Bispecific antibodies and their use

Abstract

The present invention provides a bispecific antibody and its use, the bispecific antibody comprising an IL-21 molecule, a CD16a single chain antibody and Fc, the N-terminus of the Fc being connected to the C-terminus of the single chain antibody, the CD16a single chain antibody comprising a CD16a antibody comprising a heavy chain variable region and a light chain variable region, the C-terminus of the Fc being connected to the N-terminus of the IL21 molecule. The prepared bispecific antibody can simultaneously target CD16 and IL-21 receptors to activate NK cells, has a long half-life, and exhibits stronger anti-tumor and anti-virus abilities than single target antibodies, while promoting the proliferation of NK cells and potentially being applied to culture and expand NK cells in vitro, and the expanded NK cells have good cytotoxicity.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, specifically to bispecific antibodies and their uses, and more specifically to recombinant antibodies, immune cells, nucleic acids, expression vectors, recombinant cells, compositions, the use of the above substances in drug manufacture, and reagent kits.

Background Art

[0002] A bispecific antibody is an antibody that can specifically bind two antigen sites simultaneously. NK cells are the main effector cells of the innate immune system. Compared with T cells, NK cells do not require prior stimulation and can kill tumor cells and virus-infected cells without MHC restriction by using perforin, granule enzymes and related mechanisms. NK cells express many activating receptors and can effectively kill tumor cells by effectively identifying stress ligands produced by tumor cells or infected cells. At the same time, tumor cells also secrete and produce many immunosuppressive molecules to form an immunosuppressive tumor microenvironment, which suppresses the activation of NK cells and the exertion of their anti-tumor ability in this environment, and depletes NK cells. Currently, there are also many methods to restore the depletion of NK cells in the tumor microenvironment, such as immune checkpoint blockade, activating antibodies, activating cytokines, etc. These methods can provide activation signals to NK cells, block inhibitory signals in the tumor microenvironment, restore NK cells to a normal activated state, and exert anti-tumor or anti-virus functions.

[0003] CD16a is a type of important activating receptor expressed on NK cells. When CD16a is activated by its ligand or antibody binding, it induces various cascade activation reactions, releases granule enzymes, perforin, inflammatory cytokines and chemokines, which brings about a strong activating effect and enhanced anti-tumor ability on NK cells. IL-21R (IL-21 receptor) is a cytokine receptor constitutively expressed on NK cells. When recognized, bound and activated by the corresponding ligand, IL-21 molecule, for mouse NK cells, this process promotes the increase of these NK cell particles, enhances the expression of perforin, increases the secretion of IFN-γ, promotes effector activity. For human NK cells, this process enhances IFN-γ secretion, improves proliferation and NK cell cytotoxic activity, and at the same time accelerates the maturation of NK cells and the expression of NK cell receptors. Therefore, developing a highly targeted bispecific antibody has extremely great value for tumor prevention and treatment.

Summary of the Invention

Problems to be Solved by the Invention

[0004] This application is created based on the inventor's discovery and recognition of the following facts and problems.

[0005] In the present invention, the inventor combines a CD16a antibody and an IL-21 molecule to design a recombinant bispecific antibody. The recombinant bispecific recombinant antibody can specifically bind to CD16a and the IL-21 receptor (IL-21R), and further bring the IL-21 molecule and the CD16a antibody specifically to the vicinity of NK cells. By binding to CD16a and / or IL-21R on NK cells, it induces various cascade activation reactions, activates NK cells, restores the depletion of NK cells in the tumor microenvironment, and at the same time enhances IFN-γ secretion, improves proliferation and NK cell cytotoxic activity, and at the same time accelerates the maturation of NK cells and the expression of NK cell receptors to kill tumor cells or virus-infected cells. The recombinant bispecific antibody has high CD16a and IL-21R binding activities and high anti-tumor and anti-virus abilities.

Means for Solving the Problem

[0006] Therefore, in the first aspect of the present invention, the present invention provides a recombinant antibody. According to an embodiment of the present invention, it comprises an IL-21 molecule and a CD16a antibody. The CD16a antibody comprises a CD16a single-chain antibody and an Fc region. The N-terminus of the Fc region is connected to the C-terminus of the single-chain antibody. The CD16a single-chain antibody comprises a heavy-chain variable region and a light-chain variable region. The C-terminus of the FC is connected to the N-terminus of the IL21 molecule. The recombinant antibody according to an embodiment of the present invention can effectively bind to CD16a and IL-21R, has strong in vivo and in vitro binding activities, can effectively activate NK cells, has a long in vivo half-life, and has significant anti-tumor and anti-viral effects.

[0007] In the second aspect of the present invention, 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 effectively bind to CD16a and IL-21R, has strong in vivo and in vitro binding activities, can effectively activate NK cells, has a long in vivo half-life, and has significant anti-tumor and anti-viral effects.

[0008] In the third aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid described in the second aspect. The expression vector may include selectable control sequences, and the control sequences are operably connected to the nucleic acid molecule. The control sequences are one or more control sequences capable of directing the expression of the nucleic acid molecule in a host. The expression vector proposed in the embodiment of the present invention can efficiently express the recombinant antibody in a suitable host cell, and further can effectively activate NK cells and is used for the treatment or prevention of diseases caused by tumors or viral infections.

[0009] In a fourth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant antibody described in the first aspect. The recombinant cell is obtained by transfection or transformation of the expression vector. According to an embodiment of the present invention, the recombinant cell can efficiently express the above recombinant antibody under appropriate conditions, and the recombinant cell can be effectively used for the treatment or prevention of diseases caused by tumors or viral infections, and can effectively activate NK cells.

[0010] In a fifth aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, it includes the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect. As described above, the recombinant antibody according to the embodiment of the present invention can effectively bind to the CD16a and IL-21R protein molecules, and further specifically recognize NK cells that highly express the IL-21R and / or CD16a, effectively activate NK cells, and the composition containing the recombinant antibody also plays a significant role in the treatment or prevention of diseases caused by tumors or viral infections, and its anti-tumor and anti-viral abilities are remarkable.

[0011] In a sixth aspect of the present invention, the present invention provides the use of the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the composition described in the fifth aspect in the manufacture of a drug. According to an embodiment of the present invention, the drug is used for treating or preventing tumors or viral infections. As described above, the recombinant antibody according to the embodiment of the present invention can effectively bind to the CD16a and IL-21R protein molecules, and further specifically recognize NK cells that highly express the IL-21R and / or CD16a, and the drug manufactured using the recombinant antibody and its corresponding series of substances also plays a significant role in the treatment or prevention of tumors or viral infections, and its anti-tumor and anti-viral abilities are remarkable.

[0012] In a seventh aspect of the present invention, the present invention provides the use of the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, and the recombinant cell described in the fourth aspect in the culture amplification of NK cells. The recombinant antibody, nucleic acid molecule, expression vector and the recombinant cell according to the examples of the present invention can be used to promote the proliferation and amplification of the NK cells in vitro, and the amplified NK cells have good cytotoxicity.

[0013] In an eighth aspect of the present invention, the present invention provides a drug. According to the examples of the present invention, it contains the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect or the composition described in the fifth aspect, and the drug is used for treating or preventing tumors or viral infections. As described above, the recombinant antibody according to the examples of the present invention can effectively bind to CD16a and IL-21R proteins, and can further specifically recognize NK cells that highly express the IL-21R and / or CD16a. The drug manufactured using the recombinant antibody and its corresponding series of substances can similarly play a significant role in the treatment or prevention of tumors or viral infections, and its anti-tumor and anti-viral abilities are remarkable.

[0014] In a ninth aspect of the present invention, the present invention provides a reagent kit. According to the examples of the present invention, it contains the recombinant antibody described in the first aspect. The recombinant antibody according to the examples of the present invention can effectively bind to CD16a and IL-21R protein molecules, and can further specifically recognize NK cells that specifically and highly express the IL-21R or CD16a. Therefore, the recombinant antibody can be used to manufacture a reagent kit for detecting CD16a and / or IL-21R, and the reagent kit can be used in scientific research such as qualitatively or quantitatively detecting CD16a and / or IL-21R protein molecules in a biological sample and obtaining a biological sample that meets the requirements.

[0015] In a tenth aspect of the present invention, the present invention provides the use of the recombinant antibody, nucleic acid molecule, expression vector, recombinant cell, composition or drug described above in the treatment or prevention of tumors or viral infections. As described above, the recombinant antibody can specifically recognize the above CD16a and IL-21R proteins, and further can specifically recognize NK cells that specifically and highly express the IL-21R or CD16a, effectively activate NK cells, enhance IFN-γ secretion, improve proliferation and NK cell cytotoxic activity, effectively kill tumor cells, and at the same time, accelerate the maturation of NK cells and the expression of NK cell receptors, and can restore the depletion of NK cells in the tumor microenvironment. Therefore, the recombinant antibody according to the examples of the present invention, the composition, drug containing the recombinant antibody, or a series of substances capable of expressing the recombinant antibody under appropriate conditions, such as the above nucleic acid molecule, expression vector, recombinant cell, etc., can all effectively activate NK cells, and further can treat or prevent tumors or viral infections.

[0016] In the 11th aspect of the present invention, the present invention provides a method for treating or preventing tumors or viral infections. According to some specific examples of the present invention, a subject is administered with at least one of 1) the recombinant antibody described above, 2) the nucleic acid molecule described above, 3) the expression vector described above, 4) the recombinant cell described above, 5) the composition described above, or 6) the drug described above. As described above, the recombinant antibody can specifically recognize the above CD16a and IL-21R proteins, and further can specifically recognize NK cells that specifically highly express the IL-21R or CD16a, effectively activate NK cells, enhance IFN-γ secretion, improve proliferation and NK cell cytotoxic activity, effectively kill tumor cells, and at the same time accelerate the maturation of NK cells and the expression of NK cell receptors, and can restore the depletion of NK cells in the tumor microenvironment. Therefore, the recombinant antibody according to the examples of the present invention, the composition containing the recombinant antibody, the drug or a series of substances that can express the recombinant antibody under appropriate conditions, such as the above nucleic acid molecule, expression vector, recombinant cell, etc. can all effectively activate NK cells and further treat or prevent tumors or viral infections.

[0017] Additional aspects and advantages of the present invention will be shown in part in the following description, will become apparent in part from the following description, or can be understood through the practice of the present invention.

Brief Description of the Drawings

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and easy to understand from the description of the examples combined with the following drawings.

Figure 1

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Figure 7

Mode for Carrying Out the Invention

[0019] Hereinafter, embodiments of the present invention will be described in detail, and examples of the above embodiments are shown in the drawings. The embodiments described below with reference to the drawings are exemplary and are only used for interpreting the present invention and should not be understood as a limitation to the present invention.

[0020] Note that terms such as "first" and "second" are merely for the purpose of explanation and cannot be construed as indicating relative importance, implicitly indicating it, or implicitly indicating a number for clarifying technical features. Therefore, features limited as "first" and "second" can explicitly or implicitly include one or more of such features. In the description of the present invention, unless specifically and clearly limited, the concept of "a plurality" means at least two, for example, two or three.

[0021] Recombinant antibody

[0022] In one aspect of the present invention, the present invention provides a recombinant antibody, which comprises an IL-21 molecule and a CD16a antibody. The CD16a antibody comprises a CD16a single-chain antibody and an Fc region. The N-terminus of the Fc region is connected to the C-terminus of the single-chain antibody. The CD16a single-chain antibody comprises a heavy-chain variable region and a light-chain variable region. The C-terminus of the FC is connected to the N-terminus of the IL21 molecule. The recombinant antibody according to the examples of the present invention can effectively bind to CD16a and IL-21R, has strong in vivo and in vitro binding activities, can effectively activate NK cells, has a long in vivo half-life, and has significant anti-tumor and anti-viral effects.

[0023] According to some specific embodiments of the present invention, the above recombinant antibody can further comprise at least one of the following additional technical features.

[0024] According to some specific embodiments of the present invention, it further comprises a linking peptide 1. The N-terminus of the linking peptide 1 is connected to the C-terminus of the heavy-chain variable region of the CD16a single-chain antibody, and the C-terminus of the linking peptide 1 is connected to the N-terminus of the light-chain variable region of the CD16a single-chain antibody.

[0025] According to some specific embodiments of the present invention, the linking peptide 1 has an amino acid sequence represented by SEQ ID NO: 1. GGGGSGGGGSGGGGS(SEQ ID NO: 1).

[0026] According to some specific embodiments of the present invention, it further comprises a linking peptide 2, the N-terminus of the linking peptide 2 is connected to the C-terminus of the IL-21 molecule, and the C-terminus of the linking peptide 2 is connected to the N-terminus of the Fc region.

[0027] According to some specific embodiments of the present invention, the linking peptide 2 has an amino acid sequence represented by SEQ ID NO: 2. GGGGSGGGGSGGGGS (SEQ ID NO: 2).

[0028] According to some specific embodiments of the present invention, at least a part of the Fc region is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody or a variant thereof.

[0029] According to some specific embodiments of the present invention, at least a part of the Fc region is derived from human IgG or a variant thereof.

[0030] According to some specific embodiments of the present invention, at least a part of the Fc region is derived from Fcγ4 or a variant thereof.

[0031] According to some specific embodiments of the present invention, at least a part of the Fc region is derived from human Fcγ4 or a variant thereof.

[0032] According to some specific embodiments of the present invention, the Fc region has an amino acid sequence represented by SEQ ID NO: 3. APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 3).

[0033] According to some specific embodiments of the present invention, the recombinant antibody has an amino acid sequence represented by SEQ ID NO:4. QVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSSGGGGSGGGGSGGGGSSYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVLESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO: 4).

[0034] Preventive or therapeutic composition

[0035] In one aspect of the present invention, the present invention provides a nucleic acid, and the nucleic acid encodes the recombinant antibody described above. The recombinant antibody encoded by the nucleic acid according to the examples of the present invention can effectively bind to CD16a and IL-21R, has strong in vivo and in vitro binding activities, can effectively activate NK cells, has a long in vivo half-life, and has significant anti-tumor and anti-viral effects.

[0036] According to some specific embodiments of the present invention, the nucleic acid has a nucleotide sequence represented by SEQ ID NO: 5.

[0037] In addition, for the nucleic acids mentioned in the specification and claims of the present invention, those skilled in the art should understand that in fact, either of the complementary double strands, or two of them, are included. For convenience, in this specification and the claims, in many cases, only one strand is shown, but in fact, the other strand complementary to it is also disclosed. In addition, the nucleic acid sequences in the present application include DNA form or RNA form, and disclosing one means that the other is also disclosed.

[0038] In another aspect of the present invention, the present invention provides an expression vector, and the expression vector carries the nucleic acid molecule described above. The expression vector may include a selectable control sequence. The control sequence is operably connected to the nucleic acid molecule. The control sequence is one or more control sequences capable of directing the expression of the nucleic acid molecule in a host. The expression vector proposed in the examples of the present invention can efficiently express the recombinant antibody in an appropriate host cell, and further, can effectively activate NK cells, and is used for the treatment or prevention of diseases caused by tumors or viral infections.

[0039] In another aspect of the present invention, the present invention provides a recombinant cell, and the recombinant cell carries the nucleic acid molecule, expression vector or recombinant antibody described above. The recombinant cell is obtained by transfection or transformation of the expression vector. According to some specific examples of the present invention, the recombinant cell can efficiently express the above recombinant antibody under appropriate conditions, and the recombinant cell is effectively used for the treatment or prevention of diseases caused by tumors or viral infections.

[0040] Note that the "appropriate conditions" described in the specification of the present application are conditions suitable for the expression of the recombinant antibody described in the present application. Those skilled in the art can easily understand that the conditions suitable for recombinant antibody expression include, but are not limited to, appropriate transformation or transfection methods, appropriate transformation or transfection conditions, healthy host cell states, appropriate host cell densities, appropriate cell culture environments, and appropriate cell culture times. The "appropriate conditions" are not particularly limited, and those skilled in the art can optimize the conditions for optimal recombinant antibody expression according to the specific environment of the laboratory.

[0041] In a further aspect of the present invention, the present invention provides a composition comprising the recombinant antibody, nucleic acid molecule, expression vector or recombinant cell described above. As described above, the recombinant antibody according to the examples of the present invention can effectively bind to the CD16a or IL-21R protein molecule, and can further specifically recognize NK cells that highly express the IL-21R and / or CD16a, effectively activate NK cells, and the composition containing the recombinant antibody can similarly play a significant role in the treatment or prevention of diseases caused by tumors or viral infections, and its anti-tumor and anti-viral abilities are remarkable. The composition is not particularly limited, and any of the above substances combined with any other substances, for example, a food composition or a drug composition, is within the scope of the composition described in the present application.

[0042] In one aspect of the present invention, the present invention provides a drug comprising the recombinant antibody nucleic acid molecule, expression vector, recombinant cell or composition described above, and the drug is used for treating or preventing tumors or viral infections. As described above, the recombinant antibody according to the examples of the present invention can effectively bind to the CD16a and IL-21R proteins, and can further specifically recognize NK cells that highly express the IL-21R and / or CD16a. The drug manufactured using the recombinant antibody and its corresponding series of substances can similarly play a significant role in the treatment or prevention of tumors or viral infections, and its anti-tumor and anti-viral abilities are remarkable.

[0043] According to some specific embodiments of the present invention, the tumor includes at least one of rectal cancer, lung cancer, breast cancer, liver cancer, gastric cancer, skin cancer, kidney cancer, pancreatic cancer, and ovarian cancer.

[0044] According to some specific embodiments of the present invention, the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus.

[0045] The drug according to some specific embodiments of the present invention includes a pharmaceutically acceptable vector and an effective amount of the recombinant antibody active ingredient.

[0046] As used herein, the term "effective amount" or "effective dosage" means an amount that can bring about a function or activity in a human and / or animal and is acceptable to the human and / or animal.

[0047] As used herein, a "pharmaceutically acceptable" component is a substance that can be applied to humans and / or mammals without excessive side effects (such as toxicity, irritation, and allergic reactions), that is, a substance having a reasonable benefit / risk ratio. The term "pharmaceutically acceptable vector" refers to a vector used for the administration of therapeutic agents including various excipients and diluents.

[0048] The drug of the present invention includes a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable vector. Such vectors include, but are not limited to, saline, buffer solutions, glucose, water, glycerin, ethanol, and combinations thereof. Usually, the pharmaceutical formulation should be matched with the administration method, and the dosage form of the drug of the present invention is an injection, an oral formulation (tablet, capsule, oral liquid), a transdermal agent, and a sustained release agent. For example, it can be produced by a conventional method using an aqueous solution containing physiological saline, glucose, and other adjuvants. It is desirable to produce the above drugs under aseptic conditions.

[0049] The effective amount of the active ingredient described in the present invention varies depending on factors such as the pattern of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by those skilled in the art based on various factors (e.g., clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of the above-mentioned active ingredient, such as bioavailability, metabolism, half-life, etc., the severity of the disease the patient is attempting to treat, the patient's weight, the patient's immune status, the route of administration, etc. For example, due to the urgent requirements of the treatment situation, several separate doses can be administered per day, or the dose can be proportionally reduced.

[0050] The pharmaceutically acceptable vectors described in the present invention include, but are not limited to, water, saline, liposomes, lipids, proteins, protein-antibody complexes, peptide-based substances, cellulose, nanogels, or combinations thereof. The selection of the vector should be coordinated with the mode of administration, and these are well known to those skilled in the art.

[0051] Use

[0052] In one aspect of the present invention, the present invention provides the use of the recombinant antibody, nucleic acid molecule, expression vector, and recombinant cell described above in the culture amplification of NK cells. The recombinant antibody, nucleic acid molecule, expression vector, and the recombinant cell according to some specific examples of the present invention can be used to promote the proliferation and amplification of the NK cells in vitro, and the amplified NK cells have good cytotoxicity.

[0053] According to some specific examples of the present invention, the NK cells are derived from peripheral blood or obtained by the induction of stem cells. NK cells are mainly distributed in peripheral blood, and NK cells may also be obtained by inducing stem cells.

[0054] According to some specific embodiments of the present invention, the NK cells include at least one of NK cells derived from human peripheral blood mononuclear cells, NK cells induced from human umbilical cord blood stem cells, NK cells induced from human embryonic stem cells, and NK cells induced from human iPSCs. NK cell peripheral blood mononuclear cells include lymphocytes and monocytes. According to some specific embodiments of the present invention, the recombinant antibody, nucleic acid molecule, expression vector, and the recombinant cell described above can promote the proliferation of NK cells derived from the above, have a good amplification effect, and the amplified NK cells have good cytotoxicity.

[0055] In the seventh aspect of the present invention, the present invention provides the use of the recombinant antibody, nucleic acid molecule, expression vector, recombinant cell or composition described above in the manufacture of a drug, and the drug is used for treating or preventing tumors or viral infections. As described above, the recombinant antibody according to the embodiment of the present invention can effectively bind to CD16a and IL-21R protein molecules, and further specifically recognize NK cells that highly express the IL-21R and / or CD16a. Drugs manufactured using the recombinant antibody and its corresponding series of substances can similarly play a significant role in the treatment or prevention of tumors or viral infections, and their anti-tumor and anti-viral abilities are remarkable.

[0056] According to some specific embodiments of the present invention, the above use may further include at least one of the following additional technical features.

[0057] According to some specific embodiments of the present invention, the tumor includes at least one of rectal cancer, lung cancer, breast cancer, liver cancer, gastric cancer, skin cancer, kidney cancer, pancreatic cancer, and ovarian cancer.

[0058] According to some specific embodiments of the present invention, the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus.

[0059] Reagent kit

[0060] In a last aspect of the present invention, the present invention provides a reagent kit comprising the recombinant antibody described above. The recombinant antibody according to an embodiment of the present invention can effectively bind to CD16a and IL-21R protein molecules, and further can specifically recognize NK cells that specifically highly express the IL-21R or CD16a. Therefore, the recombinant antibody can be used to manufacture a reagent kit for detecting CD16a or IL-21R, and the reagent kit is used in scientific research such as qualitatively or quantitatively detecting CD16a and / or IL-21R protein molecules in a biological sample.

[0061] According to some specific embodiments of the present invention, the reagent kit is used for detecting CD16a and / or IL-21R.

[0062] Therapeutic Use and Methods for Diseases

[0063] In one aspect of the present invention, the present invention provides the use of the recombinant antibody, nucleic acid molecule, expression vector, recombinant cell, composition or drug described above in the treatment or prevention of tumors or viral infections. As described above, the recombinant antibody can specifically recognize the above CD16a and IL-21R proteins, and further can specifically recognize NK cells that specifically highly express the IL-21R or CD16a, effectively activate NK cells, enhance IFN-γ secretion, improve proliferation and NK cytotoxic activity, effectively kill tumor cells, and at the same time accelerate the maturation of NK cells and the expression of NK cell receptors, and can restore the depletion of NK cells in the tumor microenvironment. Therefore, the recombinant antibody according to an embodiment of the present invention, the composition, drug containing the recombinant antibody or a series of substances capable of expressing the recombinant antibody under appropriate conditions, such as the above nucleic acid molecule, expression vector, recombinant cell, etc. can all effectively activate NK cells and further treat or prevent tumors or viral infections.

[0064] According to some specific embodiments of the present invention, the tumor includes at least one of rectal cancer, lung cancer, breast cancer, liver cancer, gastric cancer, skin cancer, kidney cancer, pancreatic cancer, and ovarian cancer. The viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus.

[0065] In another aspect of the present invention, the present invention provides a method for treating or preventing a tumor or viral infection. According to some specific embodiments of the present invention, the method includes administering to a subject at least one of 1) the recombinant antibody described above, 2) the nucleic acid molecule described above, 3) the expression vector described above, 4) the recombinant cell described above, 5) the composition described above, or 6) the drug described above. As described above, the recombinant antibody can specifically recognize the above CD16a and IL-21R proteins, and further can specifically recognize NK cells that specifically highly express the IL-21R or CD16a, effectively activate NK cells, enhance IFN-γ secretion, improve proliferation and NK cell cytotoxic activity, effectively kill tumor cells, and at the same time accelerate the maturation of NK cells and the expression of NK cell receptors, and restore the depletion of NK cells in the tumor microenvironment. Therefore, the recombinant antibody according to the embodiments of the present invention, the composition, drug containing the recombinant antibody, or a series of substances that can express the recombinant antibody under appropriate conditions, such as the above nucleic acid molecule, expression vector, recombinant cell, etc., can all effectively activate NK cells and further treat or prevent tumors or viral infections.

[0066] According to some specific embodiments of the present invention, the tumor includes at least one of rectal cancer, lung cancer, breast cancer, liver cancer, gastric cancer, skin cancer, kidney cancer, pancreatic cancer, and ovarian cancer, and the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus.

[0067] Hereinafter, examples will be specifically described. When specific technologies or conditions are not indicated in the examples, they need to be carried out according to the technologies or conditions described in the literature of the relevant field or according to the product specifications. When the manufacturer of the reagents or equipment used is not indicated, they are conventional products that can be purchased commercially.

[0068] Example 1 Design and construction of bispecific antibody molecule anti-CD16a-FCγ4-IL21

[0069] The specific experimental operations of this example are as follows. 1.1 Obtaining the anti-CD16a-FCγ4-IL21 fusion gene (1) Obtaining the Anti-CD16a-FCγ4 fusion gene Using the Pgapza-CD16-FC4γ4 plasmid as a template, PCR was performed using Primer 1 and Primer 2 to obtain the anti-CD16a-FCγ4 fusion gene, which has the nucleotide sequence shown in SEQ ID NO:6. PCR program: Denaturation: 98°C, 20 s; Cycle: 98°C, 10 s → 54°C, 15 s → 72°C, 40 s (30 cycles) Extension: 72°C, 2 min.

[0070] (2) Acquisition of GS-IL21 fusion gene Using the picza-rdna-FC-Y407T-IL21 plasmid as a template, PCR was performed using Primer 3 and Primer 4 to obtain the GS-IL21 fusion gene, which has the nucleotide sequence shown in SEQ ID NO:7. PCR program: Denaturation: 98°C, 20 s; Cycle: 98°C, 10 s → 54°C, 15 s → 72°C, 30 s (30 cycles); Extension: 72°C, 5 min. GGTGGTGGTGGTTCTggaGGTggcGGTTCTGGTGGTGGTGGTTCTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTAG (SEQ ID NO:7).

[0071] The sequences of the above Primers 1-4 are shown in Table 1.

[0072]

Table 1

[0073] Perform agarose gel electrophoresis on the product after the above two-step amplification, and according to the operation steps of the DNA gel recovery reagent kit, cut and recover the strips corresponding to the two target genes to obtain the Anti-CD16a-FCγ4 and GS-IL21 fusion genes, that is, obtain the first target gene fragment and the second target gene fragment.

[0074] (3) Obtaining the Anti-CD16a-FCγ4-IL21 fusion gene Using the Anti-CD16a-FCγ4 and GS-IL21 fusion genes obtained in the above steps as templates, use Primer 1 and Primer 4 to perform PCR to obtain the Anti-CD16a-FCγ4-IL21 fusion gene. PCR program: Denaturation: 98°C, 20 s; 3 cycles: 98°C, 10 s → 54°C, 20 s → 72°C, 40 s (3 cycles); Extension: 72°C, 2 min; Then, add primers: Denaturation: 98°C, 20 s; 3 cycles: 98°C, 10 s → 54°C, 20 s → 72°C, 40 s (30 cycles); Extension: 72°C, 10 min; Perform agarose gel electrophoresis on the amplification product, and recover it with a DNA gel recovery reagent kit to obtain one segment of the gene. This gene has XhoI and NotI enzyme cleavage sites at both ends and is named Anti-CD16a-FCγ4-IL21. The above Pgapza-CD16-FC4γ4 and Ppicza-rdna-FC-Y407T-IL21 plasmids were both independently constructed by the inventor according to the conventional expression vector construction method.

[0075] 1.2 Construction of the Pichia pastoris expression vector for the anti-CD16a-FCγ4-IL21 fusion protein Perform double enzyme digestion on the gene sequence (SEQ ID NO: 5) of the anti-CD16a-FCγ4-IL21 fusion protein obtained in Step 1.1 and the Pichia pastoris expression plasmid PGAPzα-rDNA-NTS stored in the laboratory using restriction endonucleases Xhol and NotI. The enzyme-digested anti-CD16a-FCγ4-IL21 fusion protein gene sequence product gene is recovered using a PCR product recovery reagent kit (Axygen), and the enzyme-digested plasmid is recovered using a DNA gel recovery reagent kit (Axygen). Refer to the reagent kit instructions for the operation procedure. The fusion protein gene after enzyme digestion and the vector after enzyme digestion obtained by recovery are connected with T4 DNA ligase, and the structure of the constructed plasmid is shown in Figure 1. The enzyme ligation product is transformed into Escherichia coli TOP10 receptor cells. The transformation operation procedure is as follows: Add 10 μL of the enzyme ligation product to 100 μL of Escherichia coli receptor, mix evenly, let it stand on ice for 15 - 30 minutes, flick evenly every 5 minutes, then perform heat shock at 42°C for 90 seconds, let it stand on ice for 3 - 5 minutes, add 400 μL of LB liquid medium without antibiotics, resuscitate and culture it on a rocking bed at 37°C for 45 minutes, and then spread it on a Zeocin-resistant plate. After the clone bacterial colonies grow on the plate, select a single colony clone and put it into a cell culture tube containing LZ liquid medium (Zeocin-resistant LB medium), culture it at 37°C for 6 - 8 h, then perform PCR identification using the bacterial liquid as a template, name the obtained positive clone PGAP-zα-anti-CD16a-FCγ4-IL21, extract the bacterial liquid corresponding to the positive clone using a plasmid miniprep reagent kit, and send it to a general biology company for sequencing. After sequencing, the sequencing result is consistent with the theoretically expected result, indicating that the clone construction is correct.

[0076] Example 2 Expression and Purification of Bispecific Antibody Molecule anti-CD16a-FCγ4-IL21

[0077] 2.1 Expression of anti-CD16a-FCγ4-IL21 Bispecific Antibody Molecule The positive clone constructed in Example 1 was expanded and cultured using 50 mL of LZ liquid medium, and the plasmid PGAP-zα-anti-CD16a-FCγ4-IL21 was extracted using a mid-volume reagent kit (Axygen). The expression plasmid PGAP-zα-anti-CD16a-FCγ4-IL21 prepared in the previous step was linearized with the endonuclease SpeI and recovered by ethanol precipitation. Ethanol precipitation step: 1) Add two volumes of anhydrous ethanol and 0.1 volume of 3M sodium acetate to the enzyme cleavage reaction system and precipitate at -20°C for 2 hours or more. 2) Centrifuge at 12,000 g for 10 minutes, and discard the supernatant. 3) Resuspend and precipitate with 300 μL of 70% ethanol, centrifuge at 12,000 g for 10 minutes, and discard the supernatant. 4) Dry at 37°C, resuspend with deionized water, measure the concentration, and adjust the concentration to 0.5 - 1.0 μg / μL.

[0078] Production of yeast receptor: Preparation steps: 1) Take the well-preserved frozen Saccharomyces cerevisiae X33 strain, streak it on a YPD plate, and incubate it at a constant temperature of 30°C in a yeast culture tank. 2) When the clone grows to a diameter of about 1 mm, transfer the clone to 4 mL of liquid YPD medium and incubate the yeast at a constant temperature of 30°C on a rocking bed. 3) Incubate for 24 - 48 hours. Inoculate 1 mL of the bacterial solution into 50 mL of fresh YPD medium. 4) When the OD600 value reaches 1 - 1.5, transfer the bacterial solution to a 50 mL centrifuge tube, centrifuge at 1500 g for 5 minutes at 4°C, and discard the supernatant. 5) Resuspend with 50 mL of ice-cold water, centrifuge at 1500 g for 5 minutes at 4°C, discard the supernatant, and repeat once. 6) Resuspend with 50 mL of 1M cold sorbitol, centrifuge at 1500 g for 5 minutes at 4°C, discard the supernatant, and repeat once. 7) Resuspend with 500 μL of 1M cold sorbitol.

[0079] Electrotransformation step: Put 100 μL of the above recipient cells into a sterile pre-cooled electroporation cuvette, add 10 μL of linearized plasmid (5 - 10 μg) into it, and mix uniformly. Set the electroporation parameters: 2000 V, 200 Ω, 25 μF. Perform electroporation. Immediately after electroporation, add 1 mL of 1 M cold sorbitol to the electroporation cuvette, let it stand on ice for 5 - 10 min, then transfer all the bacterial solution to a 50 mL centrifuge tube containing 1 mL of YPD, and resuscitate at 30 °C, 225 rpm for 2 h. After resuscitation is completed, take 100 - 1000 μL of the above bacterial solution and spread it on a YPDZ (plate containing Zeocin resistance) plate, and place the plate in a constant-temperature yeast culture tank at 30 °C for culture.

[0080] Positive expression clone screening: After yeast clones grow on the above YPDZ plate, select some clones into 2 mL of BMGY medium, and incubate the yeast at 28 °C on a rocking bed until the bacterial solution becomes milky. Freeze and store 1 mL of the bacterial solution among them to preserve the strain. After centrifuging the remaining 1 mL of the bacterial solution at 12000 g for 5 min, take the supernatant and perform an initial screening by Dot blot identification using an anti-human FCγ4 antibody to select yeast strains with relatively high expression levels. Then, spread the yeast strains with relatively high expression levels on a YPD plate containing zeocin. After clones grow, select multiple clones from them, shake the bacteria as above to obtain the bacterial solution supernatant, and perform Western Blot identification analysis using an Anti-human FCγ antibody to screen for a high-expression strain (34 - 3). Freeze and store the remaining 1 mL of the bacterial solution of the corresponding strain to preserve the strain. This high-expression strain is a Pichia yeast strain that can express the anti-CD16a-FCγ4-IL21 complex protein.

[0081] Specific experimental results are as follows: As shown in Figure 3, after screening on the YPDZ resistant culture plate, yeast expression strains of the fusion protein are obtained. Initial screening is carried out using Dot blot, and some clones with relatively high expression levels (numbers 66; 91; 98; 34 in Figure 3B) are initially obtained. Subsequently, further confirmation and comparison are carried out by Western Blot, and yeast expression clones with high expression levels can be screened, which are then used for subsequent protein expression and purification.

[0082] 2.2 Fermentation, purification and identification of anti-CD16a-FCγ4-IL21 bispecific antibody molecule (1) Fermentation Cultivation of expression strain: The fermentation of the recombinant strain is carried out according to the fermentation instruction manual of Invitrogen. The screened high-expression strain is inoculated into a cell culture tube containing 4 mL of YPD medium and cultured at 30 °C on a rocking bed for 24 - 48 hours until OD600 reaches 2 - 6, which is the primary fermentation seed solution. Take 1 mL of this seed solution and transfer it into a conical bottle containing 200 mL of BMGY medium, and culture it at 30 °C on a rocking bed for 12 - 24 hours, which is the secondary fermentation seed solution. Inoculate all 200 mL of the secondary seed solution into a fermentation tank containing 6 L of BMGY medium. Parameter setting: Monitor the culture temperature at 30 °C, pH 7.0, dissolved oxygen, rotation speed, etc., and start the fermentation culture. In the cell growth stage, BMGY medium is used. When the dissolved oxygen rises rapidly, the basic glycerol in the medium is consumed completely, and the addition of glycerol is started. The addition rate of glycerol is 90 mL / h, and the fermentation temperature is adjusted to 25 °C and continues until the end of fermentation.

[0083] Clarification of the culture solution: After the fermentation is completed, centrifuge at 10000 g for 30 minutes, collect the supernatant, filter and clarify it with 0.22 μm and 500 kDa microfilters, and then adjust the pH to 7.4.

[0084] Monitoring of each parameter in the fermentation process is shown in Figure 4. The entire fermentation process lasts for 50 hours, and the fermentation process is stable and easy to repeat. As can be seen from Figure 4B, protein expression (20 h) is initiated by inducing glycerol addition-limited culture from the end of the basic glycerol culture stage. Proteins accumulate throughout the process, and there is no significant difference in the protein accumulation amount from 43 to 50 h, and the fermentation end point is determined.

[0085] (2) Purification Protein capture: The recombinant fusion protein is captured using an affinity column Mabselect. The specific steps are as follows: 1) Wash the column with 5 column volumes by deionization. 2) Wash the column with 3 column volumes of PBS. 3) Add the sample. 4) After the sample addition is completed, wash the column with 3 column volumes of PBS. 5) Impurity washing: Wash with 5 column volumes of citric acid buffer at pH 5.5. 6) Elute the target protein. The eluent is citric acid buffer at pH 3.0, and the collected eluent is used for subsequent purification after concentration.

[0086] Fine purification: Use AKTA PURE 25 to equilibrate the Superdex 200 molecular sieve with PBS buffer. After equilibration, add the sample to the neutral eluent, wash it with PBS buffer, and then collect the eluted sample to obtain the anti-CD16a-FCγ4-IL21 recombinant protein. After centrifugal filtration and two-stage purification, through SDS-PAGE and HPLC identification, the protein purity can reach over 95% (Figure 5A, C).

[0087] (3) Identification Recombinant protein identification: SDS-PAGE, HPLC, and dynamic light scattering identification are performed on the purified protein. The specific experimental results are shown in Figure 4B. The molecular weight of the anti-CD16a-FCγ4-IL21 protein is about 135 kDa, which is as expected. Through dynamic light scattering identification, the molecular diameter of anti-CD16a-FCγ4-IL21 is about 10 nm, and the uniformity between molecules is good.

[0088] Example 3 Verification of the Function of Activating NK Cells by the Bispecific Antibody anti-CD16a-FCγ4-IL21 In Vitro

[0089] Human PBMCs are separated using the Ficoll density gradient centrifugation method. The separated PBMCs are diluted to 1.0×10 6 cells / mL using RPMI 1640 containing 10% FBS. 10 mL of the diluted cells are added to a T25 culture bottle. After culturing for 12 hours, the anti-CD16a-FCγ4-IL21 fusion protein is added to the culture bottle at a final concentration of 20 nM. After culturing for 24 hours, the phenotypic changes of NK cells are detected by flow cytometry, mainly detecting the expression status of the main activating molecules CD69, NKp44 and the killing molecules 4-1BB, TRAIL, Granzyme B on the surface of NK cells.

[0090] The specific experimental results are shown in Figure 6. In the experimental group with the addition of the anti-CD16a-FCγ4-IL21 fusion protein compared with the control group PBS (Control group), the expression levels of the main activating molecules CD69, NKp44 and the killing molecules 4-1BB, TRAIL, Granzyme B on the surface of NK cells all increased significantly. It is thus judged that NK cells are effectively activated by the anti-CD16a-FCγ4-IL21 fusion protein.

[0091] Example 4 Evaluation of the Amplification and Amplification Effect of the Bispecific Antibody Molecule anti-CD16a-FCγ4-IL21 on NK Cells In Vitro

[0092] 4.1 Detection of the Purity and Number of NK Cells Amplified by anti-CD16a-FCγ4-IL21 The T75 culture bottle is coated with the fusion protein 24 hours before. 7.5 ml of the coating solution is taken per bottle and covers the bottom of the bottle body. It is coated overnight in a refrigerator at 4°C.

[0093] After separating PBMC cells from the blood sample, count them (using a hemocytometer) and record the percentage of each cell subset in the PBMC cells. Inoculate according to a density of 1.5×10^6 / ml (20 ml culture volume). The total number of cells is 30×10^6. This is day 0. At the same time, add 1% IL2 (1000 IU / ml) and 5% of the corresponding PBMC's autologous plasma to the KBM581 medium without anti-CD16a-FCγ4-IL21. On day 3, supplement with 1% IL2 factor of the corresponding culture volume. On day 5, observe the color of the medium and the cell attachment state on the bottom wall of the T75 bottle body. If necessary, supplement with 5 - 10 mL of KBM581 medium containing anti-CD16a-FCγ4-IL21 and add 1% IL2 (1000 IU / ml) factor of the supplement volume. On day 6, sample and count, and perform cell subculture expansion (1.3×10^6 / mL). Then perform staining labeling (CD16a / CD45 / CD56), detect the percentage of the NK cell subset and the percentage of the CD56+ cell subset with a flow cytometer, calculate the total cell number, and calculate the number of NK cells from the flow purity detection results. After day 5, perform cell subculture expansion, use the KBM581 medium containing anti-CD16a-FCγ4-IL21, and add 1% IL2 and 2% autologous plasma. On day 7, observe the color of the medium and the cell attachment growth state of the bottle body. If necessary, replenish the liquid (add 1% IL2 factor of the supplement volume). On day 8, sample and count. If necessary, perform cell subculture expansion (T75 or T175 bottle, 1.4×10^6 / ml) and add 1% IL2 factor and 2% autologous plasma of the medium. On day 9, observe the color of the medium and the cell attachment growth state of the bottle body. If necessary, replenish the liquid (add 1% IL2 factor of the supplement volume). On day 10, sample and count, and perform cell subculture expansion (1.5×10^6 / ml). Then perform staining labeling (CD16a / CD45 / CD56), detect the percentage of the NK cell subset and the percentage of the CD56+ cell subset with a flow cytometer, calculate the total cell number, and calculate the number of NK cells from the flow purity detection results. On the 12th day, observe the color of the medium and the cell wall attachment growth state of the bottle body, and supplement the liquid as needed (add IL2 factor at 1% of the supplemented liquid volume). On the 13th day, sample and count, and perform subculture expansion culture of cells (1.5×10^6 / mL). Then perform staining labeling (CD16a / CD45 / CD56), detect the percentage of the NK cell subset and the percentage of the CD56+ cell subset with a flow cytometer, calculate the total cell number, and calculate the number of NK cells from the flow purity detection result. On the 15th day, sample and count. Perform cell subculture expansion (T175 flask or culture bag, 1.5×10^6 / mL) as needed, and add IL2 factor at 1% of the medium. On the 17th day, sample and count, then perform staining labeling (CD16a / CD45 / CD56), detect the percentage of the NK cell subset and the percentage of the CD56+ cell subset with a flow cytometer, calculate the total cell number, and calculate the number of NK cells from the flow purity detection result. Create a graph of the time-course changes in the proportion and number of NK cells according to the NK purity and cell number on the 0th, 6th, 10th, 13th, and 17th days.

[0094] The specific experimental results are shown in A - C in Figure 7. By amplifying NK cells derived from PBMC using anti-CD16a-FCγ4-IL21, the NK cells can be amplified, the purity and amplification multiple of NK cells can be improved, and the proportion of NK cells can reach about 65% at the highest during the whole culture process.

[0095] 4.2 Subset analysis of NK cells amplified from PBMC by bispecific antibody molecules

[0096] (1) Intracellular molecular labeling cell monensin induction: Put 4×10 6 cells (2 ml) cultured into a 6-well plate, add 10 μl of monensin (0.5 μg / μl), and induce at 37°C in a constant temperature culture tank with 5% CO2 for 4 hours. (2) Preparation of single cell suspension: 9.0×10 cells cultured from surface molecular labeled cells 6Take them and put them into a 15 ml centrifuge tube, centrifuge at 400 g for 8 minutes to collect the cells, wash twice with 10 ml of 1×PBS, and finally resuspend in 0.81 ml of 1×PBS to form a single-cell suspension. After the induction of intracellular molecularly labeled cells with monensin was completed, wash twice with 5 ml of 1×PBS, and finally resuspend in 180 μl of 1×PBS to form a single-cell suspension. (3) Blocking: Add 90 μl of mouse serum to the surface molecule-labeled cells, mix evenly, add 20 μl of mouse serum to the intracellular molecule-labeled cells, mix evenly, and let stand at room temperature for 15 - 30 minutes. (4) Antibody labeling: Dispense the surface molecule-labeled cells after blocking into 9 flow tubes at 0.09 ml per tube. When detecting multiple batches of cells simultaneously, the cells in tubes 1 - 8 can be mixed and labeled in equal amounts with multiple batches of cells. The cell usage per tube is 0.8 - 1×10 6 cells, and sample tube 1 is for surface molecule labeling, with the cell usage being 0.8 - 1×10 6 cells. Sample tube 2 is for intracellular molecule labeling, with the cell usage being 3 - 4×10 6 cells. Add the corresponding fluorescently labeled antibody, mix evenly, and then let stand at 4℃ in the dark for 30 minutes (mix once every 15 minutes in the middle). The specific experimental groupings are shown in Table 2.

[0097]

Table 2

[0098] Specific experimental results are shown in Figure 7-D. Subgroup analysis was performed on the amplified cells. After culture amplification with anti-CD16a-FCγ4-IL21, the proportion of NK cells in PBMC cells can reach 61%, the proportion of NKT is about 10%, and the total proportion of CD56-positive cells is about 71%. The proportions of the remaining cell subgroups in the amplified PBMC cells are low.

[0099] 4.3 Detection of the killing activity of bispecific antibody molecules in amplifying NK cells (1) Preparation of the target cell (K562) suspension Cell counting: Resuspend the cells in RPMI 1640 medium containing 0.5% FBS (the medium used for target cells), count them, and adjust the cell density to 1×10 6 / mL, CFSE staining: Add CFSE (working concentration 5 μM) to the cell suspension, immediately pipette and mix with a 1 mL pipette, vortex thoroughly, incubate in a 37 °C culture tank in the dark for 15 min, take out the vortex and mix once every 5 min, Termination of staining: Add 5 volumes of pre-cooled complete medium (the medium used for target cells) at 4 °C to terminate the staining, and incubate in an ice bath for 5 min. Centrifuge at 140 g, 4 °C for 5 min, Cell washing: Resuspend the cells in pre-cooled complete medium (the medium used for target cells) at 4 °C, centrifuge the cells at 140 g, 4 °C for 5 min, and repeat the washing 2 times, Counting: Resuspend and count the cells, and adjust the cell density to 2×10 5 / mL, Seeding: Add 100 μl of the K562 suspension to each well of a 96-well round-bottom plate. The final number of cells per well is 20,000 cells / well. (2) Preparation and addition of NK cells Adjust to an appropriate NK cell density, Take out the E-Plate 16 and place it on a super-clean table, Add 100 μl of NK cell suspension into the culture plate at different effector-target ratios (CD56+:K562) (1:1, 2:1, 4:1, 8:1). Additionally, three sets of controls are further established, namely, target cells with only CFSE staining (to detect the natural mortality rate of target cells), effector cells only (to prove that effector cells do not contain CFSE non-specific staining), and target cells + Tween-20 (as a positive control for target cell apoptosis). Co-incubate and add NK cells to each well in the pre-designed order. Centrifuge at 120 g at room temperature for 2 min to allow sufficient contact between the effective target cells. Return to the culture tank at 37 °C and incubate for 4 hours.

[0100] After incubation is completed, add 5 μl of PI, mix evenly, incubate for 5 min in the dark, and detect with the instrument. Result analysis: Killing percentage = [((experimental group target cell mortality rate (%) - target cell mortality rate (%)) / (100% - target cell natural mortality rate (%))) × 100%].

[0101] The specific experimental results are shown in Figure E (7-E) of Figure 7. Evaluate the cytotoxicity of the amplified cells, and the amplified cells can effectively kill K562 cells.

[0102] As described above, from the experimental results of this example, it is proved that anti-CD16a-FCγ4-IL21 is activated in vitro to amplify the activity of NK cells, and the amplified NK cells have relatively good cytotoxicity.

[0103] In the description of this specification, the description of reference terms such as "one embodiment", "several embodiments", "example", "specific example", or "several examples" means that the specific features, structures, materials, or characteristics described by combining the embodiments or examples are included in at least one embodiment or example of the present invention. In this specification, the exemplary description of the above terms does not necessarily target the same embodiment or example. Also, the specific features, structures, materials, or characteristics to be described can be combined in a suitable manner in any one or more embodiments or examples. Note that if there is no conflict with each other, those skilled in the art can combine and combine different embodiments or examples and the features of different embodiments or examples described in this specification.

[0104] Embodiments of the present invention have been presented and described. However, the above embodiments are exemplary and cannot be understood as limiting the present invention. Those skilled in the art can understand that various changes, modifications, substitutions, and variations can be made to the above embodiments within the scope of the present invention. Cross-reference to related applications

[0105] This application claims the priority of the Chinese patent application with the application number 202111564703.3, which was filed with the Chinese Patent Office on December 20, 2021, and the entire content thereof is incorporated herein by reference.

Claims

Claim 1 A recombinant antibody comprising: an IL-21 molecule; a CD16a single-chain antibody and an Fc region, wherein the N-terminus of the Fc region is connected to the C-terminus of the single-chain antibody, and the CD16a single-chain antibody comprises a CD16a antibody containing a heavy-chain variable region and a light-chain variable region; the C-terminus of the FC is connected to the N-terminus of the IL21 molecule, and the recombinant antibody has an amino acid sequence represented by SEQ ID NO:

4. Claim 2 A nucleic acid encoding the recombinant antibody according to claim 1. Claim 3 The nucleic acid according to claim 2, having a nucleotide sequence represented by SEQ ID NO:

5. Claim 4 An expression vector carrying the nucleic acid molecule according to claim 2. Claim 5 A recombinant cell carrying the expression vector according to claim 4. Claim 6 A composition comprising the recombinant antibody according to claim 1. Claim 7 Use of the recombinant antibody according to claim 1 in the manufacture of a drug for treating or preventing a tumor or a viral infection. Claim 8 The use according to claim 7, wherein the tumor comprises at least one of rectal cancer, lung cancer, breast cancer, liver cancer, gastric cancer, skin cancer, kidney cancer, pancreatic cancer, and ovarian cancer, and the viral infection comprises at least one of HPV, HBV, influenza virus, and coronavirus. Claim 9 Use of the recombinant antibody according to claim 1 in the culture and amplification of NK cells. Claim 10 The use according to claim 9, wherein the NK cells are derived from peripheral blood or obtained by stem cell induction. Claim 11 The use according to claim 9, wherein the NK cells comprise at least one of NK cells derived from human peripheral blood mononuclear cells, NK cells induced from human umbilical cord blood stem cells, NK cells induced from human embryonic stem cells, and NK cells induced from human iPSCs. Claim 12 A drug comprising the recombinant antibody according to claim 1 and used for treating or preventing a tumor or a viral infection. Claim 13 The drug according to claim 12, wherein the tumor comprises at least one of rectal cancer, lung cancer, breast cancer, liver cancer, gastric cancer, skin cancer, kidney cancer, pancreatic cancer, and ovarian cancer, and the viral infection comprises at least one of HPV, HBV, influenza virus, and coronavirus. Claim 14 A reagent kit comprising the recombinant antibody according to claim 1.

15. The reagent kit according to claim 14, characterized in that it is used for detecting CD16a and / or IL-21R.

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