Design, preparation, and use of innate super engager and complete immune antibody

Abstract

The present invention provides a design of an innate super engager (ISE) and a complete immune antibody (CIA), a preparation method therefor, and use thereof. The innate super engager comprises an antibody or ligand that connects and activates innate immune cells, or a cytokine that stimulates the proliferation of innate immune cells, or a pattern recognition receptor (PRR) agonist. The innate super engager can be used alone or in combination with other immune antibodies, or can be further linked with one or more acquired immune antibodies or ligands to form a complete immune antibody. The complete immune antibody has two forms: (1) an acquired immune antibody or a tumor antigen antibody is connected to the N terminal of the innate super engager; and (2) the innate super engager is connected to the C terminal or N terminal of the acquired immune antibody or the tumor antigen antibody.

Description

Design, preparation and application of innate immune superconnectors and complete immune antibodies Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically relates to the preparation and application of cell agonists or multispecific antibodies. Background Technology

[0002] Immunity is broadly classified into two categories: innate immunity and acquired immunity. Innate immunity is present at birth, while acquired immunity is gradually acquired during the body's survival. Innate immunity, also known as nonspecific immunity, is an innate function of the body to maintain health. This immunity instinctively repels and phagocytoses all foreign substances, pathogens, and foreign bodies. It includes the body surface barrier, blood-brain barrier, blood-placental barrier, phagocytosis, and antimicrobial substances in normal body fluids and tissues. The innate immune system constitutes the first line of defense against invading microbial pathogens and relies on pattern recognition receptors (PRRs). PRRs are germline-encoded host sensors that can detect typical molecules belonging to pathogens and play a crucial role in the normal function of the innate immune system. [1] These are proteins primarily expressed by innate immune cells (such as dendritic cells, macrophages, monocytes, neutrophils, and NK cells) and epithelial cells, used to recognize two classes of molecules: pathogen-associated molecular patterns (PAMPs), associated with microbial pathogens; and damage-associated molecular patterns (DAMPs), associated with host cellular components released during cell damage or death. Based on protein domains, PRRs in the vertebrate innate immune system can be classified into five types: Toll-like receptors (TLRs), nucleotide oligomer domain (NOD)-like receptors (NLRs), retinoic acid-induced gene-I (RIG-I)-like receptors (RLRs), type C lectin receptors (CLRs), and melanoma deficiency factor 2 (AIM2)-like receptors (ALRs). [1] TLRs are the most widely distributed and important PRRs. Currently, 13 types of TLRs have been identified: humans possess TLRs 1-10, and mice possess 12 TLRs (TLRs 1-9, TLRs 11, 12, and 13). Once activated, PRRs produce innate immune cytokines such as type I interferon, which can directly inhibit viral replication; and by activating other innate and acquired immune cells, they bridge the acquired immune system to clear pathogens and regulate immune function.

[0003] pDCs are a fundamental and crucial innate immune cell. When their endosome-expressed TLR-7 or TLR-9 receptors are activated by ligands, they produce large amounts of type I interferon, activating numerous interferon-stimulated genes (ISGs) that directly inhibit viral replication. Type I interferon is also a key cytokine in the body's immune regulation, activating all immune cells—dendritic cells, macrophages, T cells, and B cells—to achieve immune amplification and regulation. It can also inhibit tumor cell proliferation and induce tumor cell apoptosis. [2] It can regulate a series of downstream immune responses to achieve a restoration of immune balance in the body. Unmethylated DNA ODN in pathogenic microorganisms contains a CpG structural pattern: its sequence contains a cytosine deoxyribonucleotide followed by a guanine deoxyribonucleotide, with "p" referring to the phosphodiester bond between them. CpG ODN is an agonist of TLR-9, stimulating pDC to produce large amounts of type I interferon. CpG ODN has already demonstrated in clinical trials its ability to transform "cold tumors" into "hot tumors," playing a significant synergistic role in enhancing the efficacy of PD-1 therapy. [3] .

[0004] PRR-activated macrophages can engulf pathogenic microorganisms and tumor cells. Macrophages are a major component of immune cell infiltration in the tumor microenvironment, accounting for up to 50% of solid tumor masses. Although macrophages can directly engulf and kill tumor cells, they are subject to immunosuppression due to the influence of CD47 expressed by tumor cells in the tumor microenvironment. The signal regulatory protein-α (SIRPα) expressed by macrophages is a major myeloid cell inhibitory receptor and a regulatory mechanism to prevent excessive phagocytosis. [4] Its ligand is the CD47 molecule on the surface of certain cells, such as erythrocytes which express CD47. When CD47 binds to SIRPα expressed by macrophages, it transmits a "don't eat me" signal, thus preventing damage to erythrocytes. As erythrocytes age, their SIRPα levels decrease significantly, causing macrophages to lack the "don't eat me" signal and actively engulf aging erythrocytes, achieving homeostasis – a self-regulating mechanism of the body. Tumor cells also transmit the "don't eat me" signal by overexpressing CD47, thus achieving phagocytic escape. Currently, there are many monoclonal antibodies and Fc-SIRPα fusion molecules designed based on this mechanism to reactivate the function of macrophages in engulfing tumor cells by blocking the "don't eat me" signal. Examples of such products include Magrolimab (anti-CD47) and TTI-622 (Fc-SIRPα fusion molecule). These products have shown some efficacy in clinical trials for hematological malignancies such as acute myeloid leukemia (AML), but the efficacy is not significant. [5]The reason for this may be that the immune system functions are extremely heterogeneous in different tumors or at different stages of the same tumor. This manifests as a lack of certain components of innate immunity, such as macrophages, NK cells, or innate immune factors, and depletion of acquired immune functions, such as antigen-specific T cells and certain subsets of B cells. Especially in late-stage cancer patients, immune function is generally low or extremely heterogeneous, with multiple immunosuppressive functions at play. Simply increasing the phagocytic function of macrophages is no longer sufficient to translate into clinical efficacy. Furthermore, if the number of macrophages and the number of M2 subtypes are low in the cancer population during clinical trials, resulting in a lack of target cells for CD47-targeted therapy, the clinical trial may fail. However, if, through screening, the selected cancer patients have a certain level of macrophage and M1 cell counts, then the effect of CD47-targeted therapy is expected to be significant.

[0005] Natural killer cells, also known as NK cells or large granular lymphocytes (LGLs), are a type of cytotoxic lymphocyte that is crucial to the innate immune system. [6] They belong to the rapidly expanding known family of innate lymphocytes (ILCs), accounting for 5-20% of all circulating lymphocytes in humans. NK cells function similarly to cytotoxic T cells in the acquired immune response of vertebrates. NK cells provide a rapid response to virally infected cells and other intracellular pathogens approximately 3 days after infection, and they also monitor tumor formation, acting and killing tumors in their early stages. While most immune cells recognize and destroy infected or cancerous cells by sensing antigens presented on the major histocompatibility complex (MHC) on the surface of infected or cancerous cells, NK cells can recognize and kill infected or tumor cells even without antibodies and MHC, thus achieving a faster immune response. Because some tumor cells evade killing by specific cytotoxic T lymphocytes (CTLs), which belong to the acquired immune system, by not expressing MHC I, the natural killer function of NK cells becomes particularly important in tumor surveillance and killing. Besides directly killing cancer cells or infected cells, NK cells are also effector cells of the acquired immune system. Antibodies against tumors or microbial antigens can activate NK cells by binding to CD16a on the surface of NK cells, thereby killing tumor and infected cells. This function is also known as antibody-dependent cell-mediated cytotoxicity (ADCC). AFM-13 is a bispecific antibody against CD16a and CD30, which has entered mid-stage clinical trials and has shown good efficacy in the treatment of Hodgkin's lymphoma expressing CD30. The combination therapy of AFM-13 and AlloNK has entered the fast track of clinical development by the US FDA. [7] .

[0006] In summary, by activating PRR receptors such as TLR9, pDCs can be activated to produce large amounts of interferon, directly inhibiting tumor cell proliferation and inducing the growth of tumor cells with acquired immunosuppression. By blocking the "don't eat me" signal, macrophages can be activated to phagocytose and kill cancer cells. By connecting to and activating NK cells, general tumor cells and unlabeled tumor cells (MHCI-deficient) can be rapidly killed. These mechanisms can fully utilize the function of innate immunity to effectively kill tumor cells.

[0007] Connectors formed through parallel innate immune mechanisms can directly or indirectly bridge various innate immune effector cells and cancer cells, fully leveraging the tumor-killing functions of these different functional cells. This not only achieves a powerful tumor-killing effect when all innate immune cells are normal, but also, when certain immune cells (such as those with diminished macrophage function) are weak, the tumor-killing effect of other cells (such as NK cells) can achieve a certain therapeutic effect. Because the innate immune status varies in cancer patients, there may be situations where one cell-mediated innate immunity is strong while another is weak. Therefore, cell connectors that recruit only a single innate immune cell may not be effective. To address this issue, the Innate Super Engager (ISE) disclosed in this invention can simultaneously activate pDCs, macrophages, and natural killer cells, achieving the goal of effectively killing tumor cells and treating cancer under various immune states.

[0008] In a normal organism, both innate and acquired immunity play crucial roles in killing and controlling tumor cells. In the early stages of tumor formation, due to the small number of tumor cells, the suppression of immune mechanisms is absent or extremely low. At this time, any part of the human immune system can suppress tumor cell growth. For example, NK cells can rapidly kill tumor cells, regardless of whether they contain MHC1; activated macrophages can directly engulf tumor cells, also regardless of whether they contain MHC1. Activated pDCs (such as those infected by viruses or bacteria containing CpG fragments) produce large amounts of interferon, which can directly inhibit cancer cell proliferation and control tumor growth by activating numerous innate and acquired immune cells. Once acquired immunity against tumor cells is formed, its powerful tumor antigen-specific cytotoxic cells can directly and effectively kill tumor cells; the tumor antigen-specific B cells induced by these cells can produce anti-tumor antibodies, which kill tumor cells by recruiting the ADCC function of NK cells.

[0009] However, after tumor cells proliferate uncontrollably through numerous immune evasion mechanisms, the body's immune cells and functions are severely damaged, especially in patients with advanced or metastatic cancer, whose immune systems, including both innate and acquired immunity, have been impaired to varying degrees. Furthermore, the immune status of different cancer patients varies greatly at this point; some may have relatively robust acquired immune systems but weakened innate immunity, while others have weakened acquired immunity but relatively strong innate immunity. In this situation, influencing a single immune system or a single immune cell, such as regulating only innate immune cell function or only regulating a single innate immune effector cell like NK cells or macrophages, to kill and control tumors will only achieve limited results. Moreover, it is difficult to determine which immune system or cell will be activated to kill tumor cells in this context. Therefore, if the main innate and acquired immune regulation strategies can be integrated, effective immunotherapy can be achieved even when the immune system of cancer patients is damaged to varying degrees and the nature and extent of immune damage cannot be determined. The Complete Immune Antibody (CIA) disclosed in this invention can simultaneously integrate innate immune drugs, such as the innate immune superconnector (ISE) disclosed in this invention, and acquired immune drugs, such as T cell function modulators (anti-PD-1 / PD-L1 antibodies) or anti-tumor antigen antibodies (such as anti-EGFR / Her2 antibodies), to achieve the best effect of killing and controlling tumors. Summary of the Invention

[0010] This invention discloses an innate immune superconnector comprising at least two components selected from the following innate immune system components: pattern recognition receptor (PRR) agonists, macrophage activators, NK cell linkers and activators, and immune cytokines. More specifically, the innate immune superconnector disclosed herein uses a conventional antibody as its basic structure, removing the variable region of the antibody, or using a non-targeting antibody in the human body, and links components selected from the following innate immune system components to the N-terminus or C-terminus of the antibody heavy chain constant region, or the N-terminus or C-terminus of the antibody light chain constant region: PRR agonists, macrophage activators, NK cell linkers and activators, and immune cytokines. Two or more of the PRR agonists, macrophage activators, NK cell linkers and activators, and immune cytokines can bind to a backbone antibody via a linker to form an innate immune superconnector with the desired conformation. Various PRR agonists, macrophage activators, NK cell linkers and activators, and immune cytokines known in the art can be used. PRR agonists include, but are not limited to, Toll-like receptor (TLR) agonists, NOD-like receptor (NLR) agonists, RIG-I-like receptor (RLR) agonists, C-type lectin receptor (CLR) agonists, and melanoma deficiency factor 2 (AIM2)-like receptor (ALR) agonists. Macrophage activators include, but are not limited to, SIRPαD1 protein, CD40 antibody, and CD24 antibody. NK cell binding and activators include, but are not limited to, anti-CD16a antibody. Immune cytokines include, but are not limited to, IL-15, IL-18, IL-2, IL-7, IL-10, and IL-12.

[0011] The innate immune superconnector disclosed in this invention can recruit or activate innate immune cells. As an example, the ISE disclosed in this invention may have the following immune components: (1) ISE-A, containing SIRPα protein and anti-CD16a single-chain antibody; (2) ISE-B, containing SIRPα protein, anti-CD16a nanobody and immune cytokines such as IL-15; (3) ISE-C, containing SIRPα protein, anti-CD16a nanobody and immune cytokines and their receptors such as IL-15 and IL-15Rα; (4) ISE-D, containing SIRPα protein, anti-CD16a single-chain antibody and immune cytokines such as IL-15. (5) ISE-E, with a PRR agonist linked to the N-terminus of the long chain of ISE-A; (6) ISE-F, with a PRR agonist linked to the N-terminus or C-terminus of the short chain of ISE-A; (7) ISE-G, with a PRR agonist linked to the C-terminus of the long chain of ISE-A; (8) ISE-H, with an IL-2 mutant or an IFNα mutant linked to the N-terminus of the long chain of ISE-A; (9) ISE-I, with an IL-2 mutant or an IFNα mutant linked to the N-terminus of the long chain of ISE-B; (10) ISE-J, with an IL-2 mutant or an IFNα mutant linked to the N-terminus of the long chain of ISE-D. SIRPα protein can block the "Don't eat me" signal of macrophages; Anti-CD16a antibody can specifically bind to and activate NK cells; cytokines such as IL-15 can promote NK cell proliferation, so that the ability of NK cells to kill tumors is not limited by the number of cells; PRR agonists can activate the interferon production mechanism; IL-2 mutants or IFNα mutants, by introducing mutations, improve the binding ability with NK cell and CD8+ T cell receptors.

[0012] This invention also discloses a complete immune antibody (CIA) comprising an ISE and an acquired immune antibody. The acquired immune antibody includes antibodies that regulate T cell immunity, such as anti-PD-1 and anti-PD-L1 antibodies; antibodies against tumor antigens, such as anti-HER2 and anti-EGFR antibodies; and antibodies that regulate B cell function, such as anti-B cell surface receptor antibodies, such as anti-CD40 or anti-CD40L antibodies. T cell regulators can relieve immunosuppression of T cells by antagonizing immune checkpoints; anti-tumor antigen antibodies can target tumor cells and inhibit their growth; and antibodies against B cell surface antigens can increase the ability of B cells to present tumor antigens. At least one ISE and one acquired immune antibody constitute a series of complete immune antibodies (CIAs), and the possible compositions of CIA antibodies are shown in the table below:

[0013] Table 1. Complete Immune Antibody Combination Table

[0014] The key technical problems that this invention aims to solve are as follows:

[0015] (1) By innovative and reasonable ISE structural design, NK cells are recruited while the "Don't eat me" signal of macrophages is blocked, so as to exert the maximum effect of innate immunity.

[0016] (2) The immune cytokines and their receptors, such as IL-15 or IL-15Rα fragments, introduced into the structure can stimulate NK cell proliferation and thus enhance the killing function against tumor cells.

[0017] (3) When PRR is activated, it produces innate immune cell factors such as type I interferon, which can directly inhibit the reproduction of the virus; and by activating other innate immune cells and acquired immune cells, it can regulate immune function.

[0018] (4) There are two subtypes of human CD16 protein, CD16a and CD16b. CD16a is mainly expressed on NK cells, but also on monocytes and macrophages, and is an activating receptor. CD16b is expressed on neutrophils. The two subtypes are highly homologous. This invention ensures anti-CD16a specificity and does not bind to CD16b on the surface of neutrophils, thus avoiding side effects caused by non-specific binding. [8] .

[0019] (5) When used alone, ISE has the effect of killing tumor cells. Due to the non-specificity of ISE, it can be widely used in different tumors.

[0020] (6) ISE becomes a complete immune antibody (CIA) by linking acquired immune antibodies, which can combine innate immunity and acquired immunity to fully enhance the killing effect on tumor cells.

[0021] (7) CIA can replace the target antibody of the acquired immune system to achieve specific targeted killing of different tumors.

[0022] (8) By innovating and rationally designing the ISE and CIA structures, the toxic side effects of cytokines have been reduced.

[0023] To achieve the above objectives, the technical solution of the present invention is as follows.

[0024] One object of the present invention is to provide an innate immune superconnector and a complete immune antibody, wherein the innate immune superconnector comprises two or more of the following components: (a) an anti-CD16a antibody, (b) a SIRPαD1 protein, (c) an immune cytokine, and (d) a pattern recognition receptor agonist. The complete immune antibody is based on the structure of an ISE (immune-associated antibody) with the addition of an acquired immune antibody or a tumor antigen antibody.

[0025] In one particular implementation, a conventional antibody is used as the basic structure, the variable region of the antibody is removed, or a human-free target antibody is used. An anti-CD16a single-chain antibody or SIRPαD1 protein is linked to the N-terminus or C-terminus of the antibody heavy chain constant region via a linker, and a SIRPαD1 protein or anti-CD16a single-chain antibody is linked to the N-terminus or C-terminus of the antibody light chain constant region via a linker.

[0026] In one specific implementation, a conventional antibody is used as the basic structure, with the variable region of the antibody removed, or a non-targeting antibody in vivo is used. Human immune cytokines and / or human immune cytokine receptors and anti-CD16a VHH are linked to the N-terminus or C-terminus of the antibody heavy chain constant region via linkers, and SIRPαD1 protein is linked to the N-terminus or C-terminus of the antibody light chain constant region via linkers. Alternatively, human immune cytokines and / or human immune cytokine receptors and anti-CD16a VHH are linked to the N-terminus or C-terminus of the antibody light chain constant region via linkers, and SIRPαD1 protein is linked to the N-terminus or C-terminus of the antibody heavy chain constant region via linkers.

[0027] In one specific implementation, a conventional antibody is used as the basic structure, with the variable region of the antibody removed, or a human-free antibody is used. Human immune cytokines such as IL-15 and anti-CD16a single-chain antibodies are linked to the N-terminus or C-terminus of the antibody heavy chain constant region via linkers, and SIRPαD1 protein is linked to the N-terminus or C-terminus of the antibody light chain constant region via linkers. Alternatively, human immune cytokines such as IL-15 and anti-CD16a single-chain antibodies are linked to the N-terminus or C-terminus of the antibody light chain constant region via linkers, and SIRPαD1 protein is linked to the N-terminus or C-terminus of the antibody heavy chain constant region via linkers.

[0028] In a further implementation, the receptor agonist is recognized at the N-terminus or C-terminus of the ISE via a linker connection pattern.

[0029] In a further embodiment, human IL-2 or IFNα or its mutants are linked to the N-terminus or C-terminus of the ISE via a linker.

[0030] In one particular implementation, the immune cytokines in the ISE include, but are not limited to, IL-15, IL-18, IL-2, IL-7, IL-10, IL-12, etc.

[0031] In one specific implementation, an ISE is used as the basic structure, and a variable region of acquired immune antibody or tumor-associated antigen antibody is added to the N-terminus of the ISE to form a complete immune antibody.

[0032] In one specific implementation, an acquired immune antibody or tumor-associated antigen antibody is used as the basic backbone, with SIRPαD1 protein linked to the N-terminus / C-terminus of the antibody light chain via a linker, and anti-CD16a antibody or anti-CD16a antibody, as well as human immune cytokines and / or human immune cytokine receptors linked to the N-terminus / C-terminus of the antibody heavy chain via a linker.

[0033] In one particular implementation, the target portion of the complete immune antibody may be a tumor-associated antigen antibody, including but not limited to antibodies against PD-L1, Her2, VEGF, EGFR, CD38, Claudin18.2, Claudin6, CD19, MSLN, Trop-2, CD20, Nectin-4, c-Met, MUC16, MUC17, 5T4, DLL3, or CD30.

[0034] In one particular implementation, the target portion of the complete immune antibody may be an acquired immune antibody, including but not limited to antibodies against PD-1, CTLA-4, B7-H4, B7-H3, 4-1BB, LAG-3, TIGIT, TIM-3, LILRB2, CD24, CD40, or CD40L.

[0035] In one embodiment, the basic backbone is an anti-hen egg lysozyme HEL antibody. In a specific embodiment, the amino acid sequence of the long chain of the monoclonal antibody HELAB01 is SEQ ID NO:20, and the amino acid sequence of the short chain is SEQ ID NO:21. The amino acid sequence of the long chain of the bispecific antibody HELAB02, used for mechanism validation, is SEQ ID NO:22, and the amino acid sequence of the short chain is SEQ ID NO:23. The amino acid sequence of the long chain of the bispecific antibody HELAB03, used for mechanism validation, is SEQ ID NO:24, and the amino acid sequence of the short chain is SEQ ID NO:25. The amino acid sequence of the long chain of the trispecific antibody HELAB04, used for mechanism validation, is SEQ ID NO:26, and the amino acid sequence of the short chain is SEQ ID NO:27. The amino acid sequence of the long chain of the trispecific antibody HELAB05, used for mechanism validation, is SEQ ID NO:28, and the amino acid sequence of the short chain is SEQ ID NO:29.

[0036] In some embodiments, the antibody’s Fc segment is introduced with L234A and L235A mutations, or further with M252Y, S254T and / or T256E mutations.

[0037] Another object of the present invention is to provide a method for preparing the above-mentioned innate immune superconnector and complete immune antibody, comprising the following steps:

[0038] (1) The structure of this invention is shown in Figures 1-3, and is a symmetrical structure. The two strands of ISE or CIA were amplified by PCR technology respectively; M252Y / S254T / T256E mutations were introduced into the Fc segment of the antibody to prolong the half-life of the protein;

[0039] (2) Using conventional molecular biology methods, clone the DNA fragment of one strand obtained in step (1) into an appropriate position in a pcDNA series vector or other vector for mammalian cell expression system, and clone the DNA fragment of the other strand obtained in step (1) into another pcDNA series vector or other vector for mammalian cell expression system, including but not limited to the appropriate position in the vector of the expression system, which includes a fusion DNA sequence linked with suitable transcription and translation regulatory sequences.

[0040] (3) The recombinant vector obtained in step (2) is transfected into mammalian cells to express the fusion protein. After one-step affinity chromatography, purified ISE or CIA is obtained. The transfection method can be chemical transfection or electroporation transfection. The mammalian cells can be HEK293 cells or CHO (Chinese Hamster Ovary) cells or derived cells of the above cells or other expression systems (such as E. coli and yeast).

[0041] Invention Details

[0042] I. Innate Immune Superconnector

[0043] The innate immune superconnector described herein encompasses various components of the innate immune system, including but not limited to pattern recognition receptor (PRR) agonists, macrophage activators, NK cell linkers and activators, and immune cytokines. Various PRR agonists, macrophage activators, NK cell linkers and activators, and immune cytokines known in the art can be employed. The pattern recognition receptors include Toll-like receptors (TLRs), nucleotide oligomer domain (NOD)-like receptors (NLRs), retinoic acid-induced gene-I (RIG-I)-like receptors (RLRs), C-type lectin receptors (CLRs), and melanoma deficiency factor 2 (AIM2)-like receptors (ALRs). The PRR agonists used herein include, but are not limited to, Toll-like receptor (TLR) agonists, NOD-like receptor (NLR) agonists, RIG-I-like receptor (RLR) agonists, C-type lectin receptor (CLR) agonists, and melanoma deficiency factor 2 (AIM2)-like receptor (ALR) agonists. The macrophage activators used in this study include, but are not limited to, SIRPαD1 protein, CD40 antibody, and CD24 antibody. The NK cell binding and activators used in this study include, but are not limited to, CD16a antibody. The immune cytokines used in this study include, but are not limited to, IL-15, IL-18, IL-2, IL-7, IL-10, and IL-12.

[0044] The innate immune superconnector described in this article uses a suitable basic framework to connect various components of the innate immune system. This basic framework may include conventional antibodies with their variable regions removed, such as IgG, IgA, IgM, IgE, and IgD with their variable regions removed. It may also include non-target antibodies found in the human body, such as anti-egg white lysozyme antibodies, and antibodies against pathogenic microorganisms such as antiviral and bacterial antibodies.

[0045] In some embodiments, the innate immune superconnector of this application comprises one or more of macrophage activators, NK cell linkers and activators, PRR agonists, and immune cytokines to activate macrophages, natural killer cells, and / or dendritic cells. In some embodiments, the innate immune superconnector of this application comprises macrophage activators, NK cell linkers and activators, wherein the macrophage activator is preferably SIRPαD1 protein, and the NK cell linker and activator is preferably an anti-CD16a antibody. The anti-CD16a antibody may be an anti-CD16a single-chain antibody or an anti-CD16a nanobody.

[0046] In some embodiments, the innate immune superconnector of this application comprises two or more of the following components: (a) anti-CD16a antibody, (b) SIRPαD1 protein, (c) immune cytokines, and (d) pattern recognition receptor agonists to activate pDCs, macrophages, and / or natural killer cells.

[0047] In some embodiments, the innate immune superlinker of this application uses a conventional antibody with its variable region removed or a human-derived non-targeting antibody as its basic backbone, linking a macrophage activator, an NK cell linker, and an activator. In a preferred embodiment, the innate immune superlinker of this application uses a conventional antibody with its variable region removed or a human-derived non-targeting antibody as its basic backbone, linking an anti-CD16a antibody and a SIRPαD1 protein, for example, see the ISE-A configurations of A1 and A2 in Figure 1. In some embodiments, a conventional antibody is used as the basic structure, with the variable region of the antibody removed, or a human-derived non-targeting antibody is used as the basic structure. An anti-CD16a antibody (e.g., an anti-CD16a single-chain antibody or an anti-CD16a nanobody) or a SIRPαD1 protein is linked to the N-terminus or C-terminus of the antibody heavy chain constant region via a linker, and a SIRPαD1 protein or an anti-CD16a antibody (e.g., an anti-CD16a single-chain antibody or an anti-CD16a nanobody) is linked to the N-terminus or C-terminus of the antibody light chain constant region via a linker. In some embodiments, the innate immune superconnector of this application uses a conventional antibody with its variable region removed or a human-derived non-targeting antibody as its basic framework, connecting macrophage activators, NK cell linkers and activators, human immune cytokines and / or human immune cytokine receptors. In a preferred embodiment, the innate immune superconnector of this application uses a conventional antibody with its variable region removed or a human-derived non-targeting antibody as its basic framework, connecting anti-CD16a antibody, SIRPαD1 protein, human immune cytokines and / or human immune cytokine receptors, for example, see the ISE-B, ISE-C, and ISE-D configurations of B1 / B2, C1 / C2, and D1 / D2 in Figure 1. In some embodiments, a conventional antibody is used as the basic structure, with the variable region of the antibody removed, or a human-target-free antibody is used as the basic structure. Human immune cytokines and / or human immune cytokine receptors, anti-CD16a antibodies (e.g., anti-CD16a single-chain antibodies or anti-CD16a VHH), and SIRPαD1 protein are linked via linkers at the N-terminus and / or C-terminus of the antibody heavy chain constant region. In other embodiments, a conventional antibody is used as the basic structure, with the variable region of the antibody removed, or a human-target-free antibody is used as the basic structure. Human immune cytokines and / or human immune cytokine receptors, anti-CD16a antibodies (e.g., anti-CD16a single-chain antibodies or anti-CD16a VHH), and SIRPαD1 protein are linked via linkers at the N-terminus and / or C-terminus of the antibody light chain constant region.In some embodiments, using a conventional antibody as the basic structure, the variable region of the antibody is removed, or using a non-targeting antibody in the human body as the basic structure, (1) human immune cytokines and / or human immune cytokine receptors and anti-CD16a antibodies (e.g., anti-CD16a single-chain antibodies or anti-CD16a VHH) are linked to the N-terminus and / or C-terminus of the antibody heavy chain constant region via linkers, and (2) SIRPαD1 protein is linked to the N-terminus or C-terminus of the antibody light chain constant region via linkers. In the above embodiments, human immune cytokines include, but are not limited to, IL-15, IL-18, IL-2, IL-7, IL-10, and IL-12. In the above embodiments, human immune cytokine receptors include, but are not limited to, IL-15 receptor, IL-18 receptor, IL-2 receptor, IL-7 receptor, IL-10 receptor, and IL-12 receptor.

[0048] In one specific implementation, a conventional antibody is used as the basic structure, with the variable region of the antibody removed or a human-targeted antibody used. Human immune cytokines (e.g., human IL-15) and anti-CD16a antibodies (e.g., anti-CD16a single-chain antibodies or anti-CD16a VHH) are linked at the C-terminus of the antibody heavy chain constant region via linkers, and the SIRPαD1 protein is linked at the N-terminus of the antibody heavy chain constant region via linkers, as shown in Figure 1, for example, B1 and D1. In another specific implementation, a conventional antibody is used as the basic structure, with the variable region of the antibody removed or a human-targeted antibody used. Human immune cytokines (e.g., human IL-15), human immune cytokine receptors (e.g., human IL-15 receptors), and anti-CD16a antibodies (e.g., anti-CD16a single-chain antibodies or anti-CD16a VHH) are linked at the C-terminus of the antibody heavy chain constant region via linkers, and the SIRPαD1 protein is linked at the N-terminus of the antibody heavy chain constant region via linkers, as shown in Figure 1, C1.

[0049] In one specific implementation, a conventional antibody is used as the basic structure, with the variable region of the antibody removed or a human-targeted antibody used. Human immune cytokines (e.g., human IL-15) and anti-CD16a antibodies (e.g., anti-CD16a single-chain antibodies or anti-CD16a VHH) are linked to the C-terminus of the antibody heavy chain constant region via linkers. The SIRPαD1 protein is linked to the C-terminus of the antibody light chain constant region via linkers, as shown in Figure 1, for example, B2 and D2. In another specific implementation, a conventional antibody is used as the basic structure, with the variable region of the antibody removed or a human-targeted antibody used. Human immune cytokines (e.g., human IL-15), human immune cytokine receptors (e.g., human IL-15 receptors), and anti-CD16a antibodies (e.g., anti-CD16a single-chain antibodies or anti-CD16a VHH) are linked to the C-terminus of the antibody heavy chain constant region via linkers. The SIRPαD1 protein is linked to the C-terminus of the antibody light chain constant region via linkers, as shown in Figure 1, C2. In any of the above embodiments, the macrophage activator, NK cell linker and activator, human immune cytokines, and human immune cytokine receptors can be linked in any order. In any of the above embodiments, the immune cytokines, immune cytokine receptors, anti-CD16a antibody, and SIRPαD1 protein can be linked in any order.

[0050] In some embodiments, the innate immune superconnector of this application uses a conventional antibody with its variable region removed or a human-derived non-targeting antibody as its basic backbone, linking a macrophage activator, an NK cell linker and activator, and a PRR agonist. In a preferred embodiment, the innate immune superconnector of this application uses a conventional antibody with its variable region removed or a human-derived non-targeting antibody as its basic backbone, linking an anti-CD16a antibody (e.g., an anti-CD16a single-chain antibody or an anti-CD16a VHH), a SIRPαD1 protein, and a PRR agonist, for example, see the ISE-E, ISE-F, and ISE-G configurations of E1 / E2, F1 / F2, and G1 / G2 in Figure 1. In some implementations, a conventional antibody is used as the basic structure, with the variable region of the antibody removed, or a human non-targeting antibody is used as the basic structure. An anti-CD16a antibody (e.g., an anti-CD16a single-chain antibody or an anti-CD16a VHH), a PRR agonist, and / or a SIRPαD1 protein are linked to the N-terminus or C-terminus of the antibody heavy chain constant region via a linker. A SIRPαD1 protein and / or an anti-CD16a antibody (e.g., an anti-CD16a single-chain antibody or an anti-CD16a VHH), a PRR agonist, are linked to the N-terminus or C-terminus of the antibody light chain constant region via a linker.

[0051] In one specific embodiment, a conventional antibody is used as the basic structure, with the variable region of the antibody removed or a human-free target antibody used. An anti-CD16a antibody (e.g., an anti-CD16a single-chain antibody or anti-CD16a VHH) and / or a PRR agonist are linked via linkers at the N-terminus and / or C-terminus of the antibody heavy chain constant region, and the SIRPαD1 protein is linked via linkers at the N-terminus and / or C-terminus of the antibody light chain constant region, as shown in Figure 1, e.g., E1 / E2 and G1 / G2. In another specific embodiment, a conventional antibody is used as the basic structure, with the variable region of the antibody removed or a human-free target antibody used. An anti-CD16a antibody (e.g., an anti-CD16a single-chain antibody or anti-CD16a VHH) is linked via linkers at the N-terminus and / or C-terminus of the antibody heavy chain constant region, and the SIRPαD1 protein and / or a PRR agonist are linked via linkers at the N-terminus and / or C-terminus of the antibody light chain constant region, as shown in Figure 1, e.g., F1 and F2. In any of the above embodiments, the macrophage activator, NK cell linker and activator, and PRR agonist can be linked in any order. In any of the above embodiments, the anti-CD16a antibody, PRR agonist, and SIRPαD1 protein can be linked in any order.

[0052] Based on the above-mentioned ISE-A, ISE-B, ISE-C and ISE-D configurations, the innate immune superconnector described in this paper can further connect IL-2 or IFNα or their mutants.

[0053] In some implementations, IL-2 or IFNα or a mutant thereof is linked to the ISE-A configuration, for example, see the ISE-H configuration of H1 / H2 in Figure 1. For example, based on the ISE-A configuration, an IL-2 mutant or an IFNα mutant is linked to the N-terminus of the antibody heavy chain constant region, wherein the SIRPαD1 protein can be linked to the N-terminus and / or C-terminus of the antibody light chain constant region via a linker.

[0054] In some implementations, IL-2 or IFNα or a mutant thereof is linked to the ISE-B conformation, for example, see the ISE-I conformation of I1 / I2 in Figure 1. For example, based on the ISE-B conformation, an IL-2 mutant or an IFNα mutant is linked to the N-terminus of the antibody heavy chain constant region, wherein the SIRPαD1 protein can be linked to the N-terminus and / or C-terminus of the antibody light chain constant region via a linker.

[0055] In some embodiments, IL-2 or IFNα or its mutants are linked to the ISE-D conformation, for example, see the ISE-J conformation of J1 / J2 in Figure 1. For example, based on the ISE-D conformation, an IL-2 mutant or an IFNα mutant is linked to the N-terminus of the antibody heavy chain constant region, wherein the SIRPαD1 protein can be linked to the N-terminus and / or C-terminus of the antibody light chain constant region via a linker; further, the N-terminus and / or C-terminus of the antibody heavy chain constant region can also be linked to a cytokine receptor. In some embodiments, the variable region of the heavy chain of the anti-CD16a single-chain antibody includes: CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; the variable region of the light chain includes: CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6.

[0056] In a further embodiment, the heavy chain variable region of the anti-CD16a single-chain antibody contains the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:8.

[0057] In a further embodiment, the anti-CD16a single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:9.

[0058] In one embodiment, the anti-CD16a nanobody comprises: CDR1 as shown in SEQ ID NO:10.

[0059] CDR2 shown in SEQ ID NO:11 and CDR3 shown in SEQ ID NO:12.

[0060] In a further embodiment, the anti-CD16a nanobody comprises the amino acid sequence shown in SEQ ID NO:13.

[0061] In one embodiment, the SIRPαD1 protein comprises the amino acid sequence shown in SEQ ID NO:14.

[0062] In one embodiment, the human IL-15 protein fragment comprises the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:16.

[0063] In one embodiment, the human IL-15Rα protein fragment comprises the amino acid sequence shown in SEQ ID NO:17.

[0064] This invention prepared innate immune connectors with different structures, as shown in Figures 1A-J and 3, and named ISE-A, ISE-B, ISE-C, ISE-D, ISE-E, ISE-F, ISE-G, ISE-H, ISE-I and ISE-J, HELAB01, HELAB02, HELAB03, HELAB04 and HELAB05, respectively.

[0065] In some embodiments, the structure of the long chain of the innate immune connector ISE-A is: SIRPαD1 protein-linker-antibody heavy chain constant region-linker-anti-CD16a single-chain antibody, and the structure of the short chain is: antibody kappa chain constant region (see, for example, A1 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain of ISE-A1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:30, and the amino acid sequence of the short chain of ISE-A1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:31. In a more preferred embodiment, the amino acid sequence of the long chain of ISE-A1 contains the sequence shown in SEQ ID NO:30, and the amino acid sequence of the short chain of ISE-A1 contains the sequence shown in SEQ ID NO:31. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-A1 is SEQ ID NO:30, and the amino acid sequence of the short chain is SEQ ID NO:31.

[0066] In some embodiments, the structure of the long chain of the innate immune connector ISE-A is: antibody heavy chain constant region - linker - anti-CD16a single-chain antibody, and the structure of the short chain is: antibody kappa chain constant region - linker - SIRPαD1 protein (see, for example, A2 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain ISE-A2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:32, and the amino acid sequence of the short chain ISE-A2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:33. In a more preferred embodiment, the amino acid sequence of the long chain ISE-A2 contains the sequence shown in SEQ ID NO:32, and the amino acid sequence of the short chain ISE-A2 contains the sequence shown in SEQ ID NO:33. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-A2 is SEQ ID NO:32, and the amino acid sequence of the short chain is SEQ ID NO:33.

[0067] In some embodiments, the structure of the long chain of the innate immune connector ISE-B is: SIRPαD1 protein-linker-antibody heavy chain constant region-linker-human IL-15-linker-anti-CD16a nanobody, and the structure of the short chain is: antibody kappa chain constant region (see, for example, B1 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain of ISE-B1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:34, and the amino acid sequence of the short chain of ISE-B1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:35. In a more preferred embodiment, the amino acid sequence of the long chain of ISE-B1 contains the sequence shown in SEQ ID NO:34, and the amino acid sequence of the short chain of ISE-B1 contains the sequence shown in SEQ ID NO:35. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-B1 is SEQ ID NO:34, and the amino acid sequence of the short chain is SEQ ID NO:35.

[0068] In some embodiments, the structure of the long chain of the innate immune connector ISE-B is: antibody heavy chain constant region - linker - human IL-15 - linker - anti-CD16a nanobody, and the structure of the short chain is: antibody kappa chain constant region - linker - SIRPαD1 protein (see, for example, B2 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain ISE-B2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:36, and the amino acid sequence of the short chain ISE-B2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:37. In a more preferred embodiment, the amino acid sequence of the long chain ISE-B2 contains the sequence shown in SEQ ID NO:36, and the amino acid sequence of the short chain ISE-B2 contains the sequence shown in SEQ ID NO:37. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-B2 is SEQ ID NO:36, and the amino acid sequence of the short chain is SEQ ID NO:37.

[0069] In some embodiments, the structure of the long chain of the innate immune linker ISE-C is: SIRPαD1 protein-linker-antibody heavy chain constant region-linker-human IL-15Rα-linker-human IL-15-linker-anti-CD16a nanobody, and the structure of the short chain is: antibody kappa chain constant region (see, for example, C1 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain of ISE-C1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:38, and the amino acid sequence of the short chain of ISE-C1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:39. In a more preferred embodiment, the amino acid sequence of the long chain of ISE-C1 contains the sequence shown in SEQ ID NO:38, and the amino acid sequence of the short chain of ISE-C1 contains the sequence shown in SEQ ID NO:39. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-C1 is SEQ ID NO:38, and the amino acid sequence of the short chain is SEQ ID NO:39.

[0070] In some embodiments, the structure of the long chain of the innate immune linker ISE-C is: antibody heavy chain constant region - linker - human IL-15Rα - linker - human IL-15 - linker - anti-CD16a nanobody, and the structure of the short chain is: antibody kappa chain constant region - linker - SIRPαD1 protein (see, for example, C2 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain ISE-C2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:40, and the amino acid sequence of the short chain ISE-C2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:41. In a more preferred embodiment, the amino acid sequence of the long chain ISE-C2 contains the sequence shown in SEQ ID NO:40, and the amino acid sequence of the short chain ISE-C2 contains the sequence shown in SEQ ID NO:41. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-C2 is SEQ ID NO:40, and the amino acid sequence of the short chain is SEQ ID NO:41.

[0071] In some embodiments, the structure of the long chain of the innate immune connector ISE-D is: SIRPαD1 protein-linker-antibody heavy chain constant region-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the structure of the short chain is: antibody kappa chain constant region (see, for example, D1 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain of ISE-D1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:42, and the amino acid sequence of the short chain of ISE-D1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:43. In a more preferred embodiment, the amino acid sequence of the long chain of ISE-D1 contains the sequence shown in SEQ ID NO:42, and the amino acid sequence of the short chain of ISE-D1 contains the sequence shown in SEQ ID NO:43. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-D1 is SEQ ID NO:42, and the amino acid sequence of the short chain is SEQ ID NO:43.

[0072] In some embodiments, the structure of the long chain of the innate immune connector ISE-D is: antibody heavy chain constant region - linker - human IL-15 - linker - anti-CD16a single-chain antibody, and the structure of the short chain is: antibody kappa chain constant region - linker - SIRPαD1 protein (see, for example, D2 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain ISE-D2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:44, and the amino acid sequence of the short chain ISE-D2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:45. In a more preferred embodiment, the amino acid sequence of the long chain ISE-D2 contains the sequence shown in SEQ ID NO:44, and the amino acid sequence of the short chain ISE-D2 contains the sequence shown in SEQ ID NO:45. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-D2 is SEQ ID NO:44, and the amino acid sequence of the short chain is SEQ ID NO:45.

[0073] In some embodiments, the long chain of the innate immune connector ISE-E has the structure: CpG ODN-linker-antibody heavy chain constant region-linker-anti-CD16a single-chain antibody, and the short chain has the structure: antibody kappa chain constant region-linker-SIRPαD1 protein (see, for example, E1 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain of ISE-E1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:46, and the amino acid sequence of the short chain of ISE-E1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:47. In a more preferred embodiment, the amino acid sequence of the long chain of ISE-E1 contains the sequence shown in SEQ ID NO:46, and the amino acid sequence of the short chain of ISE-E1 contains the sequence shown in SEQ ID NO:47. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-E1 is SEQ ID NO:46, and the amino acid sequence of the short chain is SEQ ID NO:47.

[0074] In some embodiments, the long chain of the innate immune connector ISE-E has the structure: CpG ODN-linker-antibody heavy chain constant region-linker-anti-CD16a single-chain antibody, and the short chain has the structure: SIRPαD1 protein-linker-antibody kappa chain constant region (see, for example, E2 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain ISE-E2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:48, and the amino acid sequence of the short chain ISE-E2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:49. In a more preferred embodiment, the amino acid sequence of the long chain ISE-E2 contains the sequence shown in SEQ ID NO:48, and the amino acid sequence of the short chain ISE-E2 contains the sequence shown in SEQ ID NO:49. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-E2 is SEQ ID NO:48, and the amino acid sequence of the short chain is SEQ ID NO:49.

[0075] In some embodiments, the structure of the long chain of the innate immune connector ISE-F is: antibody heavy chain constant region - linker - anti-CD16a single-chain antibody, and the structure of the short chain is: CpG ODN - linker - antibody kappa chain constant region - linker - SIRPαD1 protein (see, for example, F1 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain of ISE-F1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:50, and the amino acid sequence of the short chain of ISE-F1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:51. In a more preferred embodiment, the amino acid sequence of the long chain of ISE-F1 contains the sequence shown in SEQ ID NO:50, and the amino acid sequence of the short chain of ISE-F1 contains the sequence shown in SEQ ID NO:51. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-F1 is SEQ ID NO:50, and the amino acid sequence of the short chain is SEQ ID NO:51.

[0076] In some embodiments, the structure of the long chain of the innate immune connector ISE-F is: antibody heavy chain constant region - linker - anti-CD16a single-chain antibody, and the structure of the short chain is: SIRPαD1 protein - linker - antibody kappa chain constant region - linker - CpG ODN (see, for example, F2 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain ISE-F2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:52, and the amino acid sequence of the short chain ISE-F2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:53. In a more preferred embodiment, the amino acid sequence of the long chain ISE-F2 contains the sequence shown in SEQ ID NO:52, and the amino acid sequence of the short chain ISE-F2 contains the sequence shown in SEQ ID NO:53. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-F2 is SEQ ID NO:52, and the amino acid sequence of the short chain is SEQ ID NO:53.

[0077] In some embodiments, the structure of the long chain of the innate immune connector ISE-G is: antibody heavy chain constant region - linker - anti-CD16a single-chain antibody - linker - CpG ODN, and the structure of the short chain is: antibody kappa chain constant region - linker - SIRPαD1 protein (see, for example, G1 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain of ISE-G1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:54, and the amino acid sequence of the short chain of ISE-G1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:55. In a more preferred embodiment, the amino acid sequence of the long chain of ISE-G1 contains the sequence shown in SEQ ID NO:54, and the amino acid sequence of the short chain of ISE-G1 contains the sequence shown in SEQ ID NO:55. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-G1 is SEQ ID NO:54, and the amino acid sequence of the short chain is SEQ ID NO:55.

[0078] In some embodiments, the structure of the long chain of the innate immune connector ISE-G is: antibody heavy chain constant region - linker - anti-CD16a single-chain antibody - linker - CpG ODN, and the structure of the short chain is: SIRPαD1 protein - linker - antibody kappa chain constant region (see, for example, G2 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain of ISE-G2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:56, and the amino acid sequence of the short chain of ISE-G2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:57. In a more preferred embodiment, the amino acid sequence of the long chain of ISE-G2 contains the sequence shown in SEQ ID NO:56, and the amino acid sequence of the short chain of ISE-G2 contains the sequence shown in SEQ ID NO:57. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-G2 is SEQ ID NO:56, and the amino acid sequence of the short chain is SEQ ID NO:57.

[0079] In some embodiments, the structure of the long chain of the innate immune connector ISE-H is: human IL-2 mutant - linker - antibody heavy chain constant region - linker - anti-CD16a single-chain antibody, and the structure of the short chain is: antibody kappa chain constant region - linker - SIRPαD1 protein (see, for example, H1 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain of ISE-H1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:58, and the amino acid sequence of the short chain of ISE-H1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:59. In a more preferred embodiment, the amino acid sequence of the long chain of ISE-H1 contains the sequence shown in SEQ ID NO:58, and the amino acid sequence of the short chain of ISE-H1 contains the sequence shown in SEQ ID NO:59. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-H1 is SEQ ID NO:58, and the amino acid sequence of the short chain is SEQ ID NO:59.

[0080] In some embodiments, the long chain of the innate immune connector ISE-H has the structure: human IL-2 mutant - linker - antibody heavy chain constant region - linker - anti-CD16a single-chain antibody, and the short chain has the structure: SIRPαD1 protein - linker - antibody kappa chain constant region (see, for example, H2 in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain ISE-H2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:58, and the amino acid sequence of the short chain ISE-H2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:60. In a more preferred embodiment, the amino acid sequence of the long chain ISE-H2 contains the sequence shown in SEQ ID NO:58, and the amino acid sequence of the short chain ISE-H2 contains the sequence shown in SEQ ID NO:60. In one specific embodiment, the amino acid sequence of the long chain of the innate immune connector ISE-H2 is SEQ ID NO:58, and the amino acid sequence of the short chain is SEQ ID NO:60.

[0081] In some embodiments, the long chain of the innate immune linker ISE-I has the structure of: human IL-2 mutant - linker - antibody heavy chain constant region - linker - human IL-15 - linker - anti-CD16a nanobody, and the short chain has the structure of: antibody kappa chain constant region - linker - SIRPαD1 protein (see, for example, I1 in Figure 1). In a preferred embodiment, the long chain amino acid sequence of ISE-I1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:61, and the short chain amino acid sequence of ISE-I1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:62. In a more preferred embodiment, the long chain amino acid sequence of ISE-I1 contains the sequence shown in SEQ ID NO:61, and the short chain amino acid sequence of ISE-I1 contains the sequence shown in SEQ ID NO:62. In one specific implementation, the amino acid sequence of the long chain of the innate immune connector ISE-I1 is SEQ ID NO:61, and the amino acid sequence of the short chain is SEQ ID NO:62.

[0082] In some embodiments, the long chain of the innate immune linker ISE-I has the structure of: human IL-2 mutant - linker - antibody heavy chain constant region - linker - human IL-15 - linker - anti-CD16a nanobody, and the short chain has the structure of: SIRPαD1 protein - linker - antibody kappa chain constant region (see, for example, I2 in Figure 1). In a preferred embodiment, the long chain amino acid sequence of ISE-I2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:63, and the short chain amino acid sequence of ISE-I2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:64. In a more preferred embodiment, the long chain amino acid sequence of ISE-I2 contains the sequence shown in SEQ ID NO:63, and the short chain amino acid sequence of ISE-I2 contains the sequence shown in SEQ ID NO:64. In one specific implementation, the amino acid sequence of the long chain of the innate immune connector ISE-I2 is SEQ ID NO:63, and the amino acid sequence of the short chain is SEQ ID NO:64.

[0083] In some embodiments, the long chain of the innate immune linker ISE-J has the following structure: human IL-2 mutant - linker - antibody heavy chain constant region - linker - human IL-15 - linker - anti-CD16a single-chain antibody, and the short chain has the following structure: antibody kappa chain constant region - linker - SIRPαD1 protein (see, for example, J1 in Figure 1). In a preferred embodiment, the long chain amino acid sequence of ISE-J1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:65, and the short chain amino acid sequence of ISE-J1 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:66. In a more preferred embodiment, the long chain amino acid sequence of ISE-J1 contains the sequence shown in SEQ ID NO:65, and the short chain amino acid sequence of ISE-J1 contains the sequence shown in SEQ ID NO:66. In one specific implementation, the amino acid sequence of the long chain of the innate immune connector ISE-J1 is SEQ ID NO:65, and the amino acid sequence of the short chain is SEQ ID NO:66.

[0084] In some embodiments, the long chain of the innate immune linker ISE-J has the structure: human IL-2 mutant - linker - antibody heavy chain constant region - linker - human IL-15 - linker - anti-CD16a single-chain antibody, and the short chain has the structure: SIRPαD1 protein - linker - antibody kappa chain constant region (see, for example, J2 in Figure 1). In a preferred embodiment, the long chain amino acid sequence of ISE-J2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:65, and the short chain amino acid sequence of ISE-J2 contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:67. In a more preferred embodiment, the long chain amino acid sequence of ISE-J2 contains the sequence shown in SEQ ID NO:65, and the short chain amino acid sequence of ISE-J2 contains the sequence shown in SEQ ID NO:67. In one specific implementation, the amino acid sequence of the long chain of the innate immune connector ISE-J2 is SEQ ID NO:65, and the amino acid sequence of the short chain is SEQ ID NO:67.

[0085] In some embodiments, the long chain of the ISE-Negative Control has the structure of an antibody heavy chain constant region, and the short chain has the structure of an antibody kappa chain constant region (see, for example, the ISE-Negative Control in Figure 1). In a preferred embodiment, the amino acid sequence of the long chain of the ISE-Negative Control contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:116, and the amino acid sequence of the short chain of the ISE-Negative Control contains a sequence having at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO:31. In a more preferred embodiment, the amino acid sequence of the long chain of the ISE-Negative Control contains the sequence shown in SEQ ID NO:116, and the amino acid sequence of the short chain of the ISE-Negative Control contains the sequence shown in SEQ ID NO:31. In one specific embodiment, the amino acid sequence of the long chain of the ISE-Negative Control is SEQ ID NO:116, and the amino acid sequence of the short chain is SEQ ID NO:31.

[0086] II. HEL antibody

[0087] To demonstrate the feasibility of the ISE concept, this application uses the ISE as a backbone and adds the variable region of an in vivo non-targeting antibody, anti-hen egg lysozyme HEL antibody, to prepare five antibodies with the main ISE structures: HELAB01, HELAB02, HELAB03, HELAB04, and HELAB05. The aim is to demonstrate the superposition function of different parts of the ISE and the function of cytokines.

[0088] The monoclonal antibody HELAB01 consists of an anti-HEL antibody heavy chain and an anti-HEL antibody light chain. Preferably, the amino acid sequence of the long chain (i.e., the anti-HEL antibody heavy chain) of HELAB01 is SEQ ID NO:20, and the amino acid sequence of the short chain (i.e., the anti-HEL antibody light chain) is SEQ ID NO:21.

[0089] The bispecific antibody HELAB02 uses an anti-HEL antibody as its basic structure, with the SIRPαD1 protein linked to either the N-terminus or C-terminus of the antibody heavy chain or the N-terminus or C-terminus of the antibody light chain via a linker. In one embodiment, the long chain structure of the bispecific antibody HELAB02 is: an anti-HEL antibody heavy chain, and the short chain structure is: an anti-HEL antibody light chain - linker - SIRPαD1 protein. In a specific embodiment, the amino acid sequence of the long chain of the bispecific antibody HELAB02 used for mechanism verification is SEQ ID NO:22, and the amino acid sequence of the short chain is SEQ ID NO:23. In an alternative embodiment, the bispecific antibody HELAB02 replaces the short chain structure with: SIRPαD1 protein - linker - anti-HEL antibody light chain; preferably, the amino acid sequence of the replacement short chain is SEQ ID NO:85.

[0090] The bispecific antibody HELAB03 uses an anti-HEL antibody as its basic structure, with an anti-CD16a single-chain antibody linked to the N- or C-terminus of the antibody heavy chain or the N- or C-terminus of the antibody light chain via a linker. In one embodiment, the long chain structure of the bispecific antibody HELAB03 is: anti-HEL antibody heavy chain - linker - anti-CD16a single-chain antibody, and the short chain structure is: anti-HEL antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the bispecific antibody HELAB03 used for mechanism verification is SEQ ID NO:24, and the amino acid sequence of the short chain is SEQ ID NO:25.

[0091] The trispecific antibody HELAB04 uses an anti-HEL antibody as its basic structure, with an anti-CD16a single-chain antibody linked to the N-terminus or C-terminus of the antibody heavy chain and a SIRPαD1 protein linked to the N-terminus or C-terminus of the antibody light chain. In one embodiment, the long chain structure of the trispecific antibody HELAB04 is: anti-HEL antibody heavy chain - linker - anti-CD16a single-chain antibody, and the short chain structure is: anti-HEL antibody light chain - linker - SIRPαD1 protein. In a specific embodiment, the amino acid sequence of the long chain of the trispecific antibody HELAB04 used for mechanism verification is SEQ ID NO:26, and the amino acid sequence of the short chain is SEQ ID NO:27. In an alternative embodiment, the trispecific antibody HELAB04 replaces the short chain structure with SIRPαD1 protein - linker - anti-HEL antibody light chain; preferably, the amino acid sequence of the replacement short chain is SEQ ID NO:85.

[0092] The trispecific antibody HELAB05 uses an anti-HEL antibody as its basic structure, with IL-15 and anti-CD16a nanobodies linked to the N- or C-terminus of the antibody heavy chain, and SIRPαD1 protein linked to the N- or C-terminus of the antibody light chain. In one embodiment, the long chain structure of the trispecific antibody HELAB05 is: anti-HEL antibody heavy chain - linker - human IL-15 - anti-CD16a nanobodies, and the short chain structure is: anti-HEL antibody light chain - linker - SIRPαD1 protein. In a specific embodiment, the amino acid sequence of the long chain of the trispecific antibody HELAB05 used for mechanism verification is SEQ ID NO:28, and the amino acid sequence of the short chain is SEQ ID NO:29. In an alternative embodiment, the bispecific antibody HELAB05 replaces the short chain structure with SIRPαD1 protein - linker - anti-HEL antibody light chain; preferably, the amino acid sequence of the replacement short chain is SEQ ID NO:85.

[0093] III. Complete Immune Antibodies

[0094] Complete immune antibodies (CIAs) can be obtained by linking the variable regions of acquired immune antibodies or tumor-associated antigen-antibody antibodies to the innate immune superconnector (ISE) described herein. As shown in Table 1, in some embodiments, the variable regions of acquired immune antibodies or tumor-associated antigen-antibody antibodies are linked to ISE-A to obtain complete immune antibodies ISE-AI or ISE-A-II. For example, see the CIA configuration of A1 / A2 in Figure 2. In some embodiments, the variable regions of acquired immune antibodies or tumor-associated antigen-antibody antibodies are linked to ISE-B to obtain complete immune antibodies ISE-BI or ISE-B-II. For example, see the CIA configuration of B1 / B2 in Figure 2. In some embodiments, the variable regions of acquired immune antibodies or tumor-associated antigen-antibody antibodies are linked to ISE-C to obtain complete immune antibodies ISE-CI or ISE-C-II. For example, see the CIA configuration of C1 / C2 in Figure 2. Similarly, by linking the variable region of acquired immune antibodies or the variable region of tumor-associated antigen antibodies to ISE-D, complete immune antibodies ISE-DI or ISE-D-II are obtained. Similarly, by linking the variable region of acquired immune antibodies or the variable region of tumor-associated antigen antibodies to ISE-E, complete immune antibodies ISE-EI or ISE-E-II are obtained. Similarly, by linking the variable region of acquired immune antibodies or the variable region of tumor-associated antigen antibodies to ISE-F, complete immune antibodies ISE-FI or ISE-F-II are obtained. Similarly, by linking the variable region of acquired immune antibodies or the variable region of tumor-associated antigen antibodies to ISE-G, complete immune antibodies ISE-GI or ISE-G-II ​​are obtained. Similarly, by linking the variable region of acquired immune antibodies or the variable region of tumor-associated antigen antibodies to ISE-H, complete immune antibodies ISE-HI or ISE-H-II are obtained. Finally, by linking the variable region of acquired immune antibodies or the variable region of tumor-associated antigen antibodies to ISE-I, complete immune antibodies ISE-II or ISE-I-II are obtained. By linking the variable region of an acquired immune antibody or a variable region of a tumor-associated antigen antibody to ISE-J, complete immune antibodies ISE-JI or ISE-J-II can be obtained. In the above embodiments, the acquired immune antibodies include, but are not limited to, antibodies such as PD-1, CTLA-4, B7-H4, B7-H3, 4-1BB, LAG-3, TIGIT, TIM-3, LILRB2, CD24, CD40, or CD40L. In the above embodiments, the tumor-associated antigen antibodies include, but are not limited to, antibodies such as PD-L1, Her2, VEGF, EGFR, CD38, 5T4, DLL3, CD30, Claudin18.2, Claudin6, CD19, MSLN, Trop-2, CD20, Nectin-4, BCMA, MUC17, MUC16, or c-Met.Those skilled in the art can select specific acquired immune antibodies or tumor-associated antigen antibodies as needed to prepare complete immune antibodies that are specific to specific targets, thereby achieving specific targeted killing of different tumors.

[0095] In another aspect, the present invention uses PD-L1, PD-1, CD38, CD20, Claudin18.2, Trop-2, CTLA-4, BCMA, Nectin-4, EGFR, VEGF and Her2 as examples to prepare complete immune antibodies that specifically bind to tumor-specific antigens, CD16a and CD47, respectively.

[0096] In one embodiment, a complete immune antibody TriAtezo01 with an anti-PD-L1 antibody as its backbone is provided. The complete immune antibody TriAtezo01 has an anti-CD16a single-chain antibody linked to the N-terminus or C-terminus of the anti-PD-L1 antibody heavy chain via a linker, and a SIRPαD1 protein linked to the N-terminus or C-terminus of the anti-PD-L1 antibody light chain via a linker. Preferably, the long chain structure of the complete immune antibody TriAtezo01 is: anti-PD-L1 antibody heavy chain - linker - anti-CD16a single-chain antibody, and the short chain structure is: anti-PD-L1 antibody light chain - linker - SIRPαD1 protein. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriAtezo01 includes or is SEQ ID NO:68, and the amino acid sequence of the short chain includes or is SEQ ID NO:69.

[0097] In one embodiment, a complete immune antibody TriAtezo02 with an anti-PD-L1 antibody as its backbone is provided. The complete immune antibody TriAtezo02 has a linker connecting human IL-15 and anti-CD16a nanobodies at the N-terminus and / or C-terminus of the anti-PD-L1 antibody heavy chain, and a linker connecting SIRPαD1 protein at the N-terminus or C-terminus of the anti-PD-L1 antibody light chain. Preferably, the long chain structure of the complete immune antibody is: anti-PD-L1 antibody heavy chain - linker - human IL-15 - linker - anti-CD16a nanobodies, and the short chain structure is: anti-PD-L1 antibody light chain - linker - SIRPαD1 protein. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriAtezo02 includes or is SEQ ID NO:70, and the amino acid sequence of the short chain includes or is SEQ ID NO:69.

[0098] In one embodiment, a complete immune antibody TriAtezo03 with an anti-PD-L1 antibody as its backbone is provided. The complete immune antibody TriAtezo03 has human IL-15Rα, human IL-15, and anti-CD16a nanobodies linked to the N-terminus and / or C-terminus of the anti-PD-L1 antibody heavy chain via linkers, and SIRPαD1 protein linked to the N-terminus or C-terminus of the anti-PD-L1 antibody light chain via linkers. Preferably, the long chain structure of the complete immune antibody is: anti-PD-L1 antibody heavy chain - linker - human IL-15Rα - linker - human IL-15 - linker - anti-CD16a nanobodies, and the short chain structure is: anti-PD-L1 antibody light chain - linker - SIRPαD1 protein. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriAtezo03 includes or is SEQ ID NO:71, and the amino acid sequence of the short chain includes or is SEQ ID NO:69.

[0099] In one embodiment, a complete immune antibody TriAtezo04 with an anti-PD-L1 antibody as its backbone is provided. The complete immune antibody TriAtezo04 has an anti-CD16a single-chain antibody linked to the N-terminus or C-terminus of the anti-PD-L1 antibody heavy chain via a linker, and a SIRPαD1 protein linked to the N-terminus or C-terminus of the anti-PD-L1 antibody light chain via a linker. Preferably, the long chain structure of the complete immune antibody is: anti-PD-L1 antibody heavy chain - linker - anti-CD16a single-chain antibody, and the short chain structure is: SIRPαD1 protein - linker - anti-PD-L1 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriAtezo04 includes or is SEQ ID NO:72, and the amino acid sequence of the short chain includes or is SEQ ID NO:73.

[0100] In one embodiment, a complete immune antibody TriAtezo05 with an anti-PD-L1 antibody as its backbone is provided. The complete immune antibody TriAtezo05 has an anti-CD16a single-chain antibody and a SIRPαD1 protein linked via a linker at the N-terminus and / or C-terminus of the anti-PD-L1 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-PD-L1 antibody heavy chain-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-PD-L1 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriAtezo05 includes or is SEQ ID NO:74, and the amino acid sequence of the short chain includes or is SEQ ID NO:75.

[0101] In one embodiment, a complete immune antibody, TriAtezo06, with an anti-PD-L1 antibody as its backbone is provided. The complete immune antibody TriAtezo06 has a linker linking SIRPαD1 protein, human IL-15, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-PD-L1 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-PD-L1 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-PD-L1 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriAtezo06 includes or is SEQ ID NO:76, and the amino acid sequence of the short chain includes or is SEQ ID NO:75.

[0102] In one embodiment, a complete immune antibody TriAtezo07 with an anti-PD-L1 antibody as its backbone is provided. The complete immune antibody TriAtezo07 has SIRPαD1 protein, human IL-15Rα, human IL-15, and anti-CD16a single-chain antibody linked via linkers at the N-terminus and / or C-terminus of the anti-PD-L1 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-PD-L1 antibody heavy chain-linker-human IL-15Rα-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-PD-L1 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriAtezo07 includes or is SEQ ID NO:77, and the amino acid sequence of the short chain includes or is SEQ ID NO:75.

[0103] In one embodiment, a complete immune antibody, TriAtezo12, with an anti-PD-L1 antibody as its backbone is provided. The complete immune antibody TriAtezo12 has a linker linking SIRPαD1 protein, human IL-2, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-PD-L1 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-PD-L1 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-PD-L1 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriAtezo12 includes or is SEQ ID NO:78, and the amino acid sequence of the short chain includes or is SEQ ID NO:75.

[0104] In one embodiment, a complete immune antibody TriCetu03 with an anti-EGFR antibody as its backbone is provided. The complete immune antibody TriCetu03 is linked to a SIRPαD1 protein and an anti-CD16a single-chain antibody via a linker at the N-terminus and / or C-terminus of the anti-EGFR antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-EGFR antibody heavy chain-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-EGFR antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriCetu03 includes or is SEQ ID NO:79, and the amino acid sequence of the short chain includes or is SEQ ID NO:80.

[0105] In one embodiment, a complete immune antibody TriCetu04 with an anti-EGFR antibody as its backbone is provided. The complete immune antibody TriCetu04 has a linker linking SIRPαD1 protein, human IL-15, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-EGFR antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-EGFR antibody heavy chain-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-EGFR antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriCetu04 includes or is SEQ ID NO:81, and the amino acid sequence of the short chain includes or is SEQ ID NO:80.

[0106] In one embodiment, a complete immune antibody TriCetu12 with an anti-EGFR antibody as its backbone is provided. The complete immune antibody TriCetu12 has a linker linking SIRPαD1 protein, human IL-2, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-EGFR antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-EGFR antibody heavy chain-linker-human IL-2-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-EGFR antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriCetu12 contains or is SEQ ID NO:120, and the amino acid sequence of the short chain contains or is SEQ ID NO:80.

[0107] In one embodiment, a complete immune antibody TriAB04 with an anti-Her2 antibody as its backbone is provided. The complete immune antibody TriAB04 has an anti-CD16a single-chain antibody linked to the N-terminus or C-terminus of the anti-Her2 antibody heavy chain via a linker, and a SIRPαD1 protein linked to the N-terminus or C-terminus of the Her2 antibody light chain via a linker. Preferably, the long chain structure of the complete immune antibody is: anti-Her2 antibody heavy chain - linker - anti-CD16a single-chain antibody, and the short chain structure is: SIRPαD1 protein - linker - anti-Her2 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriAB04 includes or is SEQ ID NO:82, and the amino acid sequence of the short chain includes or is SEQ ID NO:83. In an alternative embodiment, the complete immune antibody TriAB04 replaces the short chain structure with: anti-Her2 antibody light chain - linker - SIRPαD1 protein; preferably, the amino acid sequence of the replacement short chain is SEQ ID NO:84.

[0108] In one embodiment, a complete immune antibody TriTrastu06 with an anti-Her2 antibody as its backbone is provided. The complete immune antibody TriTrastu06 has a linker linking SIRPαD1 protein, human IL-15, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-Her2 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-Her2 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-Her2 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriTrastu06 contains or is SEQ ID NO:110, and the amino acid sequence of the short chain contains or is SEQ ID NO:111.

[0109] In one embodiment, a complete immune antibody TriTrastu12 with an anti-Her2 antibody as its backbone is provided. The complete immune antibody TriTrastu12 has a linker linking SIRPαD1 protein, human IL-2, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-Her2 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-Her2 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-Her2 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriTrastu12 contains or is SEQ ID NO:112, and the amino acid sequence of the short chain contains or is SEQ ID NO:111.

[0110] In one embodiment, a complete immune antibody TriKey06 with an anti-PD-1 antibody as its backbone is provided. The complete immune antibody TriKey06 has a linker linking SIRPαD1 protein, human IL-15, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-PD-1 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-PD-1 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-PD-1 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriKey06 includes or is SEQ ID NO:86, and the amino acid sequence of the short chain includes or is SEQ ID NO:87.

[0111] In one embodiment, a complete immune antibody TriKey12 with an anti-PD-1 antibody as its backbone is provided. The complete immune antibody TriKey12 has a linker linking SIRPαD1 protein, human IL-2, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-PD-1 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-PD-1 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-PD-1 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriKey12 includes or is SEQ ID NO:88, and the amino acid sequence of the short chain includes or is SEQ ID NO:87.

[0112] In one embodiment, a complete immune antibody TriDaratu06 with an anti-CD38 antibody as its backbone is provided. The complete immune antibody TriDaratu06 has a linker linking SIRPαD1 protein, human IL-15, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-CD38 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-CD38 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-CD38 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriDaratu06 includes or is SEQ ID NO:89, and the amino acid sequence of the short chain includes or is SEQ ID NO:90.

[0113] In one embodiment, a complete immune antibody TriDaratu12 with an anti-CD38 antibody as its backbone is provided. The complete immune antibody TriDaratu12 has a linker linking SIRPαD1 protein, human IL-2, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-CD38 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-CD38 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-CD38 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriDaratu12 includes or is SEQ ID NO:91, and the amino acid sequence of the short chain includes or is SEQ ID NO:90.

[0114] In one embodiment, a complete immune antibody TriRituxi06 with an anti-CD20 antibody as its backbone is provided. The complete immune antibody TriRituxi06 has a linker linking SIRPαD1 protein, human IL-15, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-CD20 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-CD20 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-CD20 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriRituxi06 includes or is SEQ ID NO:92, and the amino acid sequence of the short chain includes or is SEQ ID NO:93.

[0115] In one embodiment, a complete immune antibody, TriRituxi12, with an anti-CD20 antibody as its backbone is provided. The complete immune antibody TriRituxi12 has a linker linking SIRPαD1 protein, human IL-2, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-CD20 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-CD20 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-CD20 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriRituxi12 includes or is SEQ ID NO:94, and the amino acid sequence of the short chain includes or is SEQ ID NO:93.

[0116] In one embodiment, a complete immune antibody, TriZolbetu06, with an anti-Claudin18.2 antibody as its backbone is provided. The complete immune antibody TriZolbetu06 has a linker linking SIRPαD1 protein, human IL-15, and an anti-CD16a single-chain antibody to the N-terminus and / or C-terminus of the anti-Claudin18.2 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-Claudin18.2 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-Claudin18.2 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriZolbetu06 contains or is SEQ ID NO:95, and the amino acid sequence of the short chain contains or is SEQ ID NO:96.

[0117] In one embodiment, a complete immune antibody, TriZolbetu12, with an anti-Claudin18.2 antibody as its backbone is provided. The complete immune antibody TriZolbetu12 has a linker linking SIRPαD1 protein, human IL-2, and an anti-CD16a single-chain antibody to the N-terminus and / or C-terminus of the anti-Claudin18.2 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-Claudin18.2 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-Claudin18.2 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriZolbetu12 contains or is SEQ ID NO:97, and the amino acid sequence of the short chain contains or is SEQ ID NO:96.

[0118] In one embodiment, a complete immune antibody TriSacitu06 with an anti-Trop-2 antibody as its backbone is provided. The complete immune antibody TriSacitu06 has a linker linking SIRPαD1 protein, human IL-15, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-Trop-2 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-Trop-2 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-Trop-2 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriSacitu06 includes or is SEQ ID NO:98, and the amino acid sequence of the short chain includes or is SEQ ID NO:99.

[0119] In one embodiment, a complete immune antibody, TriSacitu12, with an anti-Trop-2 antibody as its backbone is provided. The complete immune antibody TriSacitu12 has a linker linking SIRPαD1 protein, human IL-2, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-Trop-2 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-Trop-2 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-Trop-2 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriSacitu12 contains or is SEQ ID NO:100, and the amino acid sequence of the short chain contains or is SEQ ID NO:99.

[0120] In one embodiment, a complete immune antibody TriIpilimu06 with an anti-CTLA-4 antibody as its backbone is provided. The complete immune antibody TriIpilimu06 has a linker linking SIRPαD1 protein, human IL-15, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-CTLA-4 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-CTLA-4 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-CTLA-4 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriIpilimu06 contains or is SEQ ID NO:101, and the amino acid sequence of the short chain contains or is SEQ ID NO:102.

[0121] In one embodiment, a complete immune antibody, TriIpilimu12, with an anti-CTLA-4 antibody as its backbone is provided. The complete immune antibody TriIpilimu12 has a linker linking SIRPαD1 protein, human IL-2, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-CTLA-4 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-CTLA-4 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-CTLA-4 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriIpilimu12 contains or is SEQ ID NO:103, and the amino acid sequence of the short chain contains or is SEQ ID NO:102.

[0122] In one embodiment, a complete immune antibody TriBelanta06 with an anti-BCMA antibody as its backbone is provided. The complete immune antibody TriBelanta06 has a linker linking SIRPαD1 protein, human IL-15, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-BCMA antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-BCMA antibody heavy chain-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-BCMA antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriBelanta06 contains or is SEQ ID NO:104, and the amino acid sequence of the short chain contains or is SEQ ID NO:105.

[0123] In one embodiment, a complete immune antibody, TriBelanta12, with an anti-BCMA antibody as its backbone is provided. The complete immune antibody TriBelanta12 has a linker linking SIRPαD1 protein, human IL-2, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-BCMA antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-BCMA antibody heavy chain-linker-human IL-2-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-BCMA antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriBelanta12 contains or is SEQ ID NO:106, and the amino acid sequence of the short chain contains or is SEQ ID NO:105.

[0124] In one embodiment, a complete immune antibody, TriEnfortu06, with an anti-Nectin-4 antibody as its backbone is provided. The complete immune antibody TriEnfortu06 has a linker linking SIRPαD1 protein, human IL-15, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-Nectin-4 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-Nectin-4 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-Nectin-4 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriEnfortu06 contains or is SEQ ID NO:107, and the amino acid sequence of the short chain contains or is SEQ ID NO:108.

[0125] In one embodiment, a complete immune antibody, TriEnfortu12, with an anti-Nectin-4 antibody as its backbone is provided. The complete immune antibody TriEnfortu12 has a linker linking SIRPαD1 protein, human IL-2, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-Nectin-4 antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-Nectin-4 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-Nectin-4 antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriEnfortu12 contains or is SEQ ID NO:109, and the amino acid sequence of the short chain contains or is SEQ ID NO:108.

[0126] In one embodiment, a complete immune antibody TriBeva06 with an anti-VEGF antibody as its backbone is provided. The complete immune antibody TriBeva06 has a linker linking SIRPαD1 protein, human IL-15, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-VEGF antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-VEGF antibody heavy chain-linker-human IL-15-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-VEGF antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriBeva06 includes or is SEQ ID NO:113, and the amino acid sequence of the short chain includes or is SEQ ID NO:114.

[0127] In one embodiment, a complete immune antibody TriBeva12 with an anti-VEGF antibody as its backbone is provided. The complete immune antibody TriBeva12 has a linker linking SIRPαD1 protein, human IL-2, and an anti-CD16a single-chain antibody at the N-terminus and / or C-terminus of the anti-VEGF antibody heavy chain. Preferably, the long chain structure of the complete immune antibody is: SIRPαD1 protein-linker-anti-VEGF antibody heavy chain-linker-human IL-2-linker-anti-CD16a single-chain antibody, and the short chain structure is: an anti-VEGF antibody light chain. In a specific embodiment, the amino acid sequence of the long chain of the complete immune antibody TriBeva12 includes or is SEQ ID NO:115, and the amino acid sequence of the short chain includes or is SEQ ID NO:114.

[0128] In any of the embodiments described herein, the heavy chain variable region of the anti-CD16a single-chain antibody comprises: CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; and the light chain variable region comprises: CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6.

[0129] In any of the embodiments described herein, the heavy chain variable region of the anti-CD16a single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8.

[0130] In any of the embodiments described herein, the anti-CD16a single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:9.

[0131] In any of the embodiments described herein, the anti-CD16a nanobody comprises: CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12.

[0132] In any of the embodiments described herein, the anti-CD16a nanobody comprises the amino acid sequence shown in SEQ ID NO:13.

[0133] In any of the embodiments described herein, the SIRPαD1 protein comprises the amino acid sequence shown in SEQ ID NO:14.

[0134] In any of the embodiments described herein, the human IL-15 protein fragment comprises the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:16.

[0135] In any of the embodiments described herein, the human IL-15Rα protein fragment comprises the amino acid sequence shown in SEQ ID NO:17.

[0136] In any of the embodiments described herein, the CpG ODN comprises the sequence shown in SEQ ID NO:117.

[0137] In any of the embodiments described herein, the IL-2 mutant comprises the amino acid sequence shown in SEQ ID NO:118.

[0138] In any of the embodiments described herein, the IFNα mutant comprises the amino acid sequence shown in SEQ ID NO:119.

[0139] In any of the embodiments described herein, a suitable linker can be used to link macrophage activators, NK cell linkers and activators, PRR agonists, and immune cytokines to a basic antibody structure. The linker can be a flexible linker or a flexible peptide linker, preferably a polypeptide with a length of at least 5 amino acids. In some embodiments, the peptide linker is PSGQAGAAASESLFVSNHAY (SEQ ID NO:19); in other embodiments, the peptide linker is (GxS)n or (GxS)nGm, where G = glycine, S = serine, and (x = 3, n = 3, 4, 5, or 6 and m = 0, 1, 2, or 3) or (x = 4 and n = 2, 3, 4, or 5 and m = 0, 1, 2, or 3), preferably x = 4 and n = 2 or 3, more preferably x = 4 and n = 3. In some preferred embodiments, the amino acid sequence of the peptide linker is (G4S)3 or (G4S)4, for example GGGGSGGGGSGGGGS (SEQ ID NO:18).

[0140] Exemplary implementation scheme:

[0141] 1. An innate immune superconnector (ISE) characterized in that it recruits or activates innate immune cells, the innate immune superconnector comprising two or more of the following components: (a) an anti-CD16a antibody, (b) a SIRPαD1 protein, (c) an immune cytokine, and (d) a pattern recognition receptor agonist.

[0142] 2. A complete immune antibody (CIA) characterized in that it recruits or activates innate and acquired immune cells to kill tumor cells, said complete immune antibody comprising (a) the ISE or a fragment thereof as described in embodiment 1, and (b) an acquired immune antibody or a tumor-associated antigen antibody.

[0143] 3. The innate immune superconnector according to embodiment 1 is characterized in that, with a traditional antibody as the basic structure, the variable region of the antibody is removed or a non-targeting antibody in the human body is used, and an anti-CD16a antibody or SIRPαD1 protein is linked to the N-terminus or C-terminus of the antibody heavy chain constant region by a linker, and a SIRPαD1 protein or anti-CD16a antibody is linked to the N-terminus or C-terminus of the antibody light chain constant region by a linker.

[0144] 4. The innate immune superconnector according to embodiment 1 is characterized in that, based on a traditional antibody as the basic structure, the variable region of the antibody is removed or a non-targeting antibody in the human body is used, and one or more of human immune cytokines, human immune cytokine receptors, SIRPαD1 protein and anti-CD16a antibody are linked to the N-terminus and / or C-terminus of the antibody heavy chain constant region and / or antibody light chain constant region via linkers.

[0145] 5. The innate immune superconnector according to any one of the foregoing embodiments, characterized in that a pattern recognition receptor agonist is connected to the N-terminus or C-terminus of the ISE via a linker, wherein the pattern recognition receptor agonist includes, but is not limited to, Toll-like receptor (TLR) agonists, NOD-like receptor (NLR) agonists, RIG-I-like receptor (RLR) agonists, C-type lectin receptor (CLR) agonists, and melanoma deficiency factor 2 (AIM2)-like receptor (ALR) agonists.

[0146] 6. The innate immune superconnector according to any one of the foregoing embodiments, characterized in that human IL-2 or IFNα or its mutants are linked at the N-terminus or C-terminus of the ISE via a linker.

[0147] 7. The complete immune antibody according to embodiment 2, characterized in that it uses ISE as the basic structure and adds a variable region of acquired immune antibody or a variable region of tumor-associated antigen antibody to the N-terminus of ISE.

[0148] 8. The complete immune antibody according to embodiment 7, characterized in that, based on an acquired immune antibody or tumor-associated antigen antibody as the basic backbone, one or more of human immune cytokines, human immune cytokine receptors, SIRPαD1 protein and anti-CD16a antibody are linked to the N-terminus and / or C-terminus of the antibody heavy chain and / or antibody light chain via linkers.

[0149] 9. The complete immune antibody according to any one of the foregoing embodiments, characterized in that the tumor-associated antigen is, for example, but not limited to, PD-L1, Her2, VEGF, EGFR, CD38, Claudin18.2, Claudin6, CD19, MSLN, Trop-2, CD20, Nectin-4, c-Met, MUC16, MUC17, 5T4, BCMA, DLL3, or CD30.

[0150] 10. The complete immune antibody according to any one of the foregoing embodiments, characterized in that the acquired immune antibody is an antibody targeting, for example but not limited to, the following targets: PD-1, CTLA-4, B7-H4, B7-H3, 4-1BB, LAG-3, TIGIT, TIM-3, LILRB2, CD24, CD40 or CD40L.

[0151] 11. The innate immune superconnector or complete immune antibody according to any one of the foregoing embodiments, characterized in that human immune cytokines such as, but not limited to, IL-15, IL-18, IL-2, IL-7, IL-10 or IL-12.

[0152] 12. The innate immune superconnector according to embodiment 11 is characterized in that it uses a traditional antibody as the basic structure, removes the variable region of the antibody or uses a human non-targeting antibody, and links one or more of anti-CD16a antibody, human IL-15 or IL-15Rα to the N-terminus or C-terminus of the antibody heavy chain constant region via a linker, and links SIRPαD1 protein to the N-terminus or C-terminus of the antibody light chain constant region via a linker.

[0153] 13. The innate immune superconnector or complete immune antibody according to any one of the foregoing embodiments, characterized in that the anti-CD16a antibody is an anti-CD16a single-chain antibody or an anti-CD16a nanobody.

[0154] 14. The innate immune superconnector or complete immune antibody according to any one of the foregoing embodiments, characterized in that the heavy chain variable region of the anti-CD16a single-chain antibody comprises: CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2 and CDR3 shown in SEQ ID NO:3; and the light chain variable region comprises: CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5 and CDR3 shown in SEQ ID NO:6.

[0155] 15. The innate immune superconnector or complete immune antibody according to any one of the foregoing embodiments, characterized in that the heavy chain variable region of the anti-CD16a single-chain antibody contains the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:8.

[0156] 16. The innate immune superconnector or complete immune antibody according to any one of the foregoing embodiments, characterized in that the anti-CD16a single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:9.

[0157] 17. The innate immune superconnector or complete immune antibody according to any one of the foregoing embodiments, characterized in that the anti-CD16a nanobody comprises: CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11 and CDR3 shown in SEQ ID NO:12.

[0158] 18. The innate immune superconnector or complete immune antibody according to any one of the foregoing embodiments, characterized in that the anti-CD16a nanobody comprises the amino acid sequence shown in SEQ ID NO:13.

[0159] 19. The innate immune superconnector or complete immune antibody according to any one of the foregoing embodiments, characterized in that the SIRPαD1 protein comprises the amino acid sequence shown in SEQ ID NO:14.

[0160] 20. The innate immune superconnector or complete immune antibody according to any one of the foregoing embodiments, characterized in that the human IL-15 protein fragment comprises the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:16, and the human IL-15Rα protein fragment comprises the amino acid sequence shown in SEQ ID NO:17.

[0161] 21. The innate immune superconnector according to embodiment 5, wherein the pattern recognition receptor agonist is CpG ODN, preferably, the CpG ODN comprises the sequence shown in SEQ ID NO:117.

[0162] 22. The innate immune superconnector according to embodiment 6, characterized in that the IL-2 mutant comprises the amino acid sequence shown in SEQ ID NO:118, and / or the IFNα mutant comprises the amino acid sequence shown in SEQ ID NO:119.

[0163] 23. The innate immune superconnector according to any one of the foregoing embodiments, characterized in that the basic framework is a human body without targeting antibodies, such as anti-hen egg lysozyme HEL antibody, anti-pathogenic microorganism antibodies such as antiviral and bacterial antibodies, etc.

[0164] 24. The innate immune superconnector according to embodiment 23 is characterized in that it uses an anti-egg white lysozyme antibody as the basic backbone, and connects one or more of human immune cytokines, human immune cytokine receptors, SIRPαD1 protein and anti-CD16a antibody to the N-terminus or C-terminus of the antibody heavy chain and / or the N-terminus or C-terminus of the antibody light chain via linkers.

[0165] 25. The innate immune superconnector according to embodiment 24, characterized in that the long amino acid sequence comprises SEQ ID NO:20 and the short amino acid sequence comprises SEQ ID NO:21, or the long amino acid sequence comprises SEQ ID NO:22 and the short amino acid sequence comprises SEQ ID NO:23, or the long amino acid sequence comprises SEQ ID NO:24 and the short amino acid sequence comprises SEQ ID NO:25, or the long amino acid sequence comprises SEQ ID NO:26 and the short amino acid sequence comprises SEQ ID NO:27, or the long amino acid sequence comprises SEQ ID NO:28 and the short amino acid sequence comprises SEQ ID NO:29.

[0166] 26. The innate immune superconnector according to any one of the foregoing embodiments, characterized in that it is an ISE-A, ISE-B, ISE-C, ISE-D, ISE-E, ISE-F, ISE-G, ISE-H, ISE-I, or ISE-J configuration, preferably, the long chain amino acid sequence of the innate immune connector ISE-A contains SEQ ID NO:30 or SEQ ID NO:32, and the short chain amino acid sequence contains SEQ ID NO:31 or SEQ ID NO:33; the long chain amino acid sequence of the innate immune connector ISE-B contains SEQ ID NO:34 or SEQ ID NO:36, and the short chain amino acid sequence contains SEQ ID NO:35 or SEQ ID NO:37; the long chain amino acid sequence of the innate immune connector ISE-C contains SEQ ID NO:38 or SEQ ID NO:40, and the short chain amino acid sequence contains SEQ ID NO:39 or SEQ ID NO:41; the long chain amino acid sequence of the innate immune connector ISE-D contains SEQ ID NO:42 or SEQ ID NO:30. NO:44, the short chain amino acid sequence contains SEQ ID NO:43 or SEQ ID NO:45; the long chain amino acid sequence of the innate immune connector ISE-E contains SEQ ID NO:46 or SEQ ID NO:48, and the short chain amino acid sequence contains SEQ ID NO:47 or SEQ ID NO:49; the long chain amino acid sequence of the innate immune connector ISE-F contains SEQ ID NO:50 or SEQ ID NO:52, and the short chain amino acid sequence contains SEQ ID NO:51 or SEQ ID NO:53; the long chain amino acid sequence of the innate immune connector ISE-G contains SEQ ID NO:54 or SEQ ID NO:56, and the short chain amino acid sequence contains SEQ ID NO:55 or SEQ ID NO:57; the long chain amino acid sequence of the innate immune connector ISE-H contains SEQ ID NO:58, and the short chain amino acid sequence contains SEQ ID NO:59 or SEQ ID NO:60; the long chain amino acid sequence of the innate immune connector ISE-I contains SEQ ID NO:61 or SEQ ID NO:63, and the short chain amino acid sequence contains SEQ ID NO:49. The long chain amino acid sequence of the innate immune connector ISE-J contains SEQ ID NO:62 or SEQ ID NO:64, and the short chain amino acid sequence contains SEQ ID NO:66 or SEQ ID NO:67.

[0167] 27. The complete immune antibody according to embodiment 7, characterized in that the long chain amino acid sequence is based on the amino acid sequence of the ISE by introducing the heavy chain variable region of the tumor-associated antigen antibody or the acquired immune antibody, and the short chain amino acid sequence is based on the amino acid sequence of the ISE by introducing the light chain variable region of the tumor-associated antigen antibody or the acquired immune antibody.

[0168] 28. The complete immune antibody according to embodiment 27, characterized in that the complete immune antibody is a complete immune antibody TriAtezo01, TriAtezo02, TriAtezo03, TriAtezo04, TriAtezo05, TriAtezo06, TriAtezo07, TriAtezo12, TriCetu03, TriCetu04 and TriCetu12, the long chain amino acid sequences of which respectively contain SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81 and SEQ ID NO:120;The short chain amino acid sequences respectively contain SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:80, SEQ ID NO:80 and SEQ ID NO:80, and / or the complete immune antibody is a complete immune antibody TriKey06, TriKey12, TriDaratu06, TriDaratu12, TriRituxi06, TriRituxi12, TriZolbetu06, TriZolbetu12, TriSacitu06, TriSacitu12, TriTrastu06, TriTrastu12, TriBeva06, TriBeva12, TriEnfortu06, TriEnfortu12, TriBelanta06, TriBelanta12, TriIpilimu06 and TriIpilimu12, and its long chain amino acid sequences respectively contain SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:80, SEQ ID NO:80 and SEQ ID NO:80, and / or the complete immune antibody is a complete immune antibody TriKey06, TriKey12, TriDaratu06, TriDaratu12, TriBeva06, TriBeva12, TriEnfortu06, TriEnfortu12, TriBelanta06, TriBelanta12, TriIpilimu06 and TriIpilimu12, and its long chain amino acid sequences respectively contain SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:101 and SEQ ID NO:103; the short chain amino acid sequences respectively include SEQ ID NO:87, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:99, SEQ ID NO:111, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:114, SEQ ID NO:108, SEQ ID NO:108, SEQ ID NO:105, SEQ ID NO:105, SEQ ID NO:102 and SEQ ID NO:102. ;

[0169] 29. The innate immune superconnector or complete immune antibody according to any one of the foregoing embodiments, characterized in that the Fc segment of the antibody introduces L234A and L235A mutations, or further introduces M252Y, S254T and T256E mutations in the Fc segment.

[0170] 30. The innate immune superconnector or complete immune antibody according to any one of the foregoing embodiments, characterized in that the linker includes, but is not limited to, PSGQAGAAASESLFVSNHAY or (G4S). n , where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0171] 31. A nucleic acid molecule encoding an innate immune superconnector or a complete immune antibody as described in any of the preceding embodiments.

[0172] 32. A carrier comprising the nucleic acid molecule described in embodiment 31.

[0173] 33. A cell comprising the nucleic acid molecule described in embodiment 31 or the vector described in embodiment 32.

[0174] 34. A method for preparing an innate immune superconnector or a complete immune antibody as described in any of the preceding embodiments, the method comprising the following steps:

[0175] (1) Amplify the two strands of ISE or CIA respectively using PCR technology; introduce mutations L234A, L235A, M252Y, S254T and T256E into the Fc fragment;

[0176] (2) The DNA fragment obtained in step (1) is cloned into pcDNA series vectors or other vectors used for mammalian cell expression systems to obtain a recombinant vector;

[0177] (3) The recombinant vector plasmid obtained in step (2) is transfected into mammalian cells to express the recombinant protein, and the purified target protein is obtained after affinity chromatography.

[0178] 35. Use of any of the foregoing embodiments of the innate immune superconnector or complete immune antibody in the preparation of a medicament for treating solid tumors and hematological malignancies in a subject.

[0179] 36. According to the use described in embodiment 35, the solid tumors and hematologic malignancies include, but are not limited to, respiratory tract tumors, digestive tract tumors, reproductive system tumors, nervous system tumors, urinary system tumors, skin tumors, head and neck tumors, and hematologic malignancies. Preferably, the solid tumors and hematologic malignancies include, but are not limited to, nasopharyngeal carcinoma, laryngeal carcinoma, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, ovarian cancer, testicular cancer, brain tumors, gliomas, kidney cancer, bladder cancer, ureteral cancer, epithelial tumors, melanoma, eye tumors, nasal tumors, ear tumors, oral tumors, leukemia, multiple myeloma, lymphoma, breast cancer, and epidermal cancer.

[0180] The sequence summarization involved in this invention:

[0181] SEQ ID NO:1 (Anti-CD16a single-chain antibody heavy chain CDR1 amino acid sequence)

[0182] SEQ ID NO:2 (anti-CD16a single-chain antibody heavy chain CDR2 amino acid sequence)

[0183] SEQ ID NO:3 (anti-CD16a single-chain antibody heavy chain CDR3 amino acid sequence)

[0184] SEQ ID NO:4 (Anti-CD16a single-chain antibody light chain CDR1 amino acid sequence)

[0185] SEQ ID NO:5 (Anti-CD16a single-chain antibody light chain CDR2 amino acid sequence)

[0186] SEQ ID NO:6 (Anti-CD16a single-chain antibody light chain CDR3 amino acid sequence)

[0187] SEQ ID NO:7 (Amino acid sequence of the variable region of the heavy chain of anti-CD16a single-chain antibody)

[0188] SEQ ID NO:8 (Amino acid sequence of the light chain variable region of anti-CD16a single-chain antibody)

[0189] SEQ ID NO:9 (anti-CD16a single-chain antibody amino acid sequence)

[0190] SEQ ID NO:10 (anti-CD16a nanobody CDR1 amino acid sequence)

[0191] SEQ ID NO:11 (anti-CD16a nanobody CDR2 amino acid sequence)

[0192] SEQ ID NO:12 (anti-CD16a nanobody CDR3 amino acid sequence)

[0193] SEQ ID NO:13 (anti-CD16a nanobody amino acid sequence)

[0194] SEQ ID NO:14 (SIRPαD1 protein amino acid sequence)

[0195] SEQ ID NO:15 (Amino acid sequence of human IL-15 protein fragment)

[0196] SEQ ID NO:16 (Amino acid sequence of human IL-15 protein fragment)

[0197] SEQ ID NO:17 (Amino acid sequence of human IL-15Rα protein fragment)

[0198] SEQ ID NO:18 (Flexible linker amino acid sequence)

[0199] SEQ ID NO:19 (Flexible linker amino acid sequence)

[0200] SEQ ID NO:20 (HELAB01 long chain amino acid sequence, anti-HEL antibody heavy chain)

[0201] SEQ ID NO:21 (HELAB01 short chain amino acid sequence, anti-HEL antibody light chain)

[0202] SEQ ID NO:22 (HELAB02 long chain amino acid sequence, anti-HEL antibody heavy chain)

[0203] SEQ ID NO:23 (HELAB02 short chain amino acid sequence, anti-HEL antibody light chain-linker-SIRPαD1 protein)

[0204] SEQ ID NO:24 (HELAB03 long chain amino acid sequence, anti-HEL antibody heavy chain-linker-anti-CD16a single chain antibody)

[0205] SEQ ID NO:25 (HELAB03 short chain amino acid sequence, anti-HEL antibody light chain)

[0206] SEQ ID NO:26 (HELAB04 long chain amino acid sequence, anti-HEL antibody heavy chain-linker-anti-CD16a single chain antibody)

[0207] SEQ ID NO:27 (HELAB04 short chain amino acid sequence, anti-HEL antibody light chain-linker-SIRPαD1 protein)

[0208] SEQ ID NO:28 (HELAB05 long chain amino acid sequence, anti-HEL antibody heavy chain-linker-human IL-15-anti-CD16a nanobody)

[0209] SEQ ID NO:29 (HELAB05 short chain amino acid sequence, anti-HEL antibody light chain-linker-SIRPαD1 protein)

[0210] SEQ ID NO:30 (ISE-A1 long chain amino acid sequence, SIRPαD1 protein-linker-antibody heavy chain constant region-linker-anti-CD16a single chain antibody)

[0211] SEQ ID NO:31 (ISE-A1 short chain amino acid sequence, constant region of antibody kappa chain)

[0212] SEQ ID NO:32 (ISE-A2 long chain amino acid sequence, antibody heavy chain constant region-linker-anti-CD16a single chain antibody)

[0213] SEQ ID NO:33 (ISE-A2 short chain amino acid sequence, antibody kappa chain constant region-linker-SIRPαD1 protein)

[0214] SEQ ID NO:34 (ISE-B1 long chain amino acid sequence, SIRPαD1 protein-linker-antibody heavy chain constant region-linker-human IL-15-linker-anti-CD16a nanobody)

[0215] SEQ ID NO:35 (ISE-B1 short chain amino acid sequence, constant region of antibody kappa chain)

[0216] SEQ ID NO:36 (ISE-B2 long chain amino acid sequence, antibody heavy chain constant region-linker-human IL-15-linker-anti-CD16a nanobody)

[0217] SEQ ID NO:37 (ISE-B2 short chain amino acid sequence, antibody kappa chain constant region-linker-SIRPαD1 protein)

[0218] SEQ ID NO:38 (ISE-C1 long chain amino acid sequence, SIRPαD1 protein-linker-antibody heavy chain constant region-linker-human IL-15Rα-linker-human IL-15-linker-anti-CD16a nanobody)

[0219] SEQ ID NO:39 (ISE-C1 short chain amino acid sequence, constant region of antibody kappa chain)

[0220] SEQ ID NO:40 (ISE-C2 long chain amino acid sequence, antibody heavy chain constant region-linker-human IL-15Rα-linker-human IL-15-linker-anti-CD16a nanobody)

[0221] SEQ ID NO:41 (ISE-C2 short chain amino acid sequence, antibody kappa chain constant region-linker-SIRPαD1 protein)

[0222] SEQ ID NO:42 (ISE-D1 long chain amino acid sequence, SIRPαD1 protein-linker-antibody heavy chain constant region-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0223] SEQ ID NO:43 (ISE-D1 short chain amino acid sequence, constant region of antibody kappa chain)

[0224] SEQ ID NO:44 (ISE-D2 long chain amino acid sequence, antibody heavy chain constant region-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0225] SEQ ID NO:45 (ISE-D2 short chain amino acid sequence, antibody kappa chain constant region-linker-SIRPαD1 protein)

[0226] SEQ ID NO:46 (ISE-E1 long chain amino acid sequence, CpG ODN-linker-antibody heavy chain constant region-linker-anti-CD16a single chain antibody)

[0227] SEQ ID NO:47 (ISE-E1 short chain amino acid sequence, antibody kappa chain constant region-linker-SIRPαD1 protein)

[0228] SEQ ID NO:48 (ISE-E2 long chain amino acid sequence, CpG ODN-linker-antibody heavy chain constant region-linker-anti-CD16a single chain antibody)

[0229] SEQ ID NO:49 (ISE-E2 short chain amino acid sequence, SIRPαD1 protein-linker-antibody kappa chain constant region)

[0230] SEQ ID NO:50 (ISE-F1 long chain amino acid sequence, antibody heavy chain constant region-linker-anti-CD16a single chain antibody)

[0231] SEQ ID NO:51 (ISE-F1 short chain amino acid sequence, CpG ODN-linker-antibody kappa chain constant region-linker-SIRPαD1 protein)

[0232] SEQ ID NO:52 (ISE-F2 long chain amino acid sequence, antibody heavy chain constant region-linker-anti-CD16a single chain antibody)

[0233] SEQ ID NO:53 (ISE-F2 short chain amino acid sequence, SIRPαD1 protein-linker-antibody kappa chain constant region-linker-CpG ODN)

[0234] SEQ ID NO:54 (ISE-G1 long chain amino acid sequence, antibody heavy chain constant region-linker-anti-CD16a single chain antibody-linker-CpG ODN)

[0235] SEQ ID NO:55 (ISE-G1 short chain amino acid sequence, antibody kappa chain constant region-linker-SIRPαD1 protein)

[0236] SEQ ID NO:56 (ISE-G2 long chain amino acid sequence, antibody heavy chain constant region-linker-anti-CD16a single chain antibody-linker-CpG ODN)

[0237] SEQ ID NO:57 (ISE-G2 short chain amino acid sequence, SIRPαD1 protein-linker-antibody kappa chain constant region)

[0238] SEQ ID NO:58 (ISE-H1 long chain amino acid sequence, human IL-2 mutant-linker-antibody heavy chain constant region-linker-anti-CD16a single chain antibody)

[0239] SEQ ID NO:59 (ISE-H1 short chain amino acid sequence, antibody kappa chain constant region-linker-SIRPαD1 protein)

[0240] SEQ ID NO:60 (ISE-H2 short chain amino acid sequence, SIRPαD1 protein-linker-antibody kappa chain constant region)

[0241] SEQ ID NO:61(ISE-I1 long chain amino acid sequence, human IL-2 mutant-linker-antibody heavy chain constant region-linker-human IL-15-linker-anti-CD16a nanobody)

[0242] SEQ ID NO:62 (ISE-I1 short chain amino acid sequence, antibody kappa chain constant region-linker-SIRPαD1 protein)

[0243] SEQ ID NO:63 (ISE-I2 long chain amino acid sequence, human IL-2 mutant-linker-antibody heavy chain constant region-linker-human IL-15-linker-anti-CD16a nanobody)

[0244] SEQ ID NO:64 (ISE-I2 short chain amino acid sequence, SIRPαD1 protein-linker-antibody kappa chain constant region)

[0245] SEQ ID NO:65(ISE-J1 long chain amino acid sequence, human IL-2 mutant-linker-antibody heavy chain constant region-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0246] SEQ ID NO:66 (ISE-J1 short chain amino acid sequence, antibody kappa chain constant region-linker-SIRPαD1 protein)

[0247] SEQ ID NO:68 (TriAtezo01 long chain amino acid sequence, anti-PD-L1 antibody heavy chain-linker-anti-CD16a single chain antibody)

[0248] SEQ ID NO:69 (TriAtezo01 short chain amino acid sequence, anti-PD-L1 antibody light chain linker-SIRPαD1 protein)

[0249] SEQ ID NO:70 (TriAtezo02 long chain amino acid sequence, anti-PD-L1 antibody heavy chain-linker-human IL-15-linker-anti-CD16a nanobody)

[0250] SEQ ID NO:71 (TriAtezo03 long chain amino acid sequence, anti-PD-L1 antibody heavy chain-linker-human IL-15Rα-linker-human IL-15-linker-anti-CD16a nanobody)

[0251] SEQ ID NO:72 (TriAtezo04 long chain amino acid sequence, anti-PD-L1 antibody heavy chain-linker-anti-CD16a single chain antibody)

[0252] SEQ ID NO:73 (TriAtezo04 short chain amino acid sequence, SIRPαD1 protein-linker-anti-PD-L1 antibody light chain)

[0253] SEQ ID NO:74 (TriAtezo05 long chain amino acid sequence, SIRPαD1 protein-linker-anti-PD-L1 antibody heavy chain-linker-anti-CD16a single chain antibody)

[0254] SEQ ID NO:75 (TriAtezo05 short chain amino acid sequence, light chain of anti-PD-L1 antibody)

[0255] SEQ ID NO:76 (TriAtezo06 long chain amino acid sequence, SIRPαD1 protein-linker-anti-PD-L1 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0256] SEQ ID NO:77 (TriAtezo07 long chain amino acid sequence, SIRPαD1 protein-linker-anti-PD-L1 antibody heavy chain-linker-human IL-15Rα-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0257] SEQ ID NO:78 (TriAtezo12 long chain amino acid sequence, SIRPαD1 protein-linker-anti-PD-L1 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single chain antibody)

[0258] SEQ ID NO:79 (TriCetu03 long chain amino acid sequence, SIRPαD1 protein-linker-anti-EGFR antibody heavy chain-linker-anti-CD16a single chain antibody)

[0259] SEQ ID NO:80 (TriCetu03 short chain amino acid sequence, light chain of anti-EGFR antibody)

[0260] SEQ ID NO:81 (TriCetu04 long chain amino acid sequence, SIRPαD1 protein-linker-anti-EGFR antibody heavy chain-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0261] SEQ ID NO:82 (TriAB04 long chain amino acid sequence, anti-Her2 antibody heavy chain linker-CD16a single chain antibody)

[0262] SEQ ID NO:83 (TriAB04 short chain amino acid sequence, SIRPαD1 protein-linker-anti-Her2 antibody light chain)

[0263] SEQ ID NO:84 (TriAB04 substituted short chain amino acid sequence, anti-Her2 antibody light chain linker-SIRPαD1 protein)

[0264] SEQ ID NO:85 (HELAB02, HELAB04 and HELAB05 substitutions for short chain amino acid sequences, SIRPαD1 protein-linker-anti-HEL antibody light chain)

[0265] SEQ ID NO:86 (TriKey06 long chain amino acid sequence, SIRPαD1 protein-linker-anti-PD-1 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0266] SEQ ID NO:87 (TriKey06 short chain amino acid sequence, light chain of anti-PD-1 antibody)

[0267] SEQ ID NO:88 (TriKey12 long chain amino acid sequence, SIRPαD1 protein-linker-anti-PD-1 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single chain antibody)

[0268] SEQ ID NO:89 (TriDaratu06 long chain amino acid sequence, SIRPαD1 protein-linker-anti-CD38 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0269] SEQ ID NO:90 (TriDaratu06 short chain amino acid sequence, light chain of anti-CD38 antibody)

[0270] SEQ ID NO:91 (TriDaratu12 long chain amino acid sequence, SIRPαD1 protein-linker-anti-CD38 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single chain antibody)

[0271] SEQ ID NO:92 (TriRituxi06 long chain amino acid sequence, SIRPαD1 protein-linker-anti-CD20 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0272] SEQ ID NO:93 (TriRituxi06 short chain amino acid sequence, light chain of anti-CD20 antibody)

[0273] SEQ ID NO:94 (TriRituxi12 long chain amino acid sequence, SIRPαD1 protein-linker-anti-CD20 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single chain antibody)

[0274] SEQ ID NO:95 (TriZolbetu06 long chain amino acid sequence, SIRPαD1 protein-linker-anti-Claudin18.2 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0275] SEQ ID NO:96 (TriZolbetu06 short chain amino acid sequence, light chain of anti-Claudin18.2 antibody)

[0276] SEQ ID NO:97 (TriZolbetu12 long chain amino acid sequence, SIRPαD1 protein-linker-anti-Claudin18.2 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single chain antibody)

[0277] SEQ ID NO:98 (TriSacitu06 long chain amino acid sequence, SIRPαD1 protein-linker-anti-Trop-2 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0278] SEQ ID NO:99 (TriSacitu06 short chain amino acid sequence, light chain of anti-Trop-2 antibody)

[0279] SEQ ID NO:100 (TriSacitu12 long chain amino acid sequence, SIRPαD1 protein-linker-anti-Trop-2 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single chain antibody)

[0280] SEQ ID NO:101(TriIpilimu06 long chain amino acid sequence, SIRPαD1 protein-linker-anti-CTLA-4 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0281] SEQ ID NO:102 (TriIpilimu06 short chain amino acid sequence, light chain of anti-CTLA-4 antibody)

[0282] SEQ ID NO:103 (TriIpilimu12 long chain amino acid sequence, SIRPαD1 protein-linker-anti-CTLA-4 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single chain antibody)

[0283] SEQ ID NO:104 (TriBelanta06 long chain amino acid sequence, SIRPαD1 protein-linker-anti-BCMA antibody heavy chain-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0284] SEQ ID NO:105 (TriBelanta06 short chain amino acid sequence, light chain of anti-BCMA antibody)

[0285] SEQ ID NO:106 (TriBelanta12 long chain amino acid sequence, SIRPαD1 protein-linker-anti-BCMA antibody heavy chain-linker-human IL-2-linker-anti-CD16a single chain antibody)

[0286] SEQ ID NO:107 (TriEnfortu06 long chain amino acid sequence, SIRPαD1 protein-linker-anti-Nectin-4 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0287] SEQ ID NO:108 (TriEnfortu06 short chain amino acid sequence, light chain of anti-Nectin-4 antibody)

[0288] SEQ ID NO:109 (TriEnfortu12 long chain amino acid sequence, SIRPαD1 protein-linker-anti-Nectin-4 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single chain antibody)

[0289] SEQ ID NO:110 (TriTrastu06 long chain amino acid sequence, SIRPαD1 protein-linker-anti-Her2 antibody heavy chain-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0290] SEQ ID NO:111 (TriTrastu06 short chain amino acid sequence, light chain of anti-Her2 antibody)

[0291] SEQ ID NO:112 (TriTrastu12 long chain amino acid sequence, SIRPαD1 protein-linker-anti-Her2 antibody heavy chain-linker-human IL-2-linker-anti-CD16a single chain antibody)

[0292] SEQ ID NO:113 (TriBeva06 long chain amino acid sequence, SIRPαD1 protein-linker-antiVEGF antibody heavy chain-linker-human IL-15-linker-anti-CD16a single chain antibody)

[0293] SEQ ID NO:114 (TriBeva06 short chain amino acid sequence, light chain of anti-VEGF antibody)

[0294] SEQ ID NO:115 (TriBeva12 long chain amino acid sequence, SIRPαD1 protein-linker-antiVEGF antibody heavy chain-linker-human IL-2-linker-anti-CD16a single chain antibody)

[0295] SEQ ID NO:116 (ISE-Negative Control long chain amino acid sequence, antibody heavy chain constant region)

[0296] SEQ ID NO:117 (CpG ODN sequence)

[0297] SEQ ID NO:118 (IL-2 mutant amino acid sequence)

[0298] SEQ ID NO:119 (IFNα mutant amino acid sequence)

[0299] SEQ ID NO:120 (TriCetu12 long chain amino acid sequence, SIRPαD1 protein-linker-anti-EGFR antibody heavy chain-linker-human IL-2-linker-anti-CD16a single chain antibody)

[0300] It is hereby noted that, within the scope of this invention, the above-described technical routes, features, and sequences of this invention, as well as the combinations thereof, can be combined with each other to form new or preferred technical solutions, as specifically described below (such as in the embodiments). Due to space limitations, these will not be elaborated upon here. Attached Figure Description

[0301] Figure 1 is a schematic diagram of the innate immune superconnector structure of the present invention;

[0302] Figure 2 is a schematic diagram of the structure of the complete immune antibody of the present invention;

[0303] Figure 3 is a schematic diagram of the HEL antibody structure of the present invention;

[0304] Figure 4 is an SDS-PAGE electrophoresis diagram of the anti-HEL antibody innate immune superconnector variant of the present invention.

[0305] Figure 5 shows the SDS-PAGE electrophoresis diagrams of the innate immune superconnector variant without variable region and the complete immune antibody variant of the present invention.

[0306] Figure 6 shows the binding of the complete immune antibody with anti-PD-L1 antibody as the backbone of the present invention to the PD-L1 protein;

[0307] Figure 7 shows the binding of the complete immune antibody with anti-PD-L1 antibody as the backbone of the present invention to CD47 protein;

[0308] Figure 8 shows the binding of the complete immune antibody with anti-PD-L1 antibody as the backbone of the present invention to CD16a protein;

[0309] Figure 9 shows the binding of the complete immune antibody with anti-PD-L1 antibody as the backbone of the present invention to CD16b protein;

[0310] Figure 10 shows the binding of the complete immune antibody with anti-PD-L1 antibody as the backbone of the present invention to FcRn protein;

[0311] Figure 11 shows the ADCC activity of the variable region-de-variable innate immune superconnector of the present invention;

[0312] Figure 12 shows the mechanism verification of the present invention, specifically the activity of the human targetless antibody-based innate immune superconnector ADCC.

[0313] Figure 13 shows the ADCC activity of the complete immune antibody based on the anti-PD-L1 antibody in this invention against PD-L1-expressing tumor cells (HCC-1954).

[0314] Figure 14 shows the effect of the complete immune antibody TriAtezo06, which uses anti-PD-L1 antibody as a backbone, on the proliferation of mouse T lymphocytes CTLL-2.

[0315] Figure 15 shows the ADCC activity of the complete immune antibody based on the anti-EGFR antibody of this invention against EGFR-expressing tumor cells (A. human epidermal cancer cells 431, B. human breast cancer cells MDA-MB-468).

[0316] Figure 16 shows the ADCC activity of the complete immune antibody based on the anti-Claudin18.2 antibody against NCI-N87 gastric cancer cells expressing Claudin18.2.

[0317] Figure 17 shows the ADCC activity of the complete immune antibody based on the anti-CTLA-4 antibody of this invention against human breast cancer cells HCC-1954.

[0318] Figure 18 shows the ADCC activity of the complete immune antibody based on anti-BCMA antibody against BCMA-expressing tumor cells (Raji human lymphoma cells) according to the present invention.

[0319] Figure 19 shows the ADCC activity of the complete immune antibody based on the anti-CD20 antibody in this invention against CD20-expressing tumor cells (human lymphoma cells Raji).

[0320] Figure 20 shows the ADCC activity of the complete immune antibody based on anti-CD38 antibody against CD38-expressing tumor cells (human lymphoma cells Raji).

[0321] Figure 21 shows the ADCC activity of the complete immune antibody based on the anti-Trop-2 antibody in this invention against Trop-2-expressing tumor cells (human gastric cancer cells NCI-N87).

[0322] Figure 22 shows the ADCC activity of the complete immune antibody based on the anti-Nectin-4 antibody in this invention against Nectin-4 expressing tumor cells (human gastric cancer cells NCI-N87).

[0323] Figure 23 shows the ADCC activity of the complete immune antibody based on anti-PD-1 antibody against human gastric cancer cells NCI-N87.

[0324] Figure 24 shows the ADCC activity of the complete immune antibody based on anti-Her2 antibody against Her2-expressing tumor cells (human gastric cancer cells NCI-N87) according to the present invention.

[0325] Figure 25 shows the efficacy study of the complete immune antibody based on anti-Her2 antibody in the ExVivo human pleural effusion model.

[0326] Figure 26 shows the efficacy study of the complete immune antibody based on anti-Her2 antibody in the ExVivo human ascites model.

[0327] Figure 27 shows the in vivo organ toxicity study of the fully immune antibody mouse based on the anti-PD-L1 antibody backbone of this invention. Example

[0328] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0329] Example 1: Structural Design of Innate Immune Superconnector and Complete Immune Antibody

[0330] In addition to CD16a antibodies targeting the surface of NK cells and SIRPα protein blocking the "don't eat me" signal of macrophages, the structure of the innate immune superconnector also incorporates immune cytokines and pattern recognition receptor agonists. The structure of the ISE is shown in Figure 1. The structure of the complete immune antibody is formed by attaching acquired immune antibodies or tumor antigen antibodies to the innate immune superconnector, and the structure of the complete immune antibody is shown in Figure 2.

[0331] Example 2: Structure and expression of non-targeting antibodies against innate immune superconnectors in the human body.

[0332] In one aspect, to demonstrate the feasibility of the innate immune superconnector concept, this application uses the innate immune superconnector as a backbone and adds the variable region of a non-targeting antibody from the human body, anti-hen egg lysozyme HEL antibody, to prepare antibodies for the main structures of the innate immune superconnector: HELAB01, HELAB02, HELAB03, HELAB04, and HELAB05. The aim is to demonstrate the superposition function of different parts of the ISE and the function of cytokines. These are, respectively, an anti-HEL specific antibody, an anti-HEL and SIRPα (anti-CD47) bispecific fusion antibody, an anti-HEL and anti-CD16a bispecific antibody, an anti-HEL, SIRPα (anti-CD47), and CD16a trispecific fusion antibody, and an anti-HEL, SIRPα (anti-CD47), and CD16a tetraspecific fusion antibody incorporating human IL-15. The antibody structures are shown in Figure 3, and the SDS-PAGE electrophoresis images (reduced and non-reduced) are shown in Figure 4. In this embodiment, the long chain amino acid sequences of HELAB01, HELAB02, HELAB03, HELAB04 and HELAB05 are SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:28, respectively; and the short chain amino acid sequences are SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29, respectively.

[0333] Example 3: Expression and purification of innate immune superconnectors and complete immune antibodies

[0334] Genes were amplified using conventional molecular biology techniques such as PCR, and the two-stranded genes were ligated into the pcDNA3.4 vector using homologous recombination. Plasmids were extracted from sequenced positive clones and transfected into Expi293F cells. Cells were cultured for 7 days at 37°C / 5% CO2 / 125 rpm. After culture, the supernatant was collected and subjected to protein A affinity chromatography to obtain purified innate immune superconnectors and complete immune antibodies. Antibody concentration was determined by the UV280 binding theoretical extinction coefficient. SDS-PAGE electrophoresis results are shown in Figure 5. In this embodiment, the amino acid sequences of the long chains of the innate immune superconnectors ISE-Negative Control, ISE-A2, and ISE-D2 are SEQ ID NO:116, SEQ ID NO:32, and SEQ ID NO:44, respectively; the amino acid sequences of the short chains are SEQ ID NO:31, SEQ ID NO:33, and SEQ ID NO:45 (Figure 5A-B: reduced and non-reduced SDS-PAGE). The amino acid sequences of the long chains of the complete immune antibodies TriAtezo01, TriAtezo02, TriAtezo03, TriAtezo04, TriAtezo05, TriAtezo06, TriAtezo07, TriAtezo12, TriCetu03, TriCetu04, and TriCetu12 are SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, and SEQ ID NO:120, respectively; the amino acid sequences of the short chains are SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:80, SEQ ID NO:80, and SEQ ID NO:120, respectively. NO:80 (Figure 5C-D Non-reduced SDS-PAGE).The long-chain amino acid sequences of the complete immune antibodies TriKey06, TriKey12, TriDaratu06, TriDaratu12, TriRituxi06, TriRituxi12, TriZolbetu06, TriZolbetu12, TriSacitu06, TriSacitu12, TriTrastu06, TriTrastu12, TriBeva06, TriBeva12, TriEnfortu06, TriEnfortu12, TriBelanta06, TriBelanta12, TriIpilimu06, and TriIpilimu12 are SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:113, and SEQ ID NO:114, respectively. The sequences are SEQ ID NO:115, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:101 and SEQ ID NO:103; the short chain amino acid sequences are SEQ ID NO:87, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:99, SEQ ID NO:111, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:114, SEQ ID NO:108, SEQ ID NO:108, SEQ ID NO:105, SEQ ID NO:105, SEQ ID NO:102 and SEQ ID NO:102; the innate immune superconnector and complete immune antibodies against different targets were successfully expressed, and the purity of SDS-PAGE after one-step affinity chromatography reached about 80%.

[0335] Example 4: ELISA detection of the binding of a complete immune antibody with an anti-PD-L1 antibody backbone to the PD-L1 protein.

[0336] 100 μL / well of 2 μg / ml PD-L1 protein was coated and incubated overnight at 4°C; the wells were then blocked with 3% skim milk at 37°C for 1 h; 100 μL each of different concentrations (10000 ng / ml, 2500 ng / ml, 625 ng / ml, 156.25 ng / ml, 39.06 ng / ml, 9.77 ng / ml, 2.44 ng / ml, 0) of anti-PD-L1 complete immunoglobulin and other control samples were added to each well, and the mixture was incubated at 37°C for 1 h; after incubation at 37°C for 1 h, the wells were washed three times with PBST; then 100 μL of 1:8000 diluted goat anti-human IgG-HRP was added, and the mixture was incubated at 37°C for 1 h, followed by six washes. 100 μL of TMB was added to each well for color development for 5 min, followed by 50 μL of stop solution. The OD450 was read using a microplate reader. The results are shown in Figure 6. As shown in the figure, the binding ability of the complete immunoglobulin to PD-L1 protein is at the same level as that of the monoclonal antibody.

[0337] Example 5: ELISA detection of the binding of a complete immune antibody with an anti-PD-L1 antibody backbone to CD47 protein.

[0338] Different concentrations of 2ug / ml CD47 protein were coated at 100ul / well and incubated overnight at 4°C. After blocking with 3% skim milk powder at 37°C for 1 hour, the samples were washed three times with PBST. 100ul of different concentrations (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.06ng / ml, 9.77ng / ml, 2.44ng / ml, 0) of anti-PDL-1 complete immune antibody and other control samples were added to each well. After incubation at 37°C for 1 hour, the samples were washed three times with PBST. Then, 100ul of 1:8000 diluted goat anti-human IgG-HRP was added. After incubation at 37°C for 1 hour, the samples were washed six times. 100ul of TMB was added to each well for color development for 5 minutes, followed by 50ul of stop solution. The OD450 was read on a microplate reader. As shown in Figure 7, the binding ability of the complete immune antibody to CD47 protein is at the same level as that of the SIRPα fusion molecule (TTI-622). The position of SIRPαD1 protein in the complete immune antibody with other target backbones is the same as that of the complete immune antibody, so its binding to CD47 protein is also at the same level, which will not be elaborated here.

[0339] Example 6: ELISA detection of the binding of a complete immune antibody with an anti-PD-L1 antibody backbone to CD16a protein.

[0340] The 158th amino acid of CD16a has two types: V (valine) with strong affinity and F (phenylalanine) with weak affinity. 20 μg / ml CD16a protein was coated at 100 μl / well and incubated overnight at 4°C. The wells were then blocked with 3% skim milk at 37°C for 1 h. 100 μl each of different concentrations (10000 ng / ml, 2500 ng / ml, 625 ng / ml, 156.25 ng / ml, 39.06 ng / ml, 9.77 ng / ml, 2.44 ng / ml, 0) of anti-PDL-1 complete immunoglobulin and other control samples were added to each well, and incubated at 37°C for 1 h. After incubation at 37°C for 1 h, the wells were washed three times with PBST. Then, 100 μl of 1:8000 diluted goat anti-human IgG-HRP was added, and the wells were incubated at 37°C for 1 h, followed by six washes. 100 μl of TMB was added to each well for color development for 5 min, and then 50 μl of stop solution was added. The OD450 was read using a microplate reader. The results are shown in Figure 8. Figure 8A shows the binding of the molecule to the CD16a-V protein, and Figure 8B shows the binding of the molecule to the CD16a-F protein. The complete immune antibody has a strong binding level to the CD16a protein. The anti-CD16a single-chain antibody in complete immune antibodies with other target backbones has the same position as this complete immune antibody, so its binding to the CD16a protein is also at the same level, which will not be elaborated here.

[0341] Example 7: ELISA detection of the binding of a complete immune antibody with an anti-PD-L1 antibody backbone to CD16b protein.

[0342] 20 μg / ml CD16b protein was coated at 100 μl / well and incubated overnight at 4°C. The wells were then blocked with 3% skim milk at 37°C for 1 h. Different concentrations (10000 ng / ml, 2500 ng / ml, 625 ng / ml, 156.25 ng / ml, 39.06 ng / ml, 9.77 ng / ml, 2.44 ng / ml, 0) of anti-PDL-1 complete immunoglobulin and other control samples were added to each well, and the mixture was incubated at 37°C for 1 h. After incubation at 37°C for 1 h, the wells were washed three times with PBST. Then, 100 μl of 1:8000 diluted goat anti-human IgG-HRP was added, and the mixture was incubated at 37°C for 1 h, followed by six washes. 100 μl of TMB was added to each well for color development for 5 min, and then 50 μl of stop solution was added. The OD450 was read using a microplate reader. The results are shown in Figure 9. The complete immunoglobulin did not bind to CD16b protein, indicating that the CD16a antibody in the complete immunoglobulin has strong specificity.

[0343] Example 8: ELISA detection of the binding of the fully immune antibody TriAtezo06 to FcRn protein.

[0344] 100 μL of 2 μg / ml FcRn protein per well was coated and incubated overnight at 4°C; the wells were then blocked with 3% skim milk powder at 37°C for 1 h; 100 μL of different concentrations (10000 ng / ml, 2500 ng / ml, 625 ng / ml, 156.25 ng / ml, 39.06 ng / ml, 9.77 ng / ml, 2.44 ng / ml, 0) of TriAtezo06 and other control samples were added to each well and incubated at 37°C for 1 h; after incubation at 37°C for 1 h, the wells were washed 3 times with PBST; then 100 μL of 1:8000 diluted goat anti-human IgG-HRP was added, and the wells were incubated at 37°C for 1 h, followed by 6 washes; 100 μL of TMB was added to each well for color development for 5 min, and then 50 μL of stop solution was added. The OD450 was read on a microplate reader. As shown in Figure 10, the complete immune antibody binds strongly to the FcRn protein compared to Atezolizumab monoclonal antibody, indicating that the complete immune antibody has a longer half-life in vivo than Atezolizumab monoclonal antibody.

[0345] Example 9: Study on ADCC activity of the innate immune superconnector with devariable region

[0346] Human breast cancer cells HCC-1954 were cultured and expanded to the required quantity. Cells were seeded at 4000 cells / well in 96-well plates and allowed to adhere overnight. Fresh human PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at a dose-response ratio of 10:1, with concentration gradients of 500 nM, 50 nM, 5 nM, 0.5 nM, 0.05 nM, 0.005 nM, and 0.0005 nM. After culturing for 5 days, the plates were washed three times with PBS. The number of viable cells was determined using the CCK-8 assay. The IC50 was calculated based on the pharmacodynamic efficacy. 50 The results are shown in Figure 11. In the absence of ISE components (SIRPαD1 protein, CD16a antibody, and cytokine IL-15), the ISE-Negative Control (Figure 1) showed no ADCC activity. In the presence of both SIRPαD1 protein and CD16a antibody, ISE-A2 exhibited weak ADCC activity only at high concentrations. In the presence of all ISE components (SIRPαD1 protein, CD16a antibody, and cytokine IL-15), ISE-D2 showed strong ADCC activity, significantly superior to that of the cytokine fusion molecule. This demonstrates the superiority of the ISE structure.

[0347] Example 10: Verification of ADCC activity of innate immune superconnector antibody using a mechanism without human-targeted antibody.

[0348] Human breast cancer cells HCC-1954 were cultured and expanded to the required quantity. Cells were seeded at 4000 cells / well in 96-well plates and allowed to adhere overnight. Fresh human PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at a dose-response ratio of 10:1, with concentration gradients of 500 nM, 50 nM, 5 nM, 0.5 nM, 0.05 nM, 0.005 nM, and 0.0005 nM. After culturing for 5 days, the plates were washed three times with PBS. The number of viable cells was determined using the CCK-8 assay. The IC50 was calculated based on the pharmacodynamic efficacy. 50 The results are shown in Figure 12. As can be seen from the figure, in the absence of ISE components (SIRPαD1 protein, CD16a antibody, and cytokine IL-15), the HEL antibody (HELAB01) showed no ADCC activity; in the presence of only SIRPαD1 protein, HELAB02 exhibited some ADCC activity; in the presence of only CD16a antibody, HELAB03 did not show significant ADCC activity, indicating that the macrophages in the donor PBMCs had strong activity, while the NK cells had weak activity; in the presence of both SIRPαD1 protein and CD16a antibody, the killing effect of HELAB04 was stronger than that of HELAB02 and HELAB03, demonstrating that recruiting multiple immune effector cells has a stronger killing effect than recruiting only a single immune effector cell; in the presence of all ISE components, the killing effect of HELAB05 was further enhanced than that of HELAB04, proving that the introduced IL-15 has a proliferative effect on immune cells.

[0349] Example 11 Study on ADCC activity of a complete immune antibody with anti-PD-L1 antibody as the backbone

[0350] PD-1 / PD-L1 in vitro ADCC activity model: Human breast cancer cells HCC-1954 were cultured and expanded to the required quantity. Cells were seeded at 4000 cells / well in 96-well plates and allowed to adhere overnight. Human PBMCs were incubated with SEB in vitro for 7 days. When PD1 expression was greater than or equal to 16%, the PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at an effector-to-target ratio of 10:1, with concentration gradients of 500 nM, 5 nM, 0.05 nM, 0.0005 nM, 0.000005 nM, 0.00000005 nM, and 0.0000000005 nM. After culturing for another 5 days, the plates were washed three times with PBS, and the number of viable cells was detected using CCK-8 assay. As shown in Figure 13-A, Atezolizumab itself has certain ADCC activity, and when combined with TTI-622 (Fc-SIRPα fusion molecule), it enhances the ADCC activity of human breast cancer cells HCC-1954 (PD-L1 positive) to a certain extent. TriAtezo01 (based on Atezolizumab, with SIRPα linked to the C-terminus of the light chain and an anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) exhibits significantly better ADCC activity than Atezolizumab. TriAtezo02 (based on Atezolizumab, with SIRPα linked to the C-terminus of the light chain and IL-15 and an anti-CD16a nanobody linked to the C-terminus of the heavy chain) shows even more significant ADCC activity than TriAtezo01. As shown in Figure 13-B, Atezolizumab itself has certain ADCC activity. TriAtezo05 (using Atezolizumab as the basic backbone, with SIRPα linked to the N-terminus of the heavy chain and an anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) exhibits significantly better ADCC activity than Atezolizumab; TriAtezo06 (using Atezolizumab as the basic backbone, with SIRPα linked to the N-terminus of the heavy chain and IL-15 and an anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) shows significantly better ADCC activity than Atezolizumab. The ADCC activity was significantly enhanced; TriAtezo07 (based on Atezolizumab, with SIRPα linked to the N-terminus of the heavy chain and IL-15 and its receptor IL-15R linked to the C-terminus of the heavy chain, along with an anti-CD16a single-chain antibody) showed superior ADCC activity compared to TriAtezo05; TriAtezo12 (based on Atezolizumab, with SIRPα linked to the N-terminus of the heavy chain and IL-2 linked to the C-terminus of the heavy chain, along with an anti-CD16a single-chain antibody) showed an even more significant increase in ADCC activity. These results indicate that the ISE components of a complete immunization antibody (SIRPα protein, anti-CD16a antibody, and IL-15 or IL-2) can significantly enhance the ADCC activity of anti-PD-L1 antibodies.

[0351] Example 12: Study on cytokine activity in TriAtezo06, a complete immune antibody with an anti-PD-L1 antibody backbone.

[0352] Mouse T lymphocytes (CTLL-2) were cultured and expanded at 5E5 cells / well in 96-well plates. The drug was administered at concentrations of 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM, and 0.0001 nM, and cultured for 72 h. Cell viability was assessed using CCK-8 assay according to the manufacturer's instructions. The results, shown in Figure 14, indicate that TriAtezo06 significantly inhibited the proliferation of mouse T lymphocytes (CTLL-2) compared to wild-type IL-15 and Fc-IL-15. This suggests that the structural design of the complete immunotherapy antibody influenced the activity of the cytokine IL-15, potentially significantly reducing its toxicity in clinical applications.

[0353] Example 13: Study on ADCC activity of a complete immune antibody with an anti-EGFR antibody as its backbone

[0354] 13.1 Study on ADCC activity against human epidermal cancer cell line A431

[0355] Cells were cultured and expanded to the required quantity, and seeded into 96-well plates at a rate of 1500 cells / well. Cells adhered overnight. Fresh human PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at an effector-to-target ratio of 10:1, with concentration gradients of 500 nM, 5 nM, 0.05 nM, 0.0005 nM, 0.000005 nM, 0.00000005 nM, and 0.000000005 nM. After culturing for 5 days, the plates were washed three times with PBS. The number of viable cells was determined using a CCK-8 assay. The IC50 was calculated based on the pharmacodynamic efficacy. 50 The results are shown in Figure 15A. In the ADCC activity assay targeting the A431 tumor cell line with high EGFR expression, Cetuximab exhibited some ADCC activity. The ADCC activities of Fc-IL-15 and Fc-IL-2 were only superior to Cetuximab at the highest concentration. However, TriCetu03 (using Cetuximab as the basic backbone, with SIRPα linked to the N-terminus of the heavy chain and an anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) showed superior ADCC activity to Cetuximab. The ADCC activity of TriCetu04 (using Cetuximab as the basic backbone, with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-15 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) and TriCetu12 (using Cetuximab as the basic backbone, with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-2 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) was significantly enhanced.

[0356] 13.2 Study on ADCC activity against human breast cancer cells MDA-MB-468

[0357] Cells were cultured and expanded to the required quantity, and seeded into 96-well plates at a rate of 4000 cells / well. Cells adhered overnight. Fresh human PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at an effector-to-target ratio of 10:1, with concentration gradients of 500 nM, 5 nM, 0.05 nM, 0.0005 nM, 0.000005 nM, 0.00000005 nM, and 0.000000005 nM. After culturing for 5 days, the plates were washed three times with PBS. The number of viable cells was determined using a CCK-8 assay. The IC50 was calculated based on the pharmacodynamic efficacy. 50 The results are shown in Figure 15B. In the ADCC activity assay targeting the EGFR-expressing tumor cell line MDA-MB-468, Cetuximab exhibited some ADCC activity. The ADCC activities of Fc-IL-15 and Fc-IL-2 were only superior to Cetuximab at the highest concentration. However, TriCetu03 (using Cetuximab as the basic backbone, with SIRPα linked to the N-terminus of the heavy chain and an anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) showed superior ADCC activity to Cetuximab. The ADCC activity of TriCetu04 (using Cetuximab as the basic backbone, with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-15 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) and TriCetu12 (using Cetuximab as the basic backbone, with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-2 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) was significantly enhanced.

[0358] The ADCC activity of the two EGFR-expressing tumor cell lines above showed that the ISE component of the complete immune antibody (SIRPαD1 protein, anti-CD16a antibody, cytokine IL-15 or IL-2) can significantly enhance the ADCC activity of anti-EGFR antibody.

[0359] Example 14: Study on ADCC activity of a complete immune antibody with anti-Claudin18.2 antibody as the backbone.

[0360] Human gastric cancer cells NCI-N87 were cultured and expanded to the required quantity. Cells were seeded at 4000 cells / well in 96-well plates and allowed to adhere overnight. Fresh human PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at an effector-to-target ratio of 10:1, with concentration gradients of 500 nM, 5 nM, 0.05 nM, 0.0005 nM, 0.000005 nM, 0.00000005 nM, and 0.0000000005 nM. After culturing for 5 days, the plates were washed three times with PBS, and the number of viable cells was detected using CCK-8 assay. The results are shown in Figure 16. The complete immune antibodies TriZolbetu06 (based on Zolbetuximab with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-15 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) and TriZolbetu12 (based on Zolbetuximab with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-2 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) targeting the Claudin18.2 tumor-associated antigen exhibited good ADCC activity against human gastric cancer cells NCI-N87 (Claudin18.2 positive).

[0361] Example 15: Study on ADCC activity of a complete immune antibody with anti-CTLA-4 antibody as the backbone

[0362] Human breast cancer cells HCC-1954 were cultured and expanded to the required quantity. Cells were seeded at 4000 cells / well in 96-well plates and allowed to adhere overnight. Fresh human PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at an effector-to-target ratio of 10:1, with concentration gradients of 500 nM, 5 nM, 0.05 nM, 0.0005 nM, 0.000005 nM, 0.00000005 nM, and 0.0000000005 nM. After culturing for 5 days, the plates were washed three times with PBS, and the number of viable cells was detected using CCK-8 assay. The results are shown in Figure 17. The complete immune antibodies TriIpilimu06 (based on ipilimumab with SIRPα linked at the N-terminus of the heavy chain and cytokine IL-15 and anti-CD16a single-chain antibody linked at the C-terminus of the heavy chain) and TriIpilimu12 (based on ipilimumab with SIRPα linked at the N-terminus of the heavy chain and cytokine IL-2 and anti-CD16a single-chain antibody linked at the C-terminus of the heavy chain) targeting the CTLA-4 acquired immune checkpoint exhibited good ADCC activity against human breast cancer cells HCC-1954.

[0363] Example 16: Study on ADCC activity of a complete immune antibody with anti-BCMA antibody as the backbone

[0364] Human Burkitt's lymphoma cells (Raji) were cultured and expanded to the required quantity. Cells were seeded at 10,000 cells / well in 96-well plates and allowed to adhere overnight. Fresh human PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at an effector-to-target ratio of 10:1, with concentration gradients of 500 nM, 5 nM, 0.05 nM, 0.0005 nM, 0.000005 nM, 0.00000005 nM, and 0.0000000005 nM. After culturing for 5 days, the plates were washed three times with PBS, and the number of viable cells was detected using CCK-8 assay. The results are shown in Figure 18. The complete immune antibodies TriBelanta06 (based on Belantamab with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-15 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) and TriBelabta12 (based on Belantamab with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-2 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) targeting BCMA tumor-associated antigens exhibited good ADCC activity against human lymphoma cells Raji (BCMA positive).

[0365] Example 17 Study on ADCC activity of a complete immune antibody with anti-CD20 antibody as the backbone

[0366] Human Burkitt's lymphoma cells (Raji) were cultured and expanded to the required quantity. Cells were seeded at a rate of 10,000 cells / well into 96-well plates and allowed to adhere overnight. Fresh human PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at an effector-to-target ratio of 10:1, with concentration gradients of 500 nM, 5 nM, 0.05 nM, 0.0005 nM, 0.000005 nM, 0.00000005 nM, and 0.0000000005 nM. After culturing for 5 days, the plates were washed three times with PBS, and the number of viable cells was detected using CCK-8 assay. The results are shown in Figure 19. The complete immune antibodies TriRituxi06 (based on Rituximab with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-15 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) and TriRituxi12 (based on Rituximab with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-2 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) targeting CD20 tumor-associated antigen showed good ADCC activity against human lymphoma cells Raji (CD20 positive).

[0367] Example 18: Study on ADCC activity of a complete immune antibody with anti-CD38 antibody as the backbone

[0368] Human Burkitt's lymphoma cells (Raji) were cultured and expanded to the required quantity. Cells were seeded at 10,000 cells / well in 96-well plates and allowed to adhere overnight. Fresh human PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at an effector-to-target ratio of 10:1, with concentration gradients of 500 nM, 5 nM, 0.05 nM, 0.0005 nM, 0.000005 nM, 0.00000005 nM, and 0.0000000005 nM. After culturing for 5 days, the plates were washed three times with PBS, and the number of viable cells was detected using CCK-8 assay. The results are shown in Figure 20. The complete immune antibodies TriDaratu06 (based on daratumumab with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-15 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) and TriDaratu12 (based on daratumumab with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-2 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) targeting the CD38 tumor-associated antigen showed good ADCC activity against human lymphoma cells Raji (CD38 positive).

[0369] Example 19: Study on ADCC activity of a complete immune antibody with anti-Trop-2 antibody as the backbone

[0370] Human gastric cancer cells NCI-N87 were cultured and expanded to the required quantity. Cells were seeded at 4000 cells / well in 96-well plates and allowed to adhere overnight. Fresh human PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at an effector-to-target ratio of 10:1, with concentration gradients of 500 nM, 5 nM, 0.05 nM, 0.0005 nM, 0.000005 nM, 0.00000005 nM, and 0.000000005 nM. After culturing for 5 days, the plates were washed three times with PBS, and the number of viable cells was detected using CCK-8 assay. The results are shown in Figure 21. The complete immune antibodies TriSacitu06 (based on Sacituzumab with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-15 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) and TriSacitu12 (based on Sacituzumab with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-2 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) targeting Trop-2 tumor-associated antigen exhibited strong ADCC activity against human gastric cancer cells NCI-N87 (Trop-2 positive).

[0371] Example 20: Study on ADCC activity of a complete immune antibody with anti-Nectin-4 antibody as the backbone

[0372] Human gastric cancer cells NCI-N87 were cultured and expanded to the required quantity. Cells were seeded at 4000 cells / well in 96-well plates and allowed to adhere overnight. Fresh human PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at an effector-to-target ratio of 10:1, with concentration gradients of 500 nM, 5 nM, 0.05 nM, 0.0005 nM, 0.000005 nM, 0.00000005 nM, and 0.0000000005 nM. After culturing for 5 days, the plates were washed three times with PBS, and the number of viable cells was detected using CCK-8 assay. The results are shown in Figure 22. The complete immune antibodies TriEnfortu06 (based on Enfortumab with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-15 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) and TriEnfortu12 (based on Enfortumab with SIRPα linked to the N-terminus of the heavy chain and cytokine IL-2 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) targeting Nectin-4 tumor-associated antigen exhibited good ADCC activity against human gastric cancer cells NCI-N87 (Nectin-4 positive).

[0373] Example 21: Study on ADCC activity of a complete immune antibody with an anti-PD-1 antibody as the backbone

[0374] Human gastric cancer cells NCI-N87 were cultured and expanded to the required quantity. Cells were seeded at 4000 cells / well in 96-well plates and allowed to adhere overnight. Human PBMCs were incubated with SEB in vitro for 7 days. When PD1 expression was greater than or equal to 16%, the PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at an effector-to-target ratio of 10:1, with concentration gradients of 500 nM, 5 nM, 0.05 nM, 0.0005 nM, 0.000005 nM, 0.00000005 nM, and 0.0000000005 nM. After culturing for another 5 days, the plates were washed three times with PBS, and the number of viable cells was detected using CCK-8 assay. The results are shown in Figure 23. The complete immune antibodies TriKey06 (based on Pembrolizumab with SIRPα linked at the N-terminus of the heavy chain and cytokine IL-15 and anti-CD16a single-chain antibody linked at the C-terminus of the heavy chain) and TriKey12 (based on Pembrolizumab with SIRPα linked at the N-terminus of the heavy chain and cytokine IL-2 and anti-CD16a single-chain antibody linked at the C-terminus of the heavy chain) targeting PD-1 acquired immunity exhibited good ADCC activity against human gastric cancer cells NCI-N87.

[0375] Example 22: Study on ADCC activity of a complete immune antibody with anti-Her2 antibody as the backbone

[0376] Human gastric cancer cells NCI-N87 were cultured and expanded to the required quantity. Cells were seeded at 4000 cells / well in 96-well plates and allowed to adhere overnight. Fresh human PBMCs were resuspended in 1640 medium. Antibody was added to the 96-well plates at an effector-to-target ratio of 10:1, with concentration gradients of 500 nM, 5 nM, 0.05 nM, 0.0005 nM, 0.000005 nM, 0.00000005 nM, and 0.000000005 nM. After culturing for 5 days, the plates were washed three times with PBS. The number of viable cells was detected using the CCK-8 assay, and the IC50 was calculated based on the pharmacodynamic efficacy. 50 The results are shown in Figure 24. The complete immune antibodies TriTrastu06 (based on Trastuzumab with SIRPα linked to the N-terminus of the heavy chain and IL-15 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) and TriTrastu12 (based on Trastuzumab with SIRPα linked to the N-terminus of the heavy chain and IL-2 and anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) targeting Her2 tumor-associated antigen exhibited good ADCC activity against human gastric cancer cells NCI-N87 (Her2 positive).

[0377] Example 23: Pharmacodynamic study of ExVivo organoids based on anti-Her2 complete immune antibodies.

[0378] 23.1 Her2 2 + Pleural effusion samples from breast cancer patients

[0379] Collect Her2 2 + In breast cancer pleural effusion samples, cells, including tumor cells and immune cells, were isolated. Candidate and control drugs were co-incubated with tumor-immune cell complexes in vitro. After 4-5 days of incubation, the culture supernatant was collected to detect tumor cell granzyme B and IFN-γ, and the tumor cells and immune cells were analyzed. In this example, the long chain amino acid sequence of the anti-Her2 complete immune antibody TriAB04 (based on Trastuzumab, with SIRPα linked to the C-terminus of the light chain and an anti-CD16a single-chain antibody linked to the C-terminus of the heavy chain) is SEQ ID NO:82, and the short chain amino acid sequence is SEQ ID NO:83. The results are shown in Figure 25. The immune cell phenotype of this sample (Figure 25-A): NK cells 6.5%; macrophages 2.11% (M1 0.06%, M2 0.33%); CD3+... + T cells 5.47%; CD4 + T cells 2.93%; CD8 +T cells accounted for 1.66%. The killing effect of anti-Her2 complete immune antibody on Her2-positive tumor cells was increased by 27% compared to baseline (Figure 25-B). The combination of anti-Her2 complete immune antibody, anti-PD-1 antibody, or Herceptin with TTI-622 (Fc-SIRPα fusion molecule) showed weak IFN-γ secretion, while the combination with anti-PD-1 antibody significantly enhanced IFN-γ secretion (Figure 25-D). Combination with anti-PD-1 antibody also significantly increased granzyme B levels (Figure 25-C). Since the combination of TTI-622 and Herceptin did not show significant tumor-killing activity, while the anti-Her2 complete immune antibody showed significant anti-tumor activity, combined with the immune cell analysis results (NK cells 6.5% (within the normal range of peripheral blood levels) and macrophages 2.11% (significantly lower than the normal level of peripheral blood), it can be inferred that the anti-CD16a single-chain antibody portion of the complete immune antibody played a key role in this tumor-killing process.

[0380] 23.2 Ascites samples from Her2-negative ovarian cancer patients

[0381] Ascites samples from Her2-negative ovarian cancer were collected, and cells, including tumor cells and immune cells, were isolated from the samples. Candidate and control drugs were co-incubated with the tumor cell and immune cell complex in vitro. After 4-5 days of incubation, the culture supernatant was collected to detect tumor cell granzyme B and IFN-γ, and the tumor cells (EpCAM+) and immune cells were analyzed. The results are shown in Figure 26. The immune cell phenotype of this sample (Figure 26-A) was as follows: NK cells 0.5%; macrophages 10.74% (M1 0.41%, M2 0.05%); CD3+. + T cells 5.98%; CD4 + T cells 4.36%; CD8 +T cell count was 0.97%. Results showed that, compared to the untreated group, anti-PD-1 antibody had no significant tumor cell killing function. The anti-Her2 complete immune antibody group or the group using Herceptin combined with TTI-622 showed significantly increased tumor cell killing ability (approximately 2.5-fold), while the combination of anti-Her2 complete immune antibody and anti-PD-1 antibody did not further improve efficacy (Figure 26-B). Anti-Her2 complete immune antibody or anti-PD-1 antibody did not increase the secretion of granzyme B (Figure 26-C) or IFN-γ (Figure 26-D), while the combination of anti-Her2 complete immune antibody and anti-PD-1 antibody significantly increased the secretion of granzyme B or IFN-γ. Since the patient's NK cell percentage was 0.5% (significantly lower than normal peripheral blood levels) and macrophage percentage was 10.74% (within normal peripheral blood levels), and the Herceptin and TTI-622 combination group demonstrated significant tumor-killing function, it can be inferred that the SIRPα component of the complete immune antibody (which blocks the "don't eat me" signal, thereby activating macrophages) plays a key role in tumor killing.

[0382] Example 24: Organ toxicity study of a complete immune antibody based on an anti-PD-L1 antibody in mice.

[0383] Seven-week-old Balb / c mice meeting the experimental conditions were divided into nine groups of three animals each. The mice were administered the drug intravenously on Day 0 and Day 4. From the start of the experiment, the animals' clinical condition, including mental state, activity level, diet, and water intake, was monitored until euthanasia. On Day 7, after euthanasia with carbon dioxide, the mice were grossly dissected, and the liver, spleen, and kidneys were weighed. The results are shown in Figure 27. Spleen weight: High-dose (16.99 mg / kg) and low-dose (8.49 mg / kg) administration of the complete immune antibody TriAtezo06 (containing cytokine IL-15) did not significantly change spleen weight after two administrations, while the spleen weight of the Fc-IL-15 administration group increased significantly. The complete immune antibody TriAtezo12 (containing cytokine IL-2) showed a slight increase in spleen weight after two administrations, while Fc-IL-2 showed a significant increase in spleen weight after two administrations (Figure 27-A). Liver weight: The liver weight of mice did not change significantly after two administrations of high-dose (16.99 mg / kg) and low-dose (8.49 mg / kg) complete immune antibody TriAtezo06 (containing cytokine IL-15) and high-dose (17.33 mg / kg) and low-dose (8.66 mg / kg) complete immune antibody TriAtezo12 (containing cytokine IL-2). The liver weight of mice in the Fc-IL-15 administration group also did not change significantly. However, the liver weight of mice in the high-dose Fc-IL-2 group (5.63 mg / kg) increased significantly after two administrations (Figure 27-B). Kidney weight: No significant changes in kidney weight were observed in mice after two administrations of the high-dose (16.99 mg / kg) and low-dose (8.49 mg / kg) complete immune antibody TriAtezo06 (containing cytokine IL-15), and the high-dose (17.33 mg / kg) and low-dose (8.66 mg / kg) complete immune antibody TriAtezo12 (containing cytokine IL-2). No significant changes in kidney weight were also observed in the Fc-IL-15 administration group. However, kidney weight significantly increased after two administrations in the high-dose (5.63 mg / kg) and low-dose (2.82 mg / kg) Fc-IL-2 groups (Figure 27-C). These organ toxicity results indicate that both the complete immune antibodies TriAtezo06 and TriAtezo12 exhibited good safety profiles compared to wild-type cytokines.

[0384] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

[0385] References:

[0386] [1]Danyang Li,Minghua Wu.Pattern recognition receptors in health and diseases.Signal Transduction and Targeted Therapy.6:291(2021)

[0387] [2]Liu YJ.IPC:professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors.Annu Rev Immunol.2005;23:275-306

[0388] [3]A.Ribas,I.Mehmi,T.Medina,C.Lao et al.Phase 1b / 11 study of the combination of SD-101 and pembrolizumab in patients with advanced melanoma who had progressive disease on or after prior anti-PD-1 therapy.Annals of Oncology.Volume 29,Supplement 8,October 2018.

[0389] [4]Yoji Murata,Yasuyuki Saito,Takenori Kotani,Takashi Matozaki.Blockade of CD47 or SIRPα:a new cancer immunotherapy.Expert Opinion on Therapeutic Targets.

[0390] [5]https: / / www.gilead.com / news-and-press / company-statements / gilead-statement-on-the-discontinuation-of-magrolimab-study-in-aml-with-tp53-mutations

[0391] [6]Cai Zhang,Yuan Hu,Chongdeng Shi.Targeting Natural Killer Cells for Tumor Immunotherapy.Frontiers in Immunology.Article 60 Volume 11 February 2020.

[0392] [7]https: / / www.affimed.com / affimed-receives-fda-fast-track-designation-for-afm13-in-combination-with-allonk-for-the-treatment-of-patients-with-relapsed-or-refractory-hodgkin-lymphoma /

[0393] [8]Yawu Jing,Zhenya Ni,Jianming Wu,LeeAnn Higgins et al.Identification of an ADAM17 cleavage region in human CD16(FcgRIII)and the engineering of a non-cleavable version of the receptors in NK cells.

Claims

1. An innate immune superconnector (ISE), characterized in that... The innate immune superconnector recruits or activates innate immune cells and comprises two or more of the following components: (a) anti-CD16a antibody, (b) SIRPαD1 protein, (c) immune cytokines, and (d) pattern recognition receptor agonists.

2. A complete immune antibody (CIA), characterized in that... Recruiting or activating innate and acquired immune cells to kill tumor cells, the complete immune antibody comprises (a) the ISE of claim 1 or a fragment thereof, and (b) an acquired immune antibody or a tumor-associated antigen antibody.

3. The innate immune superconnector according to claim 1, characterized in that, Using traditional antibodies as the basic structure, the variable region of the antibody is removed or a human-free target antibody is used. Anti-CD16a antibody or SIRPαD1 protein is linked to the N-terminus or C-terminus of the antibody heavy chain constant region via a linker, and SIRPαD1 protein or anti-CD16a antibody is linked to the N-terminus or C-terminus of the antibody light chain constant region via a linker.

4. The innate immune superconnector according to claim 1, characterized in that, Using traditional antibodies as the basic structure, the variable region of the antibody is removed or a non-targeting antibody in the human body is used. One or more of human immune cytokines, human immune cytokine receptors, SIRPαD1 protein and anti-CD16a antibodies are linked to the N-terminus and / or C-terminus of the antibody heavy chain constant region and / or antibody light chain constant region via linkers.

5. The innate immune superconnector according to any one of the preceding claims, characterized in that, Pattern recognition receptor agonists are connected to the N- or C-terminus of the ISE via linkers. These pattern recognition receptor agonists include, but are not limited to, Toll-like receptor (TLR) agonists, NOD-like receptor (NLR) agonists, RIG-I-like receptor (RLR) agonists, C-type lectin receptor (CLR) agonists, and melanoma deficiency factor 2 (AIM2)-like receptor (ALR) agonists.

6. The innate immune superconnector according to any one of the preceding claims, characterized in that, Human IL-2 or IFNα or its mutants are linked to the N-terminus or C-terminus of the ISE via a linker.

7. The complete immune antibody according to claim 2, characterized in that, Using ISE as the basic structure, a variable region of acquired immune antibodies or a variable region of tumor-associated antigen antibodies is added to the N-terminus of ISE.

8. The complete immune antibody according to claim 7, characterized in that, Using acquired immune antibodies or tumor-associated antigen antibodies as the basic backbone, one or more of human immune cytokines, human immune cytokine receptors, SIRPαD1 protein, and anti-CD16a antibodies are linked to the N-terminus and / or C-terminus of the antibody heavy chain and / or antibody light chain via linkers.

9. The complete immune antibody according to any one of the preceding claims, characterized in that, Tumor-associated antigens such as, but not limited to, PD-L1, Her2, VEGF, EGFR, CD38, Claudin18.2, Claudin6, CD19, MSLN, Trop-2, CD20, Nectin-4, c-Met, MUC16, MUC17, 5T4, BCMA, DLL3, or CD30.

10. The complete immune antibody according to any one of the preceding claims, characterized in that, Acquired immune antibodies are antibodies that target, for example but not limited to, the following targets: PD-1, CTLA-4, B7-H4, B7-H3, 4-1BB, LAG-3, TIGIT, TIM-3, LILRB2, CD24, CD40, or CD40L.

11. The innate immune superconnector or complete immune antibody according to any one of the preceding claims, characterized in that, Human immune cytokines such as, but not limited to, IL-15, IL-18, IL-2, IL-7, IL-10, or IL-12.

12. The innate immune superconnector according to claim 11, characterized in that, Using traditional antibodies as the basic structure, the variable region of the antibody is removed or a human-free target antibody is used. One or more of anti-CD16a antibody, human IL-15 or IL-15Rα are linked to the N-terminus or C-terminus of the antibody heavy chain constant region via linkers, and SIRPαD1 protein is linked to the N-terminus or C-terminus of the antibody light chain constant region via linkers.

13. The innate immune superconnector or complete immune antibody according to any one of the preceding claims, characterized in that, The anti-CD16a antibody is an anti-CD16a single-chain antibody or an anti-CD16a nanobody.

14. The innate immune superconnector or complete immune antibody according to any one of the preceding claims, characterized in that, The heavy chain variable region of the anti-CD16a single-chain antibody includes: CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; the light chain variable region includes: CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:

6.

15. The innate immune superconnector or complete immune antibody according to any one of the preceding claims, characterized in that, The heavy chain variable region of the anti-CD16a single-chain antibody contains the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:

8.

16. The innate immune superconnector or complete immune antibody according to any one of the preceding claims, characterized in that, The anti-CD16a single-chain antibody contains the amino acid sequence shown in SEQ ID NO:

9.

17. The innate immune superconnector or complete immune antibody according to any one of the preceding claims, characterized in that, The anti-CD16a nanobody comprises: CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:

12.

18. The innate immune superconnector or complete immune antibody according to any one of the preceding claims, characterized in that, The anti-CD16a nanobody contains the amino acid sequence shown in SEQ ID NO:

13.

19. The innate immune superconnector or complete immune antibody according to any one of the preceding claims, characterized in that, The SIRPαD1 protein contains the amino acid sequence shown in SEQ ID NO:

14.

20. The innate immune superconnector or complete immune antibody according to any one of the preceding claims, characterized in that, The human IL-15 protein fragment contains the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:16, and the human IL-15Rα protein fragment contains the amino acid sequence shown in SEQ ID NO:

17.

21. The innate immune superconnector according to claim 5, characterized in that, The pattern recognition receptor agonist is CpG ODN, preferably, the CpG ODN contains the sequence shown in SEQ ID NO:

117.

22. The innate immune superconnector according to claim 6, characterized in that, The IL-2 mutant contains the amino acid sequence shown in SEQ ID NO:118, and / or the IFNα mutant contains the amino acid sequence shown in SEQ ID NO:

119.

23. The innate immune superconnector according to any one of the preceding claims, characterized in that, The basic framework is antibodies that do not target the human body, such as anti-hen egg lysozyme HEL antibody, and antibodies against pathogenic microorganisms such as antiviral and bacterial antibodies.

24. The innate immune superconnector according to claim 23, characterized in that, Using anti-egg white lysozyme antibody as the basic backbone, one or more of human immune cytokines, human immune cytokine receptors, SIRPαD1 protein and anti-CD16a antibody are linked to the N- or C-terminus of the antibody heavy chain and / or the N- or C-terminus of the antibody light chain via linkers.

25. The innate immune superconnector according to claim 24, characterized in that, The long-chain amino acid sequence contains SEQ ID NO:20, and the short-chain amino acid sequence contains SEQ ID NO:21, or the long-chain amino acid sequence contains SEQ ID NO:22, and the short-chain amino acid sequence contains SEQ ID NO:23, or the long-chain amino acid sequence contains SEQ ID NO:24, and the short-chain amino acid sequence contains SEQ ID NO:25, or the long-chain amino acid sequence contains SEQ ID NO:26, and the short-chain amino acid sequence contains SEQ ID NO:27, or the long-chain amino acid sequence contains SEQ ID NO:28, and the short-chain amino acid sequence contains SEQ ID NO:

29.

26. The innate immune superconnector according to any one of the preceding claims, characterized in that, It has the configurations ISE-A, ISE-B, ISE-C, ISE-D, ISE-E, ISE-F, ISE-G, ISE-H, ISE-I, or ISE-J. Preferably, the long chain amino acid sequence of the innate immune connector ISE-A contains SEQ ID NO:30 or SEQ ID NO:32, and the short chain amino acid sequence contains SEQ ID NO:31 or SEQ ID NO:33; the long chain amino acid sequence of the innate immune connector ISE-B contains SEQ ID NO:34 or SEQ ID NO:36, and the short chain amino acid sequence contains SEQ ID NO:35 or SEQ ID NO:37; the long chain amino acid sequence of the innate immune connector ISE-C contains SEQ ID NO:38 or SEQ ID NO:40, and the short chain amino acid sequence contains SEQ ID NO:39 or SEQ ID NO:41; the long chain amino acid sequence of the innate immune connector ISE-D contains SEQ ID NO:42 or SEQ ID NO:44, and the short chain amino acid sequence contains SEQ ID NO:43 or SEQ ID NO:

44. NO:45, the long chain amino acid sequence of the innate immune connector ISE-E includes SEQ ID NO:46 or SEQ ID NO:48, and the short chain amino acid sequence includes SEQ ID NO:47 or SEQ ID NO:49; the long chain amino acid sequence of the innate immune connector ISE-F includes SEQ ID NO:50 or SEQ ID NO:52, and the short chain amino acid sequence includes SEQ ID NO:51 or SEQ ID NO:53; the long chain amino acid sequence of the innate immune connector ISE-G includes SEQ ID NO:54 or SEQ ID NO:56, and the short chain amino acid sequence includes SEQ ID NO:55 or SEQ ID NO:57; the long chain amino acid sequence of the innate immune connector ISE-H includes SEQ ID NO:58, and the short chain amino acid sequence includes SEQ ID NO:59 or SEQ ID NO:60; the long chain amino acid sequence of the innate immune connector ISE-I includes SEQ ID NO:61 or SEQ ID NO:63, and the short chain amino acid sequence includes SEQ ID NO:62 or SEQ ID NO:

63. NO:64, the long chain amino acid sequence of the innate immune connector ISE-J contains SEQ ID NO:65, and the short chain amino acid sequence contains SEQ ID NO:66 or SEQ ID NO:

67.

27. The complete immune antibody according to claim 7, characterized in that, The long-chain amino acid sequence is based on the amino acid sequence of the ISE, with the introduction of heavy chain variable regions of tumor-associated antigens / antibodies or acquired immune antibodies. The short-chain amino acid sequence is based on the amino acid sequence of the ISE, with the introduction of light chain variable regions of tumor-associated antigens / antibodies or acquired immune antibodies.

28. The complete immune antibody according to claim 27, characterized in that, The complete immune antibodies are TriAtezo01, TriAtezo02, TriAtezo03, TriAtezo04, TriAtezo05, TriAtezo06, TriAtezo07, TriAtezo12, TriCetu03, TriCetu04, and TriCetu12. Their long chain amino acid sequences include SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, and SEQ ID NO:120, respectively; their short chain amino acid sequences include SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:80, and SEQ ID NO:120, respectively. SEQ ID NO:80 and SEQ ID NO:80, and / or the complete immune antibody is a complete immune antibody TriKey06, TriKey12, TriDaratu06, TriDaratu12, TriRituxi06, TriRituxi12, TriZolbetu06, TriZolbetu12, TriSacitu06, TriSacitu12, TriTrastu06, TriTrastu12, TriBeva06, TriBeva12, TriEnfortu06, TriEnfortu12, TriBelanta06, TriBelanta12, TriIpilimu06 and TriIpilimu12, whose long chain amino acid sequences respectively contain SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:80, SEQ ID NO:80, and SEQ ID NO:

80. NO:110, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:101 and SEQ ID NO:103;The short-chain amino acid sequences include SEQ ID NO:87, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:99, SEQ ID NO:111, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:114, SEQ ID NO:108, SEQ ID NO:108, SEQ ID NO:105, SEQ ID NO:105, SEQ ID NO:102, and SEQ ID NO:102, respectively.

29. The innate immune superconnector or complete immune antibody according to any one of the preceding claims, characterized in that, The antibody may have L234A and L235A mutations introduced into its Fc segment, or further M252Y, S254T and T256E mutations introduced into its Fc segment.

30. The innate immune superconnector or complete immune antibody according to any one of the preceding claims, characterized in that, The connectors include, but are not limited to, PSGQAGAAASESLFVSNHAY or (G4S). n , where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

31. A nucleic acid molecule encoding an innate immune superconnector or a complete immune antibody as described in any of the preceding claims.

32. A carrier comprising the nucleic acid molecule of claim 31.

33. A cell comprising the nucleic acid molecule of claim 31 or the vector of claim 32.

34. A method for preparing an innate immune superconnector or a complete immune antibody as described in any of the preceding claims, the method comprising the following steps: (1) Amplify the two strands of ISE or CIA respectively using PCR technology; introduce mutations L234A, L235A, M252Y, S254T and T256E into the Fc fragment; (2) The DNA fragment obtained in step (1) is cloned into pcDNA series vectors or other vectors used for mammalian cell expression systems to obtain a recombinant vector; (3) The recombinant vector plasmid obtained in step (2) is transfected into mammalian cells to express the recombinant protein, and the purified target protein is obtained after affinity chromatography.

35. Use of any of the preceding claims of the innate immune superconnector or complete immune antibody in the preparation of a medicament for treating solid tumors and hematological malignancies in a subject.

36. The use according to claim 35, wherein the solid tumors and hematologic malignancies include, but are not limited to, respiratory tract tumors, digestive tract tumors, reproductive system tumors, nervous system tumors, urinary system tumors, skin tumors, head and neck tumors, and hematologic malignancies; preferably, the solid tumors and hematologic malignancies include, but are not limited to, nasopharyngeal carcinoma, laryngeal carcinoma, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, ovarian cancer, testicular cancer, brain tumors, gliomas, kidney cancer, bladder cancer, ureteral cancer, epithelial tumors, melanoma, eye tumors, nasal tumors, ear tumors, oral tumors, leukemia, multiple myeloma, lymphoma, breast cancer, and epidermal cancer.

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