The recombinant antibody can simultaneously bind to CD3 and CD155, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor ability.

[0011] In a sixth aspect, the present invention provides a composition. According to embodiments of the present invention, the composition comprises: the recombinant antibody described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fifth aspect. As previously described, the recombinant antibody of the embodiments of the present invention can effectively bind to CD3 and CD155 protein molecules, prompting T cells to selectively locate to the tumor site rather than circulating in the peripheral circulation, thus avoiding systemic activation. Compositions containing the recombinant antibody, such as food compositions and pharmaceutical compositions, also have significant therapeutic or preventative effects against tumors.

[0012] In a seventh aspect, the present invention provides the use of the recombinant antibody described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect in the preparation of a medicament for the treatment or prevention of cancer. As previously described, the recombinant antibody of the embodiments of the present invention can effectively bind to CD3 and CD155 protein molecules, prompting T cells to selectively locate to the tumor site rather than in the peripheral circulation, avoiding systemic activation. Medicaments containing a series of substances including the recombinant antibody also have significant therapeutic or preventative effects against cancer.

[0013] In an eighth aspect, the present invention provides a medicament. According to embodiments of the present invention, the medicament comprises: the recombinant antibody described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, the recombinant cells described in the fifth aspect, or the composition described in the sixth aspect. According to embodiments of the present invention, the medicament is used to treat cancer. As previously stated, the recombinant antibody of the present invention can effectively bind to CD3 and CD155 protein molecules, prompting T cells to selectively localize to the tumor site rather than circulating in the peripheral circulation, avoiding systemic activation. Therefore, the medicament comprising the recombinant antibody also has a significant therapeutic or preventative effect against cancer.

[0014] In a ninth aspect of the invention, the recombinant antibody described in the first aspect is proposed for use in the preparation of a kit. According to embodiments of the invention, the kit is used to detect CD3 and / or CD155. The recombinant antibody can bind to CD3 and / or CD155 proteins; therefore, a kit containing the recombinant antibody can be used for the efficient detection of CD3 and / or CD155. The kit can be used in scientific research, such as for the qualitative or quantitative detection of CD3 and / or CD155 proteins in biological samples.

[0015] In a tenth aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit comprises the recombinant antibody described in the first aspect. The recombinant antibody provided according to the embodiment of the invention can bind to CD3 and / or CD155 proteins; therefore, the kit comprising the recombinant antibody can be used for the effective detection of CD3 and / or CD155. The kit can be used in scientific research, such as for the qualitative or quantitative detection of CD3 and / or CD155 proteins in biological samples. Furthermore, it can also be used to assess an individual's condition, such as determining whether the individual's PD-L1 level is excessively high or low compared to normal levels after obtaining the individual's PD-L1 level.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0017] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0018] Figure 1 This is a schematic diagram of the structure of a recombinant bispecific antibody according to an embodiment of the present invention. The linking position of the linking peptide is not shown. The first Fc region (left side) and the second Fc region (right side) of the recombinant bispecific antibody are linked by a knock-into-hole structure.

[0019] Figure 2 This is a graph showing the detection results of the binding ability of the recombinant bispecific antibody to Jurkat cells according to an embodiment of the present invention;

[0020] Figure 3 This is a graph showing the detection results of the binding ability of the recombinant bispecific antibody to CHO-K1-CD155 according to an embodiment of the present invention;

[0021] Figure 4 This is a graph showing the detection results of the binding ability of the recombinant bispecific antibody to lung cancer NCI-H1299 cells according to an embodiment of the present invention;

[0022] Figure 5This is a graph showing the detection results of recombinant bispecific antibody against NCI-H1299 lung cancer cells by PBMCs according to an embodiment of the present invention;

[0023] Figure 6 This is a graph showing the detection results of recombinant bispecific antibody against PBMCs killing lung cancer NCI-H358 cells according to an embodiment of the present invention; and

[0024] Figure 7 This is a diagram showing the results of the recombinant bispecific antibody according to an embodiment of the present invention killing HepG2 liver cancer cells by PBMCs. Detailed Implementation

[0025] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0026] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0027] The endpoints and any values ​​of the ranges disclosed herein 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 the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0028] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0029] In this article, "operable ligation" refers to ligating a foreign gene to a vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene.

[0030] In this article, "double-chain antibody" refers to a peptide chain that can specifically recognize different protein molecules and is connected to two chains in the Fc region, wherein the two chains in the Fc region are connected through a knot-in-hole structure.

[0031] In this paper, the "knob into hole structure" refers to the formation of a button (hole) mutation in the CH3 region of the antibody heavy chain Fc, which facilitates heavy chain interlocking and the formation of a heterodimer. For example, in this application, it is achieved by mutating the amino acids in the CH3 domain of human IgG1-Fc (one chain has T366S, L368A, Y407V mutations, i.e., "hole"; the other chain has T366W mutations, i.e., "knob").

[0032] In a first aspect, the present invention provides a recombinant antibody, which is a double-chain antibody comprising: a first peptide chain containing a CD3 single-chain antibody and a first Fc region, wherein the CD3 single-chain antibody includes a heavy chain variable region and a light chain variable region; and a second peptide chain containing a TIGIT extracellular region and a second Fc region; wherein the C-terminus of the CD3 single-chain antibody is linked to the N-terminus of the first Fc region, and the C-terminus of the TIGIT extracellular region is linked to the N-terminus of the second Fc region. The recombinant antibody according to embodiments of the present invention can simultaneously bind to CD3 and CD155, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor ability.

[0033] According to some specific embodiments of the present invention, the above-mentioned recombinant antibody may further include at least one of the following additional technical features:

[0034] According to some specific embodiments of the present invention, it further includes a linker peptide 1, wherein the N-terminus of the linker peptide 1 is connected to the C-terminus of the heavy chain variable region, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the light chain variable region.

[0035] According to some specific embodiments of the present invention, the linker peptide 1 comprises the amino acid sequence shown in SEQ ID NO: 14.

[0036] GGGGSGGGGSGGGGS (SEQ ID NO: 14).

[0037] According to some specific embodiments of the present invention, it further includes a linker peptide 2, wherein the N-terminus of the linker peptide 2 is connected to the C-terminus of the light chain variable region, and the C-terminus of the linker peptide 2 is connected to the N-terminus of the first Fc region.

[0038] According to some specific embodiments of the present invention, the linker peptide 2 comprises the amino acid sequence shown in SEQ ID NO: 15.

[0039] GGGGS (SEQ ID NO: 15).

[0040] According to some specific embodiments of the present invention, it further includes a linker peptide 3, wherein the N-terminus of the linker peptide 3 is connected to the C-terminus of the extracellular region of the TIGIT, and the C-terminus of the linker peptide 3 is connected to the N-terminus of the second Fc region.

[0041] According to some specific embodiments of the present invention, the linker peptide 3 comprises the amino acid sequence shown in SEQ ID NO: 18.

[0042] GGGGS (SEQ ID NO: 18).

[0043] According to some specific embodiments of the present invention, the first Fc region and the second Fc region are connected by a knot into hole structure.

[0044] According to some specific embodiments of the present invention, the first Fc region has at least one of the following mutations compared to the wild-type IgG1 Fc region: lack of CH1 region and T366W mutation; the second Fc region has at least one of the following mutations compared to the wild-type IgG1 Fc region: lack of CH1 region, T366S mutation, L368A mutation, and Y407V mutation.

[0045] According to some specific embodiments of the present invention, at least a portion of the first Fc region and the second Fc region are derived from at least one of mouse antibodies, human antibodies, primate antibodies or mutants thereof.

[0046] According to some specific embodiments of the present invention, at least a portion of the first Fc region and the second Fc region are derived from mouse antibodies, human antibodies, primate IgG or mutants thereof.

[0047] According to some specific embodiments of the present invention, at least a portion of the first Fc region and the second Fc region are derived from mouse antibodies, human antibodies, primate IgG1 or mutants thereof.

[0048] According to some specific embodiments of the present invention, at least a portion of the first Fc region and the second Fc region are derived from human IgG1 or its mutants.

[0049] According to some specific embodiments of the present invention, the first antibody Fc region has an amino acid sequence as shown in SEQ ID NO:16, and the second antibody Fc region has an amino acid sequence as shown in SEQ ID NO:19.

[0050] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 16).

[0051] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 19).

[0052] According to some specific embodiments of the present invention, the recombinant antibody has the amino acid sequences shown in SEQ ID NO: 3 and 4.

[0053] EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3)。

[0054] MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFcIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:4)。

[0055] In a second aspect, the present invention provides a nucleic acid encoding the recombinant antibody described in the first aspect. The recombinant antibody encoded by the nucleic acid according to embodiments of the present invention can simultaneously bind to CD3 and CD155, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor-suppressive ability.

[0056] According to some specific embodiments of the present invention, the nucleic acid has the nucleotide sequences shown in SEQ ID NO: 1 and 2.

[0057]

[0058]

[0059] It should be noted that those skilled in the art should understand that the nucleic acids mentioned in this specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed as well. In addition, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one means that the other is also disclosed.

[0060] In a third aspect, the present invention provides an expression vector carrying the nucleic acid described in the second aspect. The expression vector may include an optional control sequence operatively linked to the nucleic acid molecule. The control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The expression vector proposed in the embodiments of the present invention can efficiently express the recombinant antibody in suitable host cells. The recombinant antibody can simultaneously bind to CD3 and CD155, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor ability.

[0061] In a fourth aspect, the present invention provides a method for preparing the recombinant antibody described in the first aspect, comprising: introducing the expression vector described in the third aspect into cells; and culturing the cells under conditions suitable for protein expression and secretion to obtain the recombinant antibody. The method proposed in the embodiments of the present invention can effectively obtain the recombinant antibody, which can simultaneously bind to CD3 and CD155, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor ability.

[0062] According to some specific embodiments of the present invention, the cells are eukaryotic cells.

[0063] According to some specific embodiments of the present invention, the eukaryotic cells are mammalian cells.

[0064] According to some specific embodiments of the present invention, the eukaryotic cells do not include germ cells, fertilized eggs, or embryonic stem cells.

[0065] In a fifth aspect, the present invention provides a recombinant cell carrying the nucleic acid described in the second aspect or the expression vector described in the third aspect. The recombinant cell is obtained by transfection or transformation of the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently express the aforementioned recombinant antibody under suitable conditions. The recombinant antibody can simultaneously bind to CD3 and CD155, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor ability.

[0066] It should be noted that the recombinant cells described in this invention 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 can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, Trichoderma, etc.; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include germ cells, fertilized eggs, or embryonic stem cells.

[0067] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the recombinant antibody described in this application. Those skilled in the art will readily understand that suitable conditions for recombinant antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the recombinant antibody based on the specific environment of their laboratory.

[0068] In a sixth aspect, the present invention provides a composition comprising: the recombinant antibody described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect. As previously described, the recombinant antibody of the embodiments of the present invention can effectively bind to CD3 protein molecules on the surface of immune cells and CD155 protein molecules on the surface of tumor cells, prompting T cells to selectively locate to the tumor site rather than circulating in the peripheral circulation, thus avoiding systemic activation. Compositions containing the recombinant antibody, such as food compositions and pharmaceutical compositions, also have significant therapeutic or preventive effects on tumors.

[0069] It should be noted that the compositions include combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the invention. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0070] In a seventh aspect, the present invention provides the use of the recombinant antibody described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect in the preparation of a medicament for the treatment or prevention of cancer. As previously described, the recombinant antibody of the embodiments of the present invention can effectively bind to CD3 and CD155 protein molecules, thereby specifically recognizing T cells and tumor cells, and prompting T cells to selectively locate to the tumor site rather than circulating in the peripheral circulation, avoiding systemic activation. Medicaments containing a series of substances including the recombinant antibody also have significant therapeutic or preventative effects against tumors.

[0071] According to some specific embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0072] According to some specific embodiments of the present invention, the cancer includes at least one of the following: 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.

[0073] In an eighth aspect, the present invention provides a medicament comprising: the recombinant antibody described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect. According to embodiments of the present invention, the medicament is used to treat cancer. As previously described, the recombinant antibody of the present invention can effectively bind to CD3 and CD155 protein molecules, thereby specifically recognizing T cells and tumor cells, and prompting T cells to selectively locate to the tumor site rather than circulating in the peripheral circulation, avoiding systemic activation. Therefore, the medicament comprising the recombinant antibody also has significant therapeutic or preventative effects against cancer.

[0074] According to some specific embodiments of the present invention, the above-mentioned drug may further include at least one of the following additional technical features:

[0075] According to some specific embodiments of the present invention, the cancer includes at least one of the following: 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.

[0076] According to some specific embodiments of the present invention, a pharmaceutically acceptable carrier and an effective amount of the antibody active ingredient are included.

[0077] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0078] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0079] The medicaments of this invention contain a safe and effective amount of the active ingredient of this invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage forms of the medicaments of this invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, they are prepared using physiological saline or aqueous solutions containing glucose and other excipients by conventional methods. The medicaments are preferably manufactured under aseptic conditions.

[0080] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0081] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0082] In a ninth aspect of the invention, the recombinant antibody described in the first aspect is proposed for use in the preparation of a kit for detecting CD3 and / or CD155. The recombinant antibody can bind to CD3 and / or CD155 proteins; therefore, a kit containing the recombinant antibody can be used for the efficient detection of CD3 and / or CD155. The kit can be used in scientific research, such as for the qualitative or quantitative detection of CD3 and / or CD155 proteins in biological samples.

[0083] In a tenth aspect, the present invention provides a kit comprising the recombinant antibody described in the first aspect. The recombinant antibody provided according to embodiments of the present invention can bind to CD3 and / or CD155 proteins; therefore, the kit comprising the recombinant antibody can be used for effective diagnosis or detection of CD3 and / or CD155. The kit can be used in scientific research, such as for qualitative or quantitative detection of CD3 and / or CD155 proteins in biological samples, and can also be used to assess an individual's condition, such as determining whether an individual's PD-L1 level is excessively high or low compared to normal levels after obtaining the individual's PD-L1 level.

[0084] According to some specific embodiments of the present invention, the kit is used to detect CD3 and / or CD155.

[0085] The embodiments will be described in detail below. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0086] Example 1: Preparation of bispecific antibody molecules

[0087] This embodiment demonstrates the production of bispecific antibodies. The specific experimental procedures are as follows: ExpiCHO cells (purchased from Thermo Fisher) were cultured in ExpiCHO Expression Medium (purchased from Thermo Fisher, A2910001) and the cell concentration was adjusted to 6 × 10⁶ cells / year. 6To obtain ExpiCHO cell solutions, pTT5 vector (synthesized by Suzhou Genewise) containing the a-chain and b-chain encoding genes (as shown in SEQ ID NO: 1 and 2, respectively) was added to 2 mL of OptiSFM medium (Thermo Fisher, 12309019) to obtain solution A. The a-chain encoding gene includes nucleotide sequences encoding CD3 single-chain antibody (SEQ ID NO: 5), linker peptide 1 (SEQ ID NO: 6), linker peptide 2 (SEQ ID NO: 7), and the first Fc region (SEQ ID NO: 8). The b-chain encoding gene includes nucleotide sequences encoding the TIGIT extracellular region (SEQ ID NO: 9), linker peptide 3 (SEQ ID NO: 10), and the second Fc region (SEQ ID NO: 11). 160 μL of ExpiFectamineCHO transfection reagent (Thermo Fisher, A29130) was added to 2 mL of OptiSFM medium to obtain solution B. Solution A and solution B were then mixed to obtain the transfection mixture, which was added to 50 mL of ExpiCHO cell solution within 5 minutes. After culturing at 37°C and 5% CO2 for 1 day, 8 mL of feed and 300 μL of Enhancer (Thermo Fisher, A29130) were added, and the cells were transferred to 32°C and 5% CO2 for 9 days. The culture supernatant was harvested, with 8 mL of feed added on day 5. The bispecific antibody was affinity purified from the culture supernatant using a Protein A purification column (GE). The specific results are shown below. Figure 1 As shown, SEQ ID NO:3 and SEQ ID NO:4 illustrate the amino acid sequences of fragment a (first peptide chain) and fragment b (second peptide chain) in the bispecific antibody, respectively. Fragment a includes the amino acid sequences of CD3 single-chain antibody (SEQ ID NO:13), linker peptide 1 (SEQ ID NO:14), linker peptide 2 (SEQ ID NO:15), and first Fc region (SEQ ID NO:16). Fragment b includes the amino acid sequences of TIGIT extracellular region (SEQ ID NO:17), linker peptide 3 (SEQ ID NO:18), and second Fc region (SEQ ID NO:19).

[0088] The gene encoding the a-chain includes the nucleotide sequence shown below:

[0089]

[0090] The b-chain encoding gene includes the nucleotide sequence shown below:

[0091]

[0092] Fragment a includes the following amino acid sequence:

[0093] EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVT VSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGT KLTVLGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3).

[0094] Fragment b includes the following amino acid sequence:

[0095] MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:4).

[0096] The gene encoding the a-chain comprises the nucleotide sequence shown below:

[0097] GGAGGTTCAACTGCTCGAATCTGGAGGTGGTTTGGTGCAACCTGGTGGATCACTGAAGCTGAGCTGCGCAGCTAGCGGCTTCACCTTTAATACTTACGCTATGAATTGGGTAAGACAGGCCCCAGGAAAGGGACTGGAATGGGTGGCCAGGATTAGGTCTAAGTACAATAATTACGCAACTTATTATGCCGATTCCGTGAAAGATCGCTTCACCATTAGCCGGGATGACTCTAAAAACACCGCATATCTGCAGATGAACAATCTGAAAACCGAGGACACTGCTGTGTACTACTGCGTTCGCCACGGCAACTTCGGAAACTCTTATGTGAGTTGGTTCGCTTACTGGGGTCAGGGCACCTTGGTGACCGTCAGTAGTGAGGTGGAGGCTCAGGAGGAGGTGGATCTGGCGGAGGAGGATCAGAGCTGGTGGTGACACAGGAGCCTTCTTTGACAGTCTCTCCAGGAGGGACAGTAACACTGACATGCCGGAGCAGCACTGGAGCTGTCACCACAAGTAACTACGCTAACTGGGTACAACAAAAGCCTGGGCAGGCACCTAGGGGGTTGATTGGGGGAACTAACAAGCGCGCCCCAGGAACCCCTGCAAGATTCTCTGGCAGCTTGCTTGGCGGGAAAGCTGCACTGACCCTTTCTGGAGTGCAGCCCGAGGACGAGGCAGAATATTACTGTGCTCTGTGGTACTCAAACCTCTGGGTGTTCGGGGGAGGAACCAAGCTTACTGTGCTC(SEQ ID NO:5).

[0098] The gene encoding linker peptide 1 includes the nucleotide sequence shown below:

[0099] GAGGTGGAGGCTCAGGAGGAGGTGGATCTGGCGGAGGAGGATCA(SEQ ID NO:6).

[0100] The gene encoding linker peptide 2 includes the nucleotide sequence shown below:

[0101] GGCGGGGGAGGCAGT(SEQ ID NO:7).

[0102] The gene encoding the first Fc region includes the following nucleotide sequence:

[0103] (SEQ ID NO:8).

[0104] The gene encoding the extracellular region of TIGIT includes the following nucleotide sequence:

[0105] ATGATGACAGGCACCATTGAAACCACCGGGAACATTTCAGCCGAAAAGGTGGCAGCATCATCCTGCAGTGTCATCTGTCTAGTACTACAGCCCAGGTGACCCAGGTGAATTGGGAGCAGCAGGACCAGCTGCTGGCAATCTGTAACGCCGACCTCGGTTGGCATATTAGCCCCAGTTTCAA GGATAGGGTCGCACCCGGCCCCGGATTGGGCCTGACACTCCAGAGCCTGACCGTGAACGATACAGGTGAATACTTTTGTATTTACCACACATACCCTGACGGAACATATACTGGTAGGATATTCCTCGAGGTGCTGGAATCTTCAGTGGCCGAGCACGGGGCTCGGTTTCAGATTCCT(SEQ ID NO:9).

[0106] The gene encoding linker peptide 3 includes the nucleotide sequence shown below:

[0107] GGAGGTGGAGGGAGT (SEQ ID NO: 10).

[0108] The gene encoding the second Fc region includes the following nucleotide sequence:

[0109] CCCAAGAGTTGTGATAAGACACATACATGCCCTCCCTGCCCAGCACCCGAAGCAGCTGGAGGCCCAAGTGTGTTCCTGTTCCCTCCTAAACCTAAGGACACCCTGATGATCTCACGCACTCCTGAGGTTACCTGCGTGGTAGTGGATGTGTCCCACGAAGACCCCGAGGTGAAATTCAACTGGTACGTCGATGGAGTCGAAGTCCATAACGCCAAGACCAAGCCACGTGAGGAGCAGTACAATTCAACATACCGAGTGGTGAGTGTGCTGACCGTCCTCCACCAGGACTGGCTGAACGGGAAAGAGTACAAGTGTAAGGTGTCTAATAAAGCATTGCCCGCCCCCATCGAGAAGACAATTTCAAAGGCTAAAGGTCAGCCACGAGAACCCCAAGTTTATACCCTGCCTCCCAGCCGGGAGGAGATGACCAAGAATCAGGTCAGTCTTTCTTGTGCCGTGAAGGGTTTTTACCCCTCCGACATCGCCGTGGAGTGGGAGTCAAACGGGCAGCCCGAGAATAATTATAAGACAACACCACCAGTGCTGGACTCCGATGGAAGCTTTTTCCTGGTGTCCAAGCTCACTGTCGACAAGTCTAGGTGGCAACAGGGAAATGTGTTCTCATGTTCCGTAATGCACGAGGCCCTCCATAACCACTATACTCAAAAGAGCCTGAGCCTGAGTCCCGGCAAG(SEQ ID NO:11).

[0110] The CD3 single-chain antibody comprises the amino acid sequence shown below:

[0111] EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSG GGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL(SEQ ID NO:13).

[0112] Linker peptide 1 includes the following amino acid sequence:

[0113] GGGGSGGGGSGGGGS (SEQ ID NO: 14).

[0114] Linker peptide 2 includes the following amino acid sequence:

[0115] GGGGS (SEQ ID NO:15).

[0116] The first Fc region includes the following amino acid sequence:

[0117] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:16).

[0118] The extracellular region of TIGIT includes the following amino acid sequence:

[0119] MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGGLTLQSLTVNDTGEYFcIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP (SEQ ID NO: 17).

[0120] Linker peptide 3 includes the following amino acid sequence:

[0121] GGGGS (SEQ ID NO:18).

[0122] The second Fc region includes the following amino acid sequence:

[0123] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:19).

[0124] Example 2: Identification of the binding ability of bispecific antibodies to Jurkat T cells

[0125] This embodiment uses flow cytometry to detect the binding characteristics of bispecific antibodies, and the strength of the signal after the addition of bispecific antibodies is used to determine the binding characteristics between the bispecific antibodies and Jurkat. The specific experimental procedures are as follows:

[0126] Jurkat T cells were diluted to 1 × 10⁻⁶ with PBS. 6 / mL, 90μL / tube was added to a 1.5mL EP tube, and 10μL / tube of mouse serum was added to the same volume. The tube was then blocked at 4℃ for 30min. After blocking, a series of concentration gradients (10 μL / mL) were added to the diluted Jurkat T cells. -3 10 -2 10 -1 10 0 10 1 10 2Add 10 μL / tube of bispecific antibody or IgG (control IgG1, Biolegend, QA16A12) at 4℃ for 30 min. Add 1 mL of PBS to the EP tube, centrifuge at 100g for 5 min at 4℃, discard the supernatant, wash the precipitate once with PBS, centrifuge again and discard the supernatant, resuspend the cells in 100 μL / tube of PBS, add 1 μL / tube of Alexa-647-labeled mouse anti-human Fc antibody secondary antibody (Biolegend, HP6017) to the resuspended cells, and incubate at 4℃ in the dark for 30 min. After incubation, wash twice with PBS, centrifuge and discard the supernatant; resuspend the cells in 200 μL / tube of PBS and analyze by flow cytometry. Specific experimental results are as follows: Figure 2 As shown, this illustrates that the bispecific antibody of the present invention can bind to Jurkat T cells.

[0127] Example 3: Identification of the binding ability of bispecific antibodies to CHO-K1-CD155 cells

[0128] This embodiment uses flow cytometry to detect the binding characteristics of bispecific antibodies, and the signal strength after the addition of bispecific antibodies is used to determine the binding characteristics between bispecific antibodies and CHO-K1-CD155 cells. The specific experimental procedures are as follows:

[0129] HEK293T cells were processed at a rate of 5 × 10 5 Seed cells into 6-well plates and culture overnight in DMEM medium without antibiotics. Discard the medium before transfection and add 1 mL of fresh DMEM medium without antibiotics. A 2:1:1 ratio of pLVX-CD155-IRES-puro (the pLVX-EF1a-IRES-puro vector contains an insertion of the coding sequence for CD155 protein (SEQ ID NO: 20) between the EcoRI and BamHI restriction sites, pMD2G, and psPAX2 vector (3 μg total) was added to 200 μL of serum-free DMEM medium. 12 μg of polyetherimide (PEI, Polysciences Ltd.) was also added. The resulting CD155 protein had the amino acid sequence shown in SEQ ID NO: 12. After mixing and standing for 16 min, the entire mixture was added to a six-well plate containing HEK293T cells. After 6 h of culture, the medium was discarded, and fresh complete DMEM medium was added. 48 h after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm Millipore filter to obtain the viral supernatant. The entire viral supernatant was then added to a solution containing 1×10⁻⁶ cells. 4In a 6-well plate containing CHO-K1 cells, 4 μg / mL of polybrene (Sigma) was added and the cells were cultured for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells are CHO-K1-CD155 cells.

[0130] The gene encoding CD155 includes the following nucleotide sequence:

[0131]

[0132] The CD155 protein includes the following amino acid sequence:

[0133] MARAMAAAWPLLLVALLVLSWPPGTGDVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVP SSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVT NALGARQAELTVQVKEGPPSEHSGISRNAIIFLVLGILVFLILLGIGIYFYWSKCSREVLWHCHLCPSSTEHASASANGHVSYSAVSRENSSSQDPQTEGTR(SEQ ID NO:12).

[0134] CHO-K1-CD155 cells were diluted to 1×10⁻⁶ with PBS. 6 / mL, 90μL / tube was added to a 1.5mL EP tube, and 10μL / tube of mouse serum was added to block the cells at 4℃ for 30min. After blocking, a series of concentration gradients (10 μL / mL) were added to the CHO-K1-CD155 cells. -3 10 -2 10 -1 10 0 10 1 10 2Add 10 μL / tube of bispecific antibody or IgG (control IgG1, Biolegend, QA16A12) at 4°C for 30 min. Add 1 mL of PBS to the EP tube, centrifuge at 100 g for 5 min at 4°C, discard the supernatant, and wash once with PBS. After centrifugation, discard the supernatant, resuspend the cells in 100 μL / tube of PBS, add 1 μL / tube of Alexa-647-labeled mouse anti-human Fc antibody secondary antibody (Biolegend, HP6017), and incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μL / tube of PBS and analyze by flow cytometry. Specific experimental results are as follows: Figure 3 As shown, the bispecific antibody of the present invention can bind to CHO-K1-CD155 cells.

[0135] Example 4: Identification of the binding ability of bispecific antibodies to NCI-H1299 lung cancer cells

[0136] This embodiment uses flow cytometry to detect the binding characteristics of bispecific antibodies, and the strength of the signal after the addition of bispecific antibodies is used to determine the binding characteristics between bispecific antibodies and NCI-H1299 cells. The specific experimental procedures are as follows:

[0137] NCI-H1299 cells were diluted to 1×10⁻⁶ with PBS. 6 / mL, add 90μL / tube to a 1.5mL EP tube, add 10μL / tube of mouse serum, and block at 4℃ for 30min. After blocking, add a series of concentration gradients (10 / mL) to each tube. -3 10 -2 10 -1 10 0 10 1 10 2 Add 10 μL / tube of bispecific antibody or IgG (control IgG1, Biolegend, QA16A12) at 4°C for 30 min. After incubation, add 1 mL of PBS to the EP tube, centrifuge at 100g for 5 min at 4°C, discard the supernatant, and wash the pellet once with PBS. After centrifugation, discard the supernatant, resuspend the cells in 100 μL / tube of PBS, add 1 μL / tube of Alexa-647-labeled mouse anti-human Fc antibody secondary antibody (Biolegend, HP6017), and incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μL / tube of PBS and analyze by flow cytometry. Specific experimental results are as follows: Figure 4 As shown, the bispecific antibody of the present invention can bind to NCI-H1299 cells.

[0138] Example 5: Bispecific antibody promotes PBMC killing of tumor cells

[0139] This embodiment examines the effect of the bispecific antibody obtained in Example 1 on the killing of tumor cells (lung cancer NCI-H1299 cells, NCI-H358 cells, and liver cancer HepG2 cells) by PBMCs. The detection was performed by constructing a reaction system consisting of tumor cells + PBMCs + bispecific antibody. The specific experimental procedures are as follows:

[0140] (1) Add complete DMEM medium to a 16-well RTCA plate and calibrate it on the instrument;

[0141] (2) The above tumor cells were diluted to 2×10⁶ using complete DMEM medium. 5 / mL, added to the RTCA plate obtained in step (1), with an addition volume of 50μL / well, and then the cell coefficient was detected for 24h using the xCELLigence RTCA TP device at 37℃ and 5% CO2.

[0142] (3) After diluting the specific antibody prepared in Example 1 into a series of concentration gradients (0.001, 0.01, 0.1, 1, 10 μg / mL) with complete DMEM, add it to the RTCA plate obtained in step (2) with an addition volume of 20 μL / well;

[0143] (4) Dilute PBMCs to 1.25 × 10⁻⁶ using complete DMEM medium. 6 Add 1 cell / mL to the RTCA plate obtained in step (3), with an addition volume of 80 μL / well;

[0144] (5) Cell coefficients were detected for 72 hours at 37°C and 5% CO2 using an xCELLigence RTCA TP device.

[0145] Specific experimental results are as follows Figure 5 , Figure 6 , Figure 7 As shown, the bispecific antibody of the present invention can promote the killing of CD155-expressing lung cancer NCI-H1299 cells, NCI-H358 cells and liver cancer HepG2 cells by PBMCs.

[0146] The experimental results above show that the bispecific antibody obtained in this invention can bind to T cells and tumor cells and promote T cells to kill tumor cells.

[0147] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0148] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention. SEQUENCE LISTING <110> Hefei Tiangang Immunotherapy Co., Ltd. <120> Recombinant antibodies and their applications <130> HI5210690 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 1455 <212> DNA <213> Artificial Sequence <220> <223> 1 <400> 1 gaggttcaac tgctcgaatc tggaggtggt ttggtgcaac ctggtggatc actgaagctg 60 agctgcgcag ctagcggctt cacctttaat acttacgcta tgaattgggt aagacaggcc 120 ccaggaaagg gactggaatg ggtggccagg attaggtcta agtacaataa ttacgcaact 180 tattatgccg attccgtgaa agatcgcttc accattagcc gggatgactc taaaacacc 240 gcatatctgc agatgaacaa tctgaaaacc gaggacactg ctgtgtacta ctgcgttcgc 300 cacggcaact tcggaaactc tttgtgagt tggttcgctt actggggtca gggcaccttg 360 gtgaccgtca gtagtggagg tggaggctca ggaggaggtg gatctggcgg aggaggatca 420 gagctggtgg tgacacagga gccttctttg acagtctctc caggagggac agtaacactg 480 acatgccgga gcagcactgg agctgtcacc acaagtaact acgctaactg ggtacaacaa 540 aagcctgggc aggcacctag ggggttgatt gggggaacta acaagcgcgc cccaggaacc 600 cctgcaagat tctctggcag cttgcttggc gggaaagctg cactgaccct ttctggagtg 660 cagcccgagg acgaggcaga atattactgt gctctgtggt actcaaacct ctgggtgttc 720 gggggaggaa ccaagcttac tgtgctcggc gggggaggca gtcctaaatc atgcgacaaa 780 actcatacat gtccaccttg tcctgctcct gaagctgccg ggggaccttc agtgttcctg 840 tttccaccta agcccaaaga taccttgatg atttctcgaa ccccagaggt gacttgcgtg 900 gtagtcgatg tctcccacga agatcccgag gtgaaattca actggtacgt ggacggcgtg 960 gaggtgcaca atgctaagac aaagccccga gaggaacagt aatatagtac ctatcgcgtg 1020 gttagcgtcc ttactgtgtt gcatcaggac tggctgaatg gaaaggaata caagtgcaag 1080 gtatcaaata aggccctccc agctcccatc gagaaaacta tttccaaggc caagggtcag 1140 ccaagggagc cacaggtcta caccttgccc cctagtcggg aagaaatgac caaaaaccag 1200 gtgagtctgt ggtgcctggt taagggtttc tacccctctg atattgcagt ggagtgggag 1260 tcaaacggcc agccagagaa caactataag acaacccctc cagtgctgga ctcagacggg 1320 tctttctttc tctatagcaa actgactgtc gacaagagca ggtggcagca agggaacgtc 1380 ttcagctgct cagtgatgca tgaggcactg cataaccact atactcaaaa aagcctgtct 1440 ctgagccctg gcaaa 1455 <210> 2 <211> 1068 <212> DNA <213> Artificial Sequence <220> <223> 2 <400> 2 atgatgacag gcaccattga aaccaccggg aacatttcag ccgaaaaggg tggcagcatc 60 atcctgcagt gtcatctgtc tagtactaca gcccaggtga cccaggtgaa ttgggagcag 120 caggaccagc tgctggcaat ctgtaacgcc gacctcggtt ggcatattag ccccagtttc 180 aaggataggg tcgcacccgg ccccggattg ggcctgacac tccagagcct gaccgtgaac 240 gatacaggtg aatacttttg tatttaccac acataccctg acggaacata tactggtagg 300 atattcctcg aggtgctgga atcttcagtg gccgagcacg gggctcggtt tcagattcct 360 ggaggtggag ggagtcccaa gagttgtgat aagacacata catgccctcc ctgcccagca 420 cccgaagcag ctggaggccc aagtgtgttc ctgttccctc ctaaacctaa ggacaccctg 480 atgatctcac gcactcctga ggttacctgc gtggtagtgg atgtgtccca cgaagacccc 540 gaggtgaaat tcaactggta cgtcgatgga gtcgaagtcc ataacgccaa gaccaagcca 600 cgtgaggagc agtacaattc aacataccga gtggtgagtg tgctgaccgt cctccaccag 660 gactggctga acgggaaaga gtacaagtgt aaggtgtcta ataaagcatt gcccgccccc 720 atcgagaaga caatttcaa ggctaaggt cagccacgag aacccaagt ttataccctg 780 cctcccagcc gggaggagat gaccaagaat caggtcagtc tttctgtgc cgtgaagggt 840 ttttacccct ccgacatcgc cgtggagtgg gagtcaacg ggcagcccga gaataattat 900 aagacacac caccagtgct ggactccgat ggaagctttt tcctggtgtc caagctcact 960 gtcgacaagt ctaggtggca acagggaat gtgttctcat gttccgtaat gcacgaggcc 1020 ctccataacc actatactca aaagagccctg agcctgagtc ccggcaag 1068 <210> 3 <211> 485 <212> PRT <213> Artificial Sequence <220> <223> 3 <400> 3 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Only Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Only Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Only Thr Tyr Tyr Only Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Val 130 135 140 Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu 145 150 155 160 Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 165 170 175 Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly 180 185 190 Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu 195 200 205 Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp 210 215 220 Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Pro Lys 245 250 255 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 260 265 270 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 275 280 285 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 290 295 300 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 305 310 315 320 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 325 330 335 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 340 345 350 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 355 360 365 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 370 375 380 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 385 390 395 400 Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 405 410 415 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 420 425 430 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 435 440 445 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 450 455 460 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 465 470 475 480 Leu Ser Pro Gly Lys 485 <210> 4 <211> 356 <212> PRT <213> Artificial Sequence <220> <223> 4 <400> 4 Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys 1 5 10 15 Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30 Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45 Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60 Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn 65 70 75 80 Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95 Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110 His Gly Ala Arg Phe Gln Ile Pro Gly Gly Gly Gly Ser Pro Lys Ser 115 120 125 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 130 135 140 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 145 150 155 160 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 165 170 175 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 180 185 190 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 195 200 205 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 210 215 220 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 225 230 235 240 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 245 250 255 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 260 265 270 Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 275 280 285 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 290 295 300 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr 305 310 315 320 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 325 330 335 Put His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 340 345 350 Ser Pro Gly Lys 355 <210> 5 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> 5 <400> 5 ggaggttcaa ctgctcgaat ctggaggtgg tttggtgcaa cctggtggat cactgaagct 60 gagctgcgca gctagcggct tcacctttaa tacttacgct atgaattggg taagacaggc 120 cccaggaaag ggactggaat gggtggccag gattaggtct aagtacaata attacgcaac 180 ttattatgcc gattccgtga aagatcgctt caccattagc cgggatgact ctaaaaacac 240 cgcatatctg cagatgaaca atctgaaaac cgaggacact gctgtgtact actgcgttcg 300 ccacggcaac ttcggaaact cttatgtgag ttggttcgct tactggggtc agggcacctt 360 ggtgaccgtc agtagtgagg tggaggctca ggaggaggtg gatctggcgg aggaggatca 420 gagctggtgg tgacacagga gccttctttg acagtctctc caggagggac agtaacactg 480 acatgccgga gcagcactgg agctgtcacc acaagtaact acgctaactg ggtacaacaa 540 aagcctgggc aggcacctag ggggttgatt gggggaacta acaagcgcgc cccaggaacc 600 cctgcaagat tctctggcag cttgcttggc gggaaagctg cactgaccct ttctggagtg 660 cagcccgagg acgaggcaga atattactgt gctctgtggt actcaaacct ctgggtgttc 720 gggggaggaa ccaagcttac tgtgctc 747 <210> 6 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> 6 <400> 6 gaggtggagg ctcaggagga ggtggatctg gcggaggagg atca 44 <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> 7 <400> 7 ggcgggggag gcagt 15 <210> 8 <211> 693 <212> DNA <213> Artificial Sequence <220> <223> 8 <400> 8 cctaaatcat gcgacaaaac tcatacatgt ccaccttgtc ctgctcctga agctgccggg 60 ggaccttcag tgttcctgtt tccacctaag cccaaagata ccttgatgat ttctcgaacc 120 ccagaggtga cttgcgtggt agtcgatgtc tcccacgaag atcccgaggt gaaattcaac 180 tggtacgtgg acggcgtgga ggtgcacaat gctaagacaa agccccgaga ggaacagtat 240 aatagtacct atcgcgtggt tagcgtcctt actgtgttgc atcaggactg gctgaatgga 300 aaggaataca agtgcaaggt atcaaataag gccctcccag ctcccatcga gaaactatt 360 tccaaggcca agggtcagcc aagggagcca caggtctaca ccttgccccc tagtcgggaa 420 gaaatgacca aaaaccaggt gagtctgtgg tgcctggtta agggtttcta cccctctgat 480 attgcagtgg agtgggagtc aaacggccag ccagagaaca actataagac aacccctcca 540 gtgctggact cagacgggtc tttctttctc tagatcaaac tgactgtcga caagagcagg 600 tggcagcaag ggaacgtctt cagctgctca gtgatgcatg aggcactgca taaccactat 660 actcaaaaaa gcctgtctct gagccctggc aaa 693 <210> 9 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> 9 <400> 9 atgatgacag gcaccattga aaccaccggg aacatttcag ccgaaaaggg tggcagcatc 60 atcctgcagt gtcatctgtc tagtactaca gcccaggtga cccaggtgaa ttgggagcag 120 caggaccagc tgctggcaat ctgtaacgcc gacctcggtt ggcatattag ccccagtttc 180 aaggataggg tcgcacccgg ccccggattg ggcctgacac tccagagcct gaccgtgaac 240 gatacaggtg aatacttttg tatttaccac acataccctg acggaacata tactggtagg 300 atattcctcg aggtgctgga atcttcagtg gccgagcacg gggctcggtt tcagattcct 360 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> 10 <400> 10 ggaggtggag ggagt 15 <210> 11 <211> 693 <212> DNA <213> Artificial Sequence <220> <223> 11 <400> 11 cccaagagtt gtgataagac acatacatgc cctccctgcc cagcacccga together ggcccaagtg tgttcctgtt ccctcctaaa cctaaggaca ccctgatgat ctcacgcact 120 cctgaggtta cctgcgtggt agtggatgtg tcccacgaag accccgaggt gaaattcaac tggtacgtcg atggagtcga agtccataac gccaagacca agccacgtga ggagcagtac aattcaacat accgagtggt gagtgtgctg accgtcctcc accaggactg gctgaacggg aaagagtaca agtgtaaggt gtctaataaa gcattgcccg cccccatcga gaagacaatt tcaaaggcta aaggtcagcc acgagaaccc caagtttata ccctgcctcc cagccggggag 420. gagatgacca agaatcaggt cagtctttct tgtgccgtga agggttttta cccctccgac atcgccgtgg agtgggagtc aaacgggcag cccgagaata attack aacaccacca 540 gtgctggact ccgatggaag ctttttcctg gtgtccaagc tcactgtcga caagtctagg tggcaacagg gaaatgtgtt ctcatgttcc gtaatgcacg aggccctcca taaccactat actcaaaaga gcctgagcct gcccggc aag 693 <210> 12 <211> 417 <212> PRT <213> Artificial Sequence <220> <223> 12 <400> 12 Met Ala Arg Ala Met Ala Ala Ala Trp Pro Leu Leu Leu Val Ala Leu 1 5 10 15 Leu Val Leu Ser Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln 20 25 30 Ala Pro Thr Gln Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro 35 40 45 Cys Tyr Leu Gln Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu 50 55 60 Thr Trp Ala Arg His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln 65 70 75 80 Thr Gln Gly Pro Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala 85 90 95 Ala Arg Leu Gly Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly 100 105 110 Leu Arg Val Glu Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe 115 120 125 Pro Gln Gly Ser Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys 130 135 140 Pro Gln Asn Thr Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro 145 150 155 160 Val Pro Met Ala Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln 165 170 175 Ile Thr Trp His Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val 180 185 190 Pro Gly Phe Leu Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu 195 200 205 Val Pro Ser Ser Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu 210 215 220 His Glu Ser Phe Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val 225 230 235 240 Tyr Tyr Pro Pro Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr 245 250 255 Leu Gly Gln Asn Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro 260 265 270 Glu Pro Thr Gly Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro 275 280 285 Phe Ala Val Ala Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys 290 295 300 Pro Ile Asn Thr Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala 305 310 315 320 Arg Gln Ala Glu Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu 325 330 335 His Ser Gly Ile Ser Arg Asn Ala Ile Ile Phe Leu Val Leu Gly Ile 340 345 350 Leu Val Phe Leu Ile Leu Leu Gly Ile Gly Ile Tyr Phe Tyr Trp Ser 355 360 365 Lys Cys Ser Arg Glu Val Leu Trp His Cys His Leu Cys Pro Ser Ser 370 375 380 Thr Glu His Ala Ser Ala Ser Ala Asn Gly His Val Ser Tyr Ser Ala 385 390 395 400 Val Ser Arg Glu Asn Ser Ser Ser Gln Asp Pro Gln Thr Glu Gly Thr 405 410 415 Arg <210> 13 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> 13 <400> 13 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Val 130 135 140 Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu 145 150 155 160 Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 165 170 175 Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly 180 185 190 Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu 195 200 205 Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp 210 215 220 Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> 14 <400> 14 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 15 <400> 15 Gly Gly Gly Gly Ser 1 5 <210> 16 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> 16 <400> 16 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 1 5 10 15 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 65 70 75 80 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105 110 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 130 135 140 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 145 150 155 160 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170 175 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220 Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 17 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 17 <400> 17 Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys 1 5 10 15 Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30 Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45 Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60 Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn 65 70 75 80 Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95 Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110 His Gly Ala Arg Phe Gln Ile Pro 115 120 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 18 <400> 18 Gly Gly Gly Gly Ser 1 5 <210> 19 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> 19 <400> 19 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 1 5 10 15 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 65 70 75 80 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105 110 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 130 135 140 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 145 150 155 160 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170 175 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 180 185 190 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205 How Does Glu Ala Meet the Asn and Tyr Thr Gln Lys Ser? 210 215 220 Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 20 <211> 1251 <212> DNA <213> Artificial Sequence <220> <223> 20 <400> 20 atggctagag caatggcagc tgcttggcca ctttgttgg tagcactcct ggtgctttca 60 tgcccccac ctggaactgg cgatgtgtc gtgcaggctc ctacccaggt gcctggttc 120 ttggggtt ctgtcacact gccatgttac cttcaggttc ctacatgga ctcactcac 180 gtgtcccaac tgacatgggc taggcacggt gagtctggct ccatggccgt gttccaccag 240 acacagggtc ctagttattc agagtccaaa cgcttggagt ttgtcgccgc agactgggg 300 gctgagttga ggaatgcaag cctgcggatg ttcggacttc gggtcgaaga tgaagggaac 360 tacacctgct tgtttgttac ctttcctcag ggtagcagat ccgttgacat atggttgaga 420 gtactggcca agccacagaa tactgcagag gtccagaagg tccagttgac aggcgaacct 480 gtgccaatgg caagatgcgt ttccaccggt ggtagaccac cagctcagat tacttggcat 540 agtgacctgg gcgggatgcc taacacatct caagtgcctg gctttttgtc aggcaccgtc 600 actgtgacct ccctttggat cctggttcca agttctcagg ttgacggcaa aaacgtaaca 660 tgcaaggtgg agcatgagag ctttgagaag ccacagctcc ttaccgtgaa tctcaccgtg 720 tattaccctc ctgaggtgtc tatcagcgga tatgacaaca actggtatct cgggcagaat 780 gaggcaaccc tgacctgtga tgctcgcagt aatcctgagc ccaccgggta taattggagc 840 actaccatgg gcccattcc ccctttcgct gtggctcagg gcgcacaact gcttattcgg 900 cctgtagata agcccattaa caccactttg atctgtaacg tgactaatgc tttgggagct 960 cgccaggccg aattgaccgt acaggtgaaa gaaggtcctc cttccgagca ttccggcatt 1020 tcccgaaacg ccatcatttt tctggtgctg ggtatcctgg tgtttctgat cttgttgggg 1080 atcggtatct acttttactg gtccaaatgc agccgcgaag tcctgtggca ctgccatctg 1140 tgcccaagtt ctactgagca cgcctctgcc tccgcaaatg gtcacgtgtc ctacagtgct 1200 gtgtctagag agaactcatc tagtcaggat cctcagactg agggtactcg g 1251

Claims

1. A recombinant antibody, characterized in that, The recombinant antibody is a double-chain antibody, comprising: The first peptide chain comprises: a CD3 single-chain antibody and a first Fc region, wherein the CD3 single-chain antibody includes a heavy chain variable region and a light chain variable region; and The second peptide chain includes: the TIGIT extracellular region and the second Fc region; The C-terminus of the CD3 single-chain antibody is connected to the N-terminus of the first Fc region, and the C-terminus of the TIGIT extracellular region is connected to the N-terminus of the second Fc region. The amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO:13, and the amino acid sequence of the extracellular region of TIGIT is shown in SEQ ID NO:

17.

2. The recombinant antibody according to claim 1, characterized in that, It further includes a linker peptide 1, the N-terminus of which is connected to the C-terminus of the heavy chain variable region, and the C-terminus of which is connected to the N-terminus of the light chain variable region. Optionally, the amino acid sequence of the linker peptide 1 is shown in SEQ ID NO:

14.

3. The recombinant antibody according to claim 1 or 2, characterized in that, It further includes a linker peptide 2, the N-terminus of which is connected to the C-terminus of the light chain variable region, and the C-terminus of which is connected to the N-terminus of the first Fc region; Optionally, the amino acid sequence of the linker peptide 2 is shown in SEQ ID NO:

15.

4. The recombinant antibody according to claim 1, characterized in that, It further includes a linker peptide 3, the N-terminus of which is connected to the C-terminus of the extracellular region of TIGIT, and the C-terminus of which is connected to the N-terminus of the second Fc region. Optionally, the amino acid sequence of the linker peptide 3 is shown in SEQ ID NO:

18.

5. The recombinant antibody according to claim 1, characterized in that, The first Fc region and the second Fc region are connected by a knobinto hole structure.

6. The recombinant antibody according to claim 5, characterized in that, The first Fc region, compared with the wild-type IgG1 Fc region, has at least one of the following: lack of CH1 region or T366W mutation; the second Fc region, compared with the wild-type IgG1 Fc region, has at least one of the following: lack of CH1 region or T366S, L368A, or Y407V mutation.

7. The recombinant antibody according to claim 5, characterized in that, At least a portion of the first Fc region and the second Fc region are derived from at least one of a mouse antibody, a primate antibody, or a mutant thereof.

8. The recombinant antibody according to claim 5, characterized in that, At least a portion of the first Fc region and the second Fc region are derived from mouse antibodies, primate IgG, or mutants thereof.

9. The recombinant antibody according to claim 5, characterized in that, At least a portion of the first Fc region and the second Fc region are derived from human IgG1 or its mutants.

10. The recombinant antibody according to claim 7, characterized in that, The primate-derived antibodies include human-derived antibodies.

11. The recombinant antibody according to claim 1, characterized in that, The first Fc region has an amino acid sequence as shown in SEQ ID NO:16, and the second Fc region has an amino acid sequence as shown in SEQ ID NO:

19.

12. The recombinant antibody according to claim 1, characterized in that, The recombinant antibody consists of the amino acid sequences shown in SEQ ID NO: 3 and 4.

13. A nucleic acid, characterized in that, The nucleic acid encodes the recombinant antibody according to any one of claims 1 to 12.

14. The nucleic acid according to claim 13, characterized in that, The nucleotide sequences of the nucleic acid are shown in SEQ ID NO: 1 and 2.

15. An expression carrier, characterized in that, Carrying the nucleic acid as described in claim 13 or 14.

16. A method for preparing the recombinant antibody according to any one of claims 1 to 12, characterized in that, include: The expression vector of claim 15 is introduced into cells; The cells are cultured under conditions suitable for protein expression and secretion in order to obtain the recombinant antibody.

17. The method according to claim 16, characterized in that, The cells in question are eukaryotic cells.

18. A recombinant cell, characterized in that, The recombinant cells carry the nucleic acid as described in claim 13 or 14 or the expression vector as described in claim 15.

19. A composition, characterized in that, include: The recombinant antibody according to any one of claims 1 to 12, the nucleic acid according to claim 13 or 14, the expression vector according to claim 15, or the recombinant cell according to claim 18.

20. Use of the recombinant antibody of any one of claims 1 to 12, the nucleic acid of claim 13 or 14, the expression vector of claim 15, the recombinant cell of claim 18, or the composition of claim 19 in the preparation of a medicament for the treatment or prevention of cancer, said cancer including at least one of the following: lung cancer and liver cancer.

21. A drug, characterized in that, include: The recombinant antibody according to any one of claims 1 to 2, the nucleic acid according to claim 13 or 14, the expression vector according to claim 15, the recombinant cell according to claim 18, or the composition according to claim 19, the drug is used to treat cancer, the cancer including at least one of the following: lung cancer, liver cancer.

22. Use of the recombinant antibody according to any one of claims 1 to 12 in the preparation of a kit for detecting CD3 and / or CD155.

23. A reagent kit, characterized in that, The kit contains the recombinant antibody as described in any one of claims 1 to 12.

24. The reagent kit according to claim 23, characterized in that, The kit is used to detect CD3 and / or CD155.

Images