Triple-functional fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation, and its applications

Abstract

A trifunctional fusion protein for antigen targeting, anti-CD16A and immune effector cell activation is disclosed, which includes a CD16A binding region that specifically binds to CD16A, a TAA binding region that specifically binds to a tumor-associated antigen (TAA), an IL-15 / IL-15Rα complex formed by the binding of IL-15 to IL-15Rα, and an Fc domain. The designed tumor-targeting innate immune cell activation molecule is a trifunctional fusion protein of anti-TAA, anti-CD16A and the cytokine IL15, which targets NK cells to tumors by simultaneously binding to tumor-associated antigens (TAA) on tumor cells and CD16A on NK cells, and the NK cells release perforin and granzymes, causing apoptosis and tumor cell death. The cytokine IL15 expands and maintains the function of NK cells, stimulates the proliferation of immune cells and changes the immune microenvironment of tumors.

Description

Detailed Description of the Invention

[0001] [Technical Field] The present invention relates to the field of biomedical technology, and specifically, to a three-functional fusion protein of antigen targeting, anti-CD16A and immune effector cell activation, and its applications.

[0002] [Background Art] Natural killer (NK) cells are innate immune cells that can very effectively destroy stressed cells such as virus-infected or tumor-transformed cells. NK cells have functions such as MHC-independent cytotoxicity, cytokine production, and immune memory, and play an important role in the immune response system. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an effective cytotoxic mechanism mediated by natural killer (NK) cells. The ADCC effect by CD16a is one of the main mechanisms of the anti-tumor effect of NK cells. When a specific antibody binds to the membrane of a target tumor cell, immune cells induce cell death through this reaction.

[0003] The interaction between antibodies and immune cells occurs through the Fc receptor (FcR) family. In humans, the IgG FcR family (FcγR) consists of six receptors: FcγRI / CD64, FcγRIIa / CD32a, FcγRIIb / CD32b, FcγRIIc / CD32c, FcγRIIIa / CD16a, and FcγRIIIb / CD16b, where CD16a primarily causes NK cell-mediated ADCC. Normally, the ADCC effect activates NK cells mainly through antibodies that bind to FcγRIII (CD16). Human NK cells are divided into two main subpopulations: CD56 bright cells and CD56 dim cells. CD56 bright NK cells are potent cytokine-producing cells but lack CD16a, while CD56 dim NK cells are highly cytotoxic and express CD16a. When NK cells identify IgG opsonized target sites via CD16a, they release various cytotoxic molecules, thereby inducing the death of target cells. Such mechanisms rely on the formation of immunological synapses and the degranulation of lysing granules containing perforin and granzymes. In addition to degranulation, NK cells can also eliminate target cells by binding to target death receptors (e.g., DR4, DR5, or Fas) and their death receptor ligands (e.g., FasL and TRAIL).

[0004] CD16a is a transmembrane receptor with a short C-ter cytoplasmic tail and two extracellular Ig-like domains. CD16a interacts with IgG's CH2 in a 1:1 ratio, where the N-glycan chain at position N297 on CH2 also plays a crucial role in this interaction. Since CD16a lacks an ITAM domain in its cytoplasmic tail, it requires the assistance of two tandem intracellular chains containing the ITAM domains of CD3ζ and FcεRIγ. After CD16a binding, the Src family kinase Lck is activated and phosphorylates the ITAM domains of CD3ζ and / or FcεRIγ. Phosphorylated ITAM allows for recruitment and phosphorylation of kinases from the Syk family, such as Syk and ZAP-70, which in turn participate in subsequent signaling. Among their substrates, PI3K is highly relevant as it transforms PIP2 into PIP3, and PIP3 releases IP3 and DAG after being treated with PLC-γ. DAG activates the PKC family, which helps induce degranulation. IP3 induces calcium release from the endoplasmic reticulum, and such calcium influx is one of the main signals triggered by ADCC, causing translocation of NFAT in the nucleus and inducing transcription of its target genes. Other pathways activated by CD16a, such as the ERK2 MAPK pathway, also contribute to ADCC. After CD16a-dependent NK cell killing, CD16a is rapidly downregulated. One mechanism of CD16a elimination is mediated by disintegrin and metalloproteinase 17 (ADAM17), the latter of which is expressed in NK cells. Cleavage of CD16a by ADAM17 occurs in a cis structure, indicating that NK cells expressing ADAM17 cannot induce CD16a elimination in other NK cells. Another mechanism of post-activation CD16a downregulation is internal translocation, which occurs not only with CD16a but also with other intracellular signaling components such as CD3ζ, ZAP-70, and Syk.

[0005] NK cells, as killer cells among innate immune cells, can directly kill pathogens and tumor cells. Compared to T cells, NK cells have not only stronger tumor recognition and killing capabilities but also greater potential for ready-made applications. How to modify the constant region (FC terminus) of therapeutic antibody drugs to further activate the innate immune response and enhance antibody-dependent cell-mediated cytotoxicity (ADCC), and how to evaluate the effectiveness of bis(poly)antibodies that can specifically bind to CD16A, are interesting questions for pharmaceutical developers. Currently, strategies to improve the role of ADCC mainly focus on the following aspects:

[0006] (1) Cytokines: A simple way to enhance ADCC activity is to stimulate NK cells with pro-inflammatory cytokines. NK cell transplantation has been successfully performed in several clinical trials, including the injection of cytokine-induced memory-like (CIML) NK cells, and the most commonly used cytokine combination includes IL-12, IL-15, and IL-18. CIML NK cells express more IFN-γ than conventional NK cells and exhibit superior cytotoxicity against leukemia cells and primary acute myeloid leukemia (AML) progenitor cells. IL-12 increases the production of IFN-γ and TNF-α by NK cells, and in Phase I / II clinical trials, cetuximab and IL-12 are used in combination in patients with unresectable or recurrent head and neck squamous cell carcinoma. Compared to patients with a progression-free survival (PFS) of less than 100 days, NK cells from patients with a PFS of more than 100 days have higher ADCC activity in vitro. IL-15 is a cytokine essential for the survival and function of NK cells and CD8+ T cells. N-803 is a mutant N72D IL-15 superagonist that has shown enhanced NK cell activity in vitro and in vivo in preclinical models and clinical trials. Furthermore, it also increased the number of NK and CD8+ T cells.

[0007] (2) Inhibition of ADAM17: ADAM17 is the main driver of post-activation CD16a downregulation. One strategy to improve ADCC in NK cells is to prevent CD16a depletion by targeting ADAM17. Studies have shown that knocking out ADAM17 in NK cells using CRISPR / Cas9 technology results in better IFN-γ production, as well as ADCC activity in vivo and in vitro. However, studies have also shown that blocking ADAM17 reduces NK cell viability and CD16a-mediated serial killing, preventing cells from separating from target cells and migrating to other target cells. In any case, the benefits of ADAM17 inhibition are unclear, and the results are debatable. Whether this strategy is successful will be determined by the results of an ongoing clinical trial (NCT04023071), which will evaluate the application of iPSC-derived NK cells possessing unseverable CD16a in AML and B-cell lymphoma.

[0008] (3) Manipulated Antibodies: Manipulating the Fc portion of an antibody increases its affinity for CD16a, subsequently improving ADCC. Some mutations are very prevalent; for example, the so-called GASDALIE consists of four substitutions in the Fc: G236A / S239D / A330L / I332E. This mutation shows a significant increase in affinity for CD16a, while another mutation called mutant 18 (F243L / R292P / Y300L / V305I / P396L) increases CD32b affinity and significantly increases ADCC. These mutations are now being used clinically as the anti-HER2 antibody margetuximab, which has shown good efficacy in phase I treatment of various HER2-positive cancers. Furthermore, it is expected that glycosylation of the antibody Fc N-glycan will increase its affinity for CD16a. The most studied glycosylation is deglycosylation, such as defucosylation modification. This modification increases binding to CD16a, improving ADCC. This technique is used in the anti-CD20 antibody obinutuzumab.

[0009] (4) NK cell engagers: In addition to engineered antibodies, antibody morphologies can also be studied. Based on the success of the bispecific T cell engager (BiTE) strategy, a similar morphology for NK cells, namely the so-called bispecific killer cell engager (BiKE), is being developed. BiKE is constructed by linking two scFv sequences, with one single-chain antibody targeting CD16a and the other targeting a tumor antigen. Furthermore, there are also triplicate killer cell engagers (TRiKE) that target both tumor antigens together with CD16a, or by adding IL-15 instead of a third single-chain antibody. The use of such structures allows NK cells to relocalize to tumor cells, causing a potent ADCC effect on target cells. AFM13 is a bispecific antibody that targets CD16a and CD30 and is currently in Phase II clinical trials, used to treat peripheral T-cell lymphoma, transformed mycosis fungoides, and Hodgkin lymphoma. In a clinical trial of relapsed or refractory Hodgkin lymphoma (Ib), the combination of AFM13 and pembrolizumab resulted in an objective response rate of 88% and an overall response rate of 83%. GTB-3550 is a CD16 / CD33 / IL-15 TRiKE variant currently being studied in Phase I / II trials for multiple types of leukemia (NCT03214666).

[0010] However, the tumor microenvironment of most tumors, including colon cancer, kidney cancer, liver cancer, breast cancer, and lung cancer, contains a very small number of NK cells. Therefore, the industry is developing immunotherapies that utilize the activity of CD16A to treat cancer, such as bispecific molecules formed by antibodies specific to tumor surface antigens and antibodies specific to NK cell CD16A. Currently, bispecific antibodies for HER2 / neu and CD16A are being developed. AFM13, a bispecific antibody that has stronger antitumor activity than a single HER2 antibody and specifically binds to CD16A and CD30, binds to the CD16 molecule on the surface of NK cells or monocytes, allowing these immune effector cells to target and kill tumor cells expressing CD30. This antibody has shown tumor-clearing effects in mouse models of Hodgkin tumor and in patient therapy. However, prolonged exposure to conventional CD16A antibodies or anti-CD16A antigen-binding proteins can lead to NK cell depletion or a decrease in NK cell toxicity, thereby affecting the tumor-killing effect of NK cells. This makes it impossible to administer CD16A biantibodies for extended periods, which affects their effectiveness.

[0011] Interleukin-15 (IL-15) is a 14-15 kDa cytokine crucial for the function of NK cells, NKT cells, and memory CD8+ T cells. IL-15 binds to its receptor, IL-15Rα, to generate the highly biologically potent complex IL-15 superagonist (IL-15 SA), which is then transduced and transported to target cells. IL-15 SA potently activates cells responsive to IL-15R expression, particularly NK cells / T cells, thereby promoting antitumor and antiviral functions. IL-15 has broad immunomodulatory effects and is involved in regulating the survival, proliferation, and function of T cells, especially NK cells and memory CD8+ T cells. IL-15 and IL-2 are structurally very similar and belong to the spiral cytokine family. The heterotrimeric receptor for IL-15 shares the IL-2 receptor with the IL-2 receptor, specifically the IL-2R / IL-15 Rβ (CD122) and a common γc chain (CD132). These common receptor components and the common JAK 1 / JAK 3 / STAT 5 signaling pathway mean that IL-15 and IL-2 have similar functions, including stimulating T cell proliferation, producing cytotoxic T lymphocytes, stimulating B cells for immunoglobulin synthesis, and producing and sustaining NK cells. In many adaptive immune responses, IL-2 and IL-15 play unique and often competing roles. The four main points are: 1. IL-2 can modulate Tregs cell activation, while IL-15 cannot. 2. IL-2 inhibits the T cell response by activating inducible CD8+ effector T cell death. 3. IL-15 plays a crucial role in the differentiation of NK, effector CD8+, and memory phenotype CD8+ T cells. 4. Preclinical studies have shown that IL-15 toxicity differs from that of IL-2, with IL-15 exhibiting significantly less vascular capillary leakage compared to IL-2. These factors make IL-15 a more potential cytokine in tumor immunotherapy strategies.Currently, several IL-15 target point products are still in clinical trials. The most noteworthy among them is the IL-15 superagonist N-803, a fusion protein that binds to and co-expresses IL-15Ra and IgG1Fc after a mutation in the IL-15 protein N72D. On May 23, 2022, ImmunityBio submitted a marketing application to the FDA for the treatment of non-muscle-invasive bladder cancer (NMIBC) in combination with the BCG vaccine (BCG). Clinical data published in 2021 showed that the combination therapy of N-803 + BCG was highly effective, with a complete response (CR) rate of 71% (59 / 83) and an average CR maintenance time of 24.1 months. Phase 1 clinical trials have shown that nivolumab (PD-1 antibody, Opdivo) combined with N-803 significantly extends long-term survival in patients with metastatic non-small cell lung cancer. Genentech, in collaboration with Xencor, developed the IL-15 cytokine XmAb® 24306 and is currently conducting clinical trials of XmAb® 24306 in combination with Tecentriq. Hengrui Pharmaceuticals Co., Ltd.'s SHR-1501 (IL-15 fusion protein) was approved for clinical trials on May 14, 2019. Furthermore, SHR-1316 (PD-L1 monoclonal antibody drug) is used in combination with SHR-1501 for patients with advanced malignancies who have failed other treatments. However, the clinical use of cytokines has drawbacks, such as insufficient targeting with monotherapy, the lack of antitumor effects unless administered at high concentrations, and the resulting immunosuppressive effects and high toxicity. Furthermore, the activation of the immune system by non-targeted cytokines is systemic, leading to widespread activation and potentially fatal side effects. In addition, cytokines belong to the category of low molecular weight proteins and lack circulatory protection mechanisms like antibodies in the body. As a result, simple cytokines often have short half-lives, requiring repeated administration of high doses over a short period.Currently, clinical research drugs primarily use PEGylation or Fc fusion to extend the half-life of cytokines, but the problem of insufficient cytokine targeting despite the extended half-life remains unresolved.

[0012] [Overview of the prefecture] [Problems the invention aims to solve] The first objective of the present invention is to provide a triplicate fusion protein with novel antigen targeting, anti-CD16A, and immune effector cell activation properties in order to solve the problems of the background art.

[0013] [Means for solving the problem] The tripliquid fusion protein provided by the present invention, which is antigen-targeting, anti-CD16A, and immune effector cell activation, comprises a CD16A-binding domain that specifically binds to CD16A, a TAA-binding domain that specifically binds to tumor-associated antigens (TAAs), an IL-15 / IL-15Rα complex formed by the binding of IL-15 and IL-15Rα, and an Fc domain. The tripliquid fusion protein provided by the present invention not only simultaneously targets CD16A and TAA, but also fuses the IL-15 / IL-15Rα complex and the Fc domain, which have unique components and form a unique structure. Furthermore, the trifunctional fusion protein of the present invention uses IL-15 and IL-15Rα to replace the CL domain and CH1 domain, respectively, in one of the antibody structures of a bispecific antibody. The IL-15 / IL-15Rα structure formed by the ultra-high affinity (KD approximately 30-100 pM) between IL-15 and IL-15Rα ensures correct pairing between the light and heavy chains of the bispecific antibody, thereby resolving the problem of light chain mismatch in bispecific antibodies. Simultaneously, the trifunctional fusion protein CD16A antibody targets NK cells to tumor sites and activates NK cells near tumor cells. Exposure to conventional CD16A antibodies or anti-CD16A antigen-binding proteins for a certain period of time causes NK cell depletion or a decrease in the efficacy of NK cell toxicity, making it impossible to administer CD16A antibodies for extended periods and affecting the effectiveness of tumor treatment. However, the toxicity of damaged NK cells can be restored through stimulation by the cytokines IL-15 and IL-15Rα in the trifunctional fusion protein structure of the present invention, thereby further enhancing the tumor cell-killing effect of NK cells and resolving the problem of NK cell depletion and weakened tumor cell toxicity caused by CD16A antibodies. IL-15 and IL-15Rα stimulate the proliferation and activation of immune effector cells such as NK cells and T cells, significantly enhancing the body's immunosuppressive activity against tumors.

[0014] The CD16A binding region is selectable and contains an anti-CD16A antibody or its antigen-binding fragment. The CD16A binding region is selectable, and the CD16A-targeting Fab or Fv fragment is also selectable.

[0015] The TAA-binding region is selectable and comprises an anti-TAA antibody or its antigen-binding fragment. The TAA-binding region is selectable and is either a Fab fragment or an Fv fragment that targets TAA.

[0016] The fusion protein is selectable and further comprises a linking fragment, the amino acid sequence of which is preferably a few GS repeat sequences.

[0017] The fusion protein is selectable and contains several peptide chains, with IL-15 and IL-15Rα each located on different peptide chains.

[0018] The fusion protein is selectable and comprises a first monomer and a second monomer, wherein the first monomer comprises the TAA binding region and a first Fc chain, and the second monomer comprises the CD16A binding region, the IL-15 / IL-15Rα complex and a second Fc chain, and the first Fc chain and the second Fc chain polymerize to form the Fc domain.

[0019] The TAA binding region is selectable, and the TAA binding region is a Fab fragment that targets TAA, and the CD16A binding region is an Fv fragment that targets CD16A.

[0020] The fusion protein is selectable and comprises a first monomer and a second monomer, wherein the first monomer comprises the CD16A binding region and a first Fc chain, and the second monomer comprises the TAA binding region, the IL-15 / IL-15Rα complex and a second Fc chain, and the first Fc chain and the second Fc chain polymerize to form the Fc domain.

[0021] The CD16A binding region is selectable, and the CD16A binding region is a Fab fragment targeting CD16A, while the TAA binding region is an Fv fragment targeting TAA.

[0022] Selectable, the first monomer comprises a first peptide chain and a second peptide chain, the second monomer comprises a third peptide chain and a fourth peptide chain, the first peptide chain comprises a light chain formed by the fusion of a VL domain and a CL domain, the second peptide chain comprises a VH domain and a heavy chain formed by the fusion of a CH1 domain and a first Fc chain, the third peptide chain comprises an antibody variable region, a cytokine and a second Fc chain, the fourth peptide chain comprises an antibody variable region and a cytokine, and the VL domain of the first peptide chain The CL domain and the VH domain and CH1 domain of the second peptide chain pair to form a Fab fragment, the antibody variable region of the third peptide chain and the antibody variable region of the fourth peptide chain pair to form an Fv fragment, and the cytokine of the third peptide chain and the cytokine of the fourth peptide chain bind to form the IL-15 / IL-15Rα complex, where the Fab fragment targets TAA and the Fv fragment targets CD16A, or the Fab fragment targets CD16A and the Fv fragment targets TAA.

[0023] The selectable antibody variable regions that pair to form the Fv fragment are selected from the VH domain and the VL domain, and the cytokines are selected from IL-15 and IL-15Rα.

[0024] Selectively, between the VH domain and VL domain that pair to form the Fv fragment, there is one or more pairs of disulfide bonds, and includes one or more of the following mutation combinations, counted according to the EU.

[0025] [Table 1]

[0026] Selectable, from the N-terminus to the C-terminus of the peptide chain, the fusion order of the third peptide chain is either antibody variable region~cytokine~second Fc chain, or cytokine~antibody variable region~second Fc chain, and from the N-terminus to the C-terminus of the peptide chain, the fusion order of the fourth peptide chain is either antibody variable region~cytokine or cytokine~antibody variable region, where "~" represents a linker fragment.

[0027] Selectable, the IL-15 includes IL-15, as well as mutations, cleavages and various derivatives that can bind to its IL-15Ra, and the IL-15Ra includes IL-15Ra, as well as mutations, cleavages and various derivatives that can bind to its IL-15. Preferably, the IL-15 includes, but is not limited to, any of the following combinations of mutations. The counting method is counted as the first position based on the first amino acid of the amino acid sequence of IL-15.

[0028] [Table 2] JPEG0007880160000003.jpg70170

[0029] Alternatively, the IL-15 / IL-15Ra complex includes, but is not limited to, any of the following combinations of mutations. The counting method is counted as the first position based on the first amino acid of the amino acid sequence of IL-15 or IL-15Ra.

[0030] [Table 3]

[0031] The following TAAs are selectable: CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD217, CD239, CD274, CD276, CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, EGFR, EGFR Selected from VIII, C-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, CCR4, VEGFR-2, CD6, integrin α4, PDGFRα, NeuGcGM3, integrin αVβ3, CD51, CTAA16.88, CD22, ROR1, CSPG4, SS1, or IGFR1.

[0032] The Fc domain is selectable, and is selected from human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc or its variants, preferably selected from IgG1 Fc or human IgG4 Fc or its variants, and the Fc domain is in the form of an Fc heterodimer, preferably the Fc heterodimer includes, but is not limited to, the following combinations of mutations, the following mutations are counted according to the EU.

[0033] [Table 4] JPEG0007880160000006.jpg245170JPEG0007880160000007.jpg242170

[0034] Selectable, the Fc domain is selected to eliminate immunoeffector function and preferably includes one of the following mutational forms, the following mutations are counted according to EU.

[0035] [Table 5]

[0036] The first Fc chain is selectable, and it does not bind to protein A, preferably containing the mutation H435R or H435R / Y436F, and counted according to EU, while the second Fc chain can bind to protein A.

[0037] The CD16A binding region is selectable, and the CD16A binding region includes a VH domain as shown in SEQ ID NO:39 and a VL domain as shown in SEQ ID NO:38, or the sequence of IL-15 is as shown in SEQ ID NO:46 or 47, or the sequence of IL-15Ra is as shown in any of SEQ ID NO:48 to 54, preferably with at least one pair of disulfide bonds between the VH and VL of the CD16A binding region and / or between IL-15 and IL-15Ra.

[0038] The selectable triplicate fusion protein, which has the properties of antigen targeting, anti-CD16A, and immune effector cell activation, (1)SEQ ID NO:01, 02, 03, 04, (2)SEQ ID NO:01, 05, 03, 06, (3)SEQ ID NO:07, 08, 09, 10, (4)SEQ ID NO:01, 02, 11, 12, (5)SEQ ID NO:01, 02, 13, 14, (6)SEQ ID NO:01, 05, 11, 15, (7) Obtained by fusing with any set of sequences shown in SEQ ID NO:01,05,13,16 or with amino acid fragments having 90% or more identity thereto.

[0039] The antigens are selectable and further include infectious disease-related antigens, pathogens, and immune function-related antigens. The present invention further provides nucleic acid molecules encoding a triplicate fusion protein with the above-mentioned antigen targeting, anti-CD16A, and immune effector cell activation properties.

[0040] The present invention further provides a pharmaceutical composition comprising the above-mentioned triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation, as well as a pharmaceutically acceptable carrier.

[0041] The present invention provides the application of the above-mentioned tripfunctional fusion protein of antigen targeting, anti-CD16A, and immune effector cell activation in the preparation of drugs for inhibiting or treating cancer, infection, and immunomodulatory diseases.

[0042] The aforementioned cancers include prostate cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, melanoma, kidney cancer, oral cancer, pharyngeal cancer, pancreatic cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, or hematological cancers.

[0043] The present invention further provides a protein complex unit comprising a first variable region, a second variable region, a first cytokine, a second cytokine, and several linking fragments, wherein the first variable region is fused to the first cytokine via linking fragments, and the second variable region is fused to the second cytokine via linking fragments, the first and second variable regions are selected from VH domains or VL domains, the first and second variable regions pair to form an FV fragment that specifically binds to CD16A, the first and second cytokines are selected from IL-15 or IL-15Rα, the IL-15 and IL-15Rα are located on different peptide chains, the first and second cytokines bind to form an IL15 / IL15Rα complex, preferably having one or more pairs of disulfide bonds between the VH domain and the VL domain and / or between the IL15 and IL15Rα.

[0044] [Effects of the invention] The beneficial effects of the present invention are as follows: (1) The triphit fusion protein provided by the present invention comprises a CD16A binding domain that specifically binds to CD16A, a TAA binding domain that specifically binds to tumor-associated antigens (TAAs), an IL-15 / IL-15Rα complex formed by the binding of IL-15 and IL-15Rα, and an Fc domain. It not only targets CD16A and TAA simultaneously, but can also fuse the IL-15 / IL-15Rα complex and the Fc domain, which have unique components and form a unique structure.

[0045] (2) The TAA / CD16A / IL15 trifunctional fusion protein designed according to the present invention simultaneously binds tumor-associated antigens (TAAs) on tumor cells and CD16A on NK cells, thereby targeting NK cells to the tumor. NK cells then release perforin and granzymes, inducing apoptosis and tumor cell death. The cytokine IL15 expands and maintains NK cell function, stimulates the proliferation of immune cells, and alters the tumor's immune microenvironment.

[0046] (3) The tripfunctional fusion protein constructed according to the present invention, which has antigen-targeting, anti-CD16A, and immune effector cell activation properties, possesses IL-15 / IL-15Rα activity, targets cytokines to tumor sites, specifically expands and activates immune effector cells (NK cells and T cells) at tumor sites, increases the number of immune cells and the release of killing cytokines, and more effectively activates the immune response to kill tumor cells.

[0047] (4) IL-15 and IL-15Rα replace the CL domain and CH1 domain in the antibody structure, and utilize the high affinity of IL-15 and IL-15Rα to form an IL-15 / IL-15Rα complex, thereby achieving correct pairing of bispecific antibody light chains and resolving the problem of bispecific antibody light chain mismatch. Furthermore, by adding one or more pairs of disulfide bonds between VH and VL, and between IL-15 and IL-15Rα through amino acid sequence mutations, the binding activity between light chains and heavy chains is further enhanced. By combining this with Fc heterodimer technology to resolve the problem of bispecific antibody heavy chain mismatch, the challenges of light chain and heavy chain mismatch, high byproducts, and insufficient stability in the bispecific antibody preparation process can be more effectively overcome, resulting in the preparation of correctly paired multifunctional fusion proteins while simultaneously shortening the development cycle of multifunctional fusion proteins and reducing preparation costs.

[0048] (5) The Fc domain can be used in a form that retains its effector function or in a form that removes the immune effector. When the Fc domain is used in a form that removes the immune effector, Fc cannot bind to FcRγIII(CD16A). When the fusion protein of the present invention retains the effector function of the Fc domain, an activation effect corresponding to immune cells is also observed. [Brief explanation of the drawing]

[0049] [Figure 1] These are schematic diagrams of the structural forms of the triplicate fusion protein in different embodiments of the present invention. [Figure 2] These are schematic diagrams of the structural forms of the triplicate fusion protein in different embodiments of the present invention. [Figure 3] These are schematic diagrams of the structural forms of the triplicate fusion protein in different embodiments of the present invention. [Figure 4] These are schematic diagrams of the structural forms of the triplicate fusion protein in different embodiments of the present invention. [Figure 5]These are schematic diagrams of the structural forms of the triplicate fusion protein in different embodiments of the present invention. [Figure 6] These are schematic diagrams of the structural forms of the triplicate fusion protein in different embodiments of the present invention. [Figure 7] These are schematic diagrams of the structural forms of the triplicate fusion protein in different embodiments of the present invention. [Figure 8] These are schematic diagrams of the structural forms of the triplicate fusion protein in different embodiments of the present invention. [Figure 9] These are schematic diagrams of the structural forms of the triplicate fusion protein in different embodiments of the present invention. [Figure 10] This is a graph showing the results of FACS detection of trifunctional proteins that bind to HL60 cells. [Figure 11] This figure shows the results of FACS detection of the CD38 / CD16A / IL-15 trifunctional fusion protein that binds to Daudi cells. [Figure 12] This is a diagram showing the results of ELISA detection of trifunctional molecules that bind to the CS1 protein. [Figure 13] This figure shows the results of CCK8 detection of the TAA / CD16A / IL-15 trifunctional fusion protein in Mo7e cell proliferation experiments. [Figure 14] This figure shows the results of detecting CCK8, a TAA / CD16A / IL-15 trifunctional fusion protein, in Mo7e cell proliferation experiments. [Figure 15] This figure shows the results of detecting CCK8, a TAA / CD16A / IL-15 trifunctional fusion protein, in Mo7e cell proliferation experiments. [Figure 16] This figure shows the results of the CD33 / CD16A / IL-15 tripfunctional fusion protein-mediated ADCC effect with an effector:target ratio of 10:1. [Figure 17] This figure shows the results of the CD33 / CD16A / IL-15 tripfunctional fusion protein-mediated ADCC effect with an effector:target ratio of 10:1. [Figure 18]This figure shows the results of the CD33 / CD16A / IL-15 trifunctional fusion protein-mediated ADCC effect with an effector:target ratio of 20:1. [Figure 19] This figure shows the results of the CD38 / CD16A / IL-15 tripfunctional fusion protein-mediated ADCC effect with an effector:target ratio of 10:1. [Figure 20] This is a result diagram of CD33 / CD16A / IL-15 trifunctional fusion protein-mediated ADCP with an effector:target ratio of 5:1. [Figure 21] This is a diagram showing the results of ADCP mediated by the CD33 / CD16A / IL-15 trifunctional fusion protein. [Modes for carrying out the invention]

[0050] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and examples. For the sake of easier understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise expressly defined elsewhere in this specification, all other technical and scientific terms used herein have meanings generally understood by those skilled in the art.

[0051] term The term "fusion" refers to direct linking of components by peptide bonds, linking of components via linking fragments, non-covalent fusion by intermolecular interactions, or fusion by one or more disulfide bonds or chemically crosslinked covalent bonds. In a single peptide chain, fusion refers to direct linking by peptide bonds or linking via linking fragments. A "multifunctional fusion protein" refers to a protein containing two or more antigen-binding domains that can bind to two or more different epitopes (e.g., two, three or more different epitopes), and a multifunctional fusion protein may further contain cytokines (e.g., IL-15, IL-15Ra), etc. "Fusion site" refers to the location of a functional region or domain in a peptide chain and indicates the linking order of each functional fragment on the peptide chain.

[0052] The term "polypeptide" refers to amino acid chains of any length, including proteins and their fragments. This invention discloses polypeptides as sequences of amino acid residues. These sequences are written from left to right, from amino group terminus to carboxyl group terminus. According to standard nomenclature, amino acid residue sequences are named with three-letter or one-letter codes, such as alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), simesin (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (I1e, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0053] The term "peptide chain" refers to a molecule in which amino acids are linearly linked by peptide bonds. The term "mutant" or "mutant" refers to a polypeptide or polynucleotide that differs from the amino acids or nucleotides it contains but retains its basic properties. Typically, the differences between mutants or between mutants and parental antibodies are limited, and the amino acid sequences are generally very similar. In this specification, the antibody or antibody fragment before mutation is referred to as the parental antibody, and the antibody or antibody fragment after mutation is referred to as the mutant. The mutant still possesses antigen-binding activity.

[0054] The term "antibody" (Ab) refers to an immunoglobulin molecule (Ig) that contains at least one antigen-binding site and is capable of specifically binding to an antigen.

[0055] The term "antigen" refers to a substance that can induce an immune response in the body and specifically bind to an antibody. The binding of an antibody to an antigen is mediated by interactions between the two, including hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic bonds. The region on the antigen surface that binds to an antibody is called an "antigenic determinant" or "epitope," and generally, each antigen has multiple determinants.

[0056] The term “antibody” as used in this invention is understood in its broadest sense and includes monoclonal antibodies (full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two different antigen-binding domains. Antibodies further include mouse antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies derived from others. Antibodies of this invention may be derived from any animal, including but not limited to human, non-human primates, mice, rats, cattle, horses, chickens, camels, alpacas, and other animals. Antibodies may contain other modifications such as non-natural amino acids, mutations in Fc effector function, and mutations in glycosylation sites. Antibodies further include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to antigen-recognition sites, insofar as the antibody exhibits the desired biological activity. In other words, antibodies include immunoglobulin molecules and immunoactive fragments of immunoglobulin molecules, i.e., molecules containing at least one antigen-binding domain.

[0057] The basic structure of an antibody is a Y-shaped monomer consisting of two completely identical heavy chains (H) and two completely identical light chains (L) linked by disulfide bonds. Each chain contains 2 to 5 amino acids, totaling approximately 110 amino acids, and is composed of domains (also called functional regions) with similar sequences but different functions. In antibody molecules, the amino acid sequences of the light and heavy chains near the N-terminus change significantly, and the resulting domains are called variable regions (V regions), while the region near the C-terminus with a relatively constant amino acid sequence is called the constant region (C region).

[0058] The V regions of the heavy and light chains are called VH and VL, respectively. VH and VL each have three amino acid compositions, and their sequence order is highly variable, so they are called hypervariable regions (HVRs). These regions form a spatial structure complementary to the antigen epitope and are also called complementarity determining regions (CDRs). The three CDRs of VH are represented as VHCDR1, VHCDR2, and VHCDR3, respectively, and the three CDRs of VL are represented as VLCDR1, VLCDR2, and VLCDR3, respectively. A total of six CDRs from VH and VL together form the antigen-binding site. The diversity of amino acids in the CDR regions is the molecular basis for antibodies to specifically bind to a large number of different antigens. The amino acid composition and sequence order outside the CDRs of the V regions are relatively less variable and are called framework regions (FRs). VH and VL have four framework regions (called framework regions) represented by FR1, FR2, FR3, and FR4. VH and VL are each composed of three CDRs and four FRs, and the sequence from the amino group end to the carboxyl group is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0059] Human immunoglobulins can be classified into five categories—IgM, IgG, IgA, IgD, and IgE—according to the amino acid sequence of the constant region of the antibody heavy chain. These can be further divided into different subtypes (isotypes); for example, human IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Subtypes of IgM, IgD, and IgE have not been discovered. Based on the light chain amino acid sequence, the light chain can be classified into κ and λ chains. The antibodies of this invention may be any type (e.g., IgM, IgG, IgA, IgD, IgE) or subtype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2).

[0060] The constant regions of the heavy and light chains are called CH and CL, respectively. The heavy chain constant regions of IgG, IgA, and IgD have three domains: CH1, CH2, and CH3, while the heavy chain constant regions of IgM and IgE have four domains: CH1, CH2, CH3, and CH4.

[0061] The hinge region, located between CH1 and CH2 and containing abundant proline, is easily stretched and bent, allowing for changes in the distance between the two Y-shaped arms, which is advantageous for both arms to bind to the antigen epitope simultaneously.

[0062] The term "Fab fragment" refers to an antigen-binding fragment (Fab), an antibody fragment composed of VL, VH, CL, and CH1 domains that binds to a single antigen epitope (monovalent). Those skilled in the art know that, under certain conditions, specific portions of an antibody molecular chain can be readily hydrolyzed by proteolytic enzymes into various fragments. Papain hydrolyzes the antibody molecule from the N-terminus of the hinge region into two completely identical antigen-binding fragments (Fab) and a crystallizable fragment (Fc).

[0063] The term "Fv fragment" refers to a monovalent, small molecule consisting of the V region (VH+VL) of the L and H chains, and is the smallest functional fragment that binds to an antigen. The terms "Fc," "Fc segment," "Fc fragment," and "Fc domain" refer to crystallizable fragments that do not possess antigen-binding activity, and are the sites on which antibodies interact with effector molecules or cell surface Fc receptors (FcRs). Fc fragments bind to cells that have corresponding Fc receptors on their surface, producing different biological effects. In the ADCC effect (antibody-dependent cell-mediated cytotoxicity), the Fab segment of an antibody binds to antigenic epitopes on virus-infected cells or tumor cells, while its Fc segment binds to FcRs on the surface of toxic cells (NK cells, macrophages, etc.), directly killing target cells via the toxic cells. The Fc fragment comprises the constant region polypeptide of the antibody excluding the heavy chain constant region CH1, i.e., the two constant region domains CH2 and CH3 at the carboxyl terminus of the heavy chain constant region of human immunoglobulin IgA, IgD, and IgG, and the three constant region domains CH2, CH3, and CH4 at the carboxyl terminus of the heavy chain constant region of human immunoglobulin IgE and IgM. The Fc fragment is often selected from human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc or their variants, preferably IgG1 Fc or human IgG4 Fc or their variants.

[0064] Fc can consist of two chains, and in this specification, the two chains of an Fc fragment are described as the first Fc chain and the second Fc chain, and the first Fc chain and the second Fc chain can each be mutated, and the present invention is not limited to these. An Fc fragment can also refer to a single polypeptide chain within an Fc domain. The Fc segment of an antibody can be selected to eliminate immunoeffector function and includes, but is not limited to, combinations of mutations (counted by the EU), such as:

[0065] [Table 6]

[0066] Mutation-designed Fc mutants can form space-filling effects, electrostatic steering, hydrogen bonding, and hydrostatic interactions. These interactions favor the formation of stable heterodimers. A preferred mutation design is a "knob-in-hole" mutation design.

[0067] A "linking fragment" refers to one or more amino acid residues that are inserted into an immunoglobulin domain to ensure the correct folding of the protein and the stability of the peptide, and that also provide sufficient mobility. The "linking fragment" is preferably (GGGGS)n, where n may be 0, 1, 2, 3, 4, 5 or more. If the linking fragment sequence is too short, it may affect the folding of the higher-order structures of the two proteins and thereby cause them to interfere with each other, and if the linking peptide sequence is too long, immunogenicity problems arise because the linking peptide sequence itself is a novel antigen.

[0068] Tumor-associated antigens (TAAs) refer to antigenic molecules present in tumor cells or normal cells, including embryonic proteins, glycoprotein antigens, and squamous cell antigens, and are commonly used in the diagnosis of clinical tumors. Tumor-associated antigens are not unique to tumor cells and can be synthesized in trace amounts in normal cells, but they are called "associated antigens" because they are highly expressed when tumor cells proliferate. Tumor-associated antigens may include, for example, CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, CD276, PD-1, CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR, EGFR VIII, C-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, or Glypican-3.

[0069] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide to which it is ligated. One type of vector is a "plasmid," which refers to a circular double-stranded DNA ring to which an additional DNA segment can be ligated. Another type of vector is a viral vector, to which an additional DNA segment can be ligated to a viral genome. Certain vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell's genome after introduction into the host cell, thereby replicating together with the host genome. Furthermore, certain vectors can direct the expression of genes to which they are operably ligated. Expression vectors, which are typically useful in recombinant DNA technology, are usually in the form of plasmids. The present invention can provide vectors comprising nucleic acid fragments capable of encoding a trifunctional fusion protein.

[0070] The terms "IL-15" and "IL-15Ra" may refer to their mutants or fragments. The "IL-15" described in the present invention may be a mutant capable of binding to any IL-15 or IL-15Rα, for example, human IL-15 or IL-15 from a non-human mammal or non-mammalian organism. Examples of non-human mammals include pigs, rabbits, monkeys, orangutans, mice, and non-mammalian organisms such as chickens, and preferably human IL-15. The term "mutant capable of binding to IL-15Rα" refers to a mutant molecule in which the mutation is obtained by the substitution, increase, or deletion of one or more amino acids, which increases or decreases the affinity between IL-15 and its receptor, or increases or decreases its activity in stimulation of T cells or NK cells.

[0071] The "IL-15Rα" described in the present invention may be IL-15Rα of any species or a mutant that can bind to IL-15Rα, for example, human IL-15Rα or non-human mammalian IL-15Rα or non-mammalian IL-15Rα. Examples of non-human mammals include pigs, rabbits, monkeys, orangutans, mice, and non-mammalian chickens. Preferably, it is human IL-15Rα, more preferably a human IL-15Rα extracellular domain fragment called IL-15RαECD (Database UniProtKB, registration number Q13261, 31-205aa). The term "mutant capable of binding to IL-15Rα" refers to a functional mutant formed by one or more amino acid deletions, insertions, or substitution mutations on IL-15Rα, which has the ability to bind to its ligand molecule, such as IL-15. Preferably, it is a human IL-15Rα molecule, more preferably a truncated form of the human IL-15Rα extracellular domain fragment, i.e., a molecule having human IL-15 receptor α activity, obtained by a deletion mutation of one or more amino acids from the C-terminus of the extracellular domain fragment, preferably a molecule retaining a deletion mutation morphology of 65 to 178 amino acids, such as IL-15Rα. The IL-15 protein itself is not as stable as when complexed with the IL-15Rα protein. As is known in the art, the IL-15Rα protein contains a "sushi domain," which is the shortest region of the receptor that retains IL-15 binding activity, or one domain of the extracellular region that can retain 90% or more of the binding activity.

[0072] CS1 (also known as CD319, SLAMF7) is a member of the signaling lymphocyte activation molecule family and is a surface receptor protein that modulates the immune function of NK cells. CS1 is highly expressed in multiple myeloma (MM) cell lines but not found in healthy tissues, primary tumor tissues, or hematological and non-hematological cancer cell lines, making it an attractive target for treating this disease.

[0073] CD33 is a 67kD single-pass transmembrane glycoprotein and a member of the sialic acid-binding immunoglobulin-like lectin (Siglecs) family. Its exact biological function is unknown, but in healthy individuals, it is thought to be primarily a myeloid differentiation antigen, with low expression in myeloid progenitor cells, neutrophils, and macrophages, and high expression in circulating monocytes and dendritic cells. Importantly, CD33 has been detected in 85-90% of blast cells and leukemia stem cells in patients with acute myeloid leukemia (AML). Interestingly, CD33 expression is limited to hematopoietic cells (Paul, Taylor, Stansbury, and McVicar, 2000; Ulyanova, Blasioli, Woodford-Thomas, and Thomas, 1999), but absent in normal hematopoietic cells (Andrews, Torok-Storb, and Bernstein, 1983; Griffin, Linch, Sabbath, Larcom, and Schlossman, 1984; Jilani et al., 2002). These findings suggest that CD33 is a suitable target for antibody-based therapies in AML.

[0074] The CD38 antigen, discovered in 1980, is a 46 kDa type II transmembrane glycoprotein. Its ligand is CD31, also known as PECAM-1, a member of the Ig gene superfamily. CD31 is expressed on endothelial cells, as well as lymphoid cells (Zaka B cells and plasma cells), the lungs (alveolar ducts, alveoli, and lymphatic vessels), and the kidneys (glomerular cells). CD38 interacts with its ligand, CD31, and plays a crucial role in regulating cell migration, receptor-mediated adhesion, and signal transduction. Furthermore, the CD38 protein is a bifunctional extracellular enzyme possessing both cyclase and hydrolytic activity, and is involved in nucleotide metabolism. CD38 uses NAD+ as a substrate to form cyclic ADP-ribose (cADPR) and ADPR. These nucleotide metabolites are effective secondary messengers that regulate the recruitment of calcium ions in the cytoplasm and activate signaling pathways that control various biological processes, including lymphocyte proliferation and insulin secretion by pancreatic B cells. Studies have shown that CD38-dependent adenosine production plays a crucial role in natural killer (NK) cell-mediated immunosuppression. Normally, CD38 is expressed at low levels in normal lymphoid cells, myeloid cells, and non-hematopoietic cells, but is widely expressed in hematopoietic-derived cells (including committed stem cells). CD38 is expressed in B cells, with higher levels of CD38 expression in plasma cells (also known as effector B cells). CD38 expression is associated with a variety of diseases, including AIDS, autoimmune diseases (e.g., oliguricaneous lupus erythematosus), type 2 diabetes, osteoporosis, and cancer. Studies have shown that CD38 is highly expressed in many malignant blood cancers, particularly multiple myeloma, and for this reason, CD38 has become a target for the development of therapeutic antibody drugs for multiple myeloma.

[0075] "Effector function" refers to the biochemical event resulting from the interaction between the Fc region of an antibody and an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC. "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated response in which nonspecific cytotoxic cells expressing FcγR recognize an antibody bound to a target cell, subsequently causing lysis of the target cell. ADCC is associated with the binding of FcγRIIIa, and increased binding to FcγRIIIa increases ADCC activity. As discussed herein, many embodiments of the invention completely eliminate ADCC activity. "ADCP" or "antibody-dependent cell-mediated phagocytosis" refers to a cell-mediated response in which nonspecific cytotoxic cells expressing FcγR recognize an antibody bound to a target cell, subsequently causing phagocytosis of the target cell.

[0076] The "amino acid sequence identity percentage (%)" for a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific (parent) sequence, after aligning the sequences and introducing gaps where necessary to achieve the highest possible sequence identity percentage, and without considering conservative substitutions as part of the sequence identity. In some examples, two or more amino acid sequences are at least 80%, 85%, or 90% identical. In some examples, two or more amino acid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

[0077] The terms "first" and "second" are used for descriptive purposes only and do not indicate or imply any degree of importance. The tripfunctional fusion protein of the present invention, which has antigen-targeting, anti-CD16A, and immune effector cell activation properties, comprises a CD16A-binding domain that specifically binds to CD16A, a TAA-binding domain that specifically binds to tumor-associated antigens (TAAs), an IL-15 / IL-15Rα complex formed by the binding of IL-15 and IL-15Rα, and an Fc domain. The TAA / CD16A / IL-15 tripfunctional fusion protein of the present invention exerts therapeutic effects by simultaneously binding tumor-associated antigens (TAAs) expressed mainly on tumor cells and CD16A on NK cells, thereby cross-linking these two cells to form immune synapses, promoting the killing of tumor cells by NK cells, while IL-15 simultaneously expands and maintains the function of NK cells.

[0078] The structural form of the triplicate fusion protein of the present invention can be seen with reference to Figures 1 to 9. The fusion protein is formed by the fusion of four peptide chains, with the first and second peptide chains forming the first monomer (left side of the figure), and the third and fourth peptide chains forming the second monomer (right side of the figure). The first peptide chain includes a light chain formed by the fusion of a VL domain and a CL domain, and the second peptide chain includes a VH domain and a heavy chain formed by the fusion of a CH1 domain and a first Fc chain. The VL and CL domains of the first peptide chain, and the VH and CH1 domains of the second peptide chain, pair up to form a Fab fragment. The third peptide chain includes an antibody variable region, a cytokine, and a second Fc chain, and the fourth peptide chain includes an antibody variable region and a cytokine. The antibody variable region of the third peptide chain and the antibody variable region of the fourth peptide chain pair up to form an Fv fragment, and the cytokine of the third peptide chain and the cytokine of the fourth peptide chain bind together to form the IL-15 / IL-15Rα complex.

[0079] In the fusion proteins shown in Figures 1 to 8, the Fab fragment in the first monomer targets TAA and realizes the function of the TAA antibody shown in the figures. In the second monomer, the antibody variable region that pairs to form the Fv fragment is selected from the VH domain and the VL domain. For example, the antibody variable region in the third peptide chain is selected from the VH domain that targets CD16A, and the antibody variable region in the fourth peptide chain is selected from the VL domain that targets CD16A. The VH and VL domains pair to form the Fv fragment that targets CD16A and realize the function of the anti-CD16A antibody shown in the figures. In another example, the antibody variable region in the third peptide chain is the VL domain that targets CD16A, and the antibody variable region in the fourth peptide chain is the VH domain that targets CD16A. VH(L) in the figures represents the selection of VH or VL. The numbers "VH(L)1", "VL(H)1", "VH2", and "VL2" in the diagram are for illustrative purposes only and represent antibody variable regions targeting different antigens. VH(L)1 indicates that either VH1 or VL1 can be selected. If the antibody variable region of the third peptide chain is VH1, then the antibody variable region of the fourth peptide chain must be VL1, thereby allowing them to pair and form an Fv fragment. The cytokines in the third and fourth peptide chains are selected from IL-15 and IL-15Rα to form an IL-15 / IL-15Rα complex. In some examples, the cytokine in the third peptide chain is IL-15 and the cytokine in the fourth peptide chain is IL-15Rα. Conversely, in some examples, the third peptide chain is IL-15Rα and the fourth peptide chain is IL-15.

[0080] In the third and fourth peptide chains, the fusion order between the antibody variable region (VH or VL) and the cytokine is not limited. For example, in the fusion protein shown in Figure 1, from the N-terminus to the C-terminus of the peptide chain, the fusion order of the third peptide chain is antibody variable region ~ cytokine ~ second Fc chain, and the fusion order of the fourth peptide chain is antibody variable region ~ cytokine, where "~" represents a linking fragment. The difference between the fusion protein shown in Figure 2 and Figure 1 is that from the N-terminus to the C-terminus of the peptide chain, the fusion order of the fourth peptide chain is cytokine ~ antibody variable region. In Figures 1 to 8, the spherical and crescent shapes represent different cytokines, and below, we assume that the spherical shape represents IL-15 and the crescent shape represents IL-15Ra. Examples of possible combinations of the third and fourth peptide chains of the fusion proteins shown in Figures 1 to 8, from the N-terminus to the C-terminus of the peptide chain, are as follows.

[0081] [Table 7] JPEG0007880160000011.jpg124170

[0082] In the fusion protein shown in Figure 9, the Fab fragment of the first monomer (the left portion in the figure) targets CD16A and realizes the function of the anti-CD16A antibody shown in the figure. The Fv fragment targets TAA and realizes the function of the anti-TAA antibody shown in the figure. Similar to the fusion proteins shown in Figures 1 to 8, the fusion protein shown in Figure 9 can also have various possible combinations of the third and fourth peptide chains listed in Table 1.

[0083] The complex unit of the innovative structural protein provided by the present invention can be referred to as the upper part of the second monomer of the fusion protein shown in Figure 1, i.e., the region where the anti-CD16A Fv fragment and the IL-15 / IL-15Rα complex are fused. The complex unit includes two antibody variable regions (VH and VL of anti-CD16A), two cytokines (IL-15, IL-15Ra), and several linking fragments. The antibody variable regions are fused to the cytokines via linking fragments. The two antibody variable regions pair to form the Fv fragment, and the two cytokines bind to form the IL-15 / IL-15R complex. Preferably, there is one or more disulfide bonds between the VH and VL domains of anti-CD16A and / or between IL15 and IL15Rα, thereby forming a more stable structure. A disulfide bond can be established between VH and VL, but not between IL15 and IL15Rα, or a disulfide bond can be established between IL15 and IL15Rα, but not between VH and VL, or a disulfide bond can be established both between VH and VL and between IL15 and IL15Rα. Using the composite units provided by the present invention, those skilled in the art can construct other target proteins without being limited to the morphology of the fusion proteins shown in the examples of the present invention.

[0084] The cytokines IL-15 and IL-15Ra have extremely high affinity (KD approximately 30-100 pM). The present invention can solve the problem of light chain mismatch in bispecific antibodies and the problem of heavy chain mismatch in FC heterodimer forms by using IL-15 and IL-15Ra to substitute the CL and CH1 domains, respectively, in one of the antibody structures. In some examples, the FC terminus with Fab is mutated so that it cannot bind to protein A. Bispecific targeting antibodies are purified through a two-step process: protein A affinity chromatography that binds to the FC terminus and CH1-XL affinity chromatography that binds to the CH1 domain.

[0085] Example 1: Acquisition of TAA / CD16A / IL-15 trifunctional fusion protein, molecular cloning, transient expression, and protein purification. Molecular Cloning: In this example, multiple fusion proteins were constructed according to the table below, with TAAs selecting CD33, CD38, and CS1, respectively. Protein expression and sequence numbers are as shown in the table below.

[0086] [Table 8] JPEG0007880160000013.jpg241170JPEG0007880160000014.jpg125170

[0087] The sequence for SEQ ID NO:01 is as follows:

[0088] [ka] Here, the sequence shown by the dotted line is the VL of anti-CD16A, and the sequence shown by the wavy line is IL-15.

[0089] The sequence for SEQ ID NO:02 is as follows:

[0090] [ka] Here, the sequence shown by the dotted line is the VH variant of anti-CD16A, the sequence shown by the wavy line is IL-15Ra, and the sequence with a double underline is Fc (Knob mutation).

[0091] The sequence for SEQ ID NO:03 is as follows:

[0092] [ka] Here, the parts not underlined are the light chain sequences of the anti-CD33 molecule, and the sequence shown by the dotted line is the VL (vertical chain).

[0093] The sequence for SEQ ID NO:04 is as follows:

[0094] [ka] Here, the parts not underlined are the heavy chain sequence of anti-CD33, and the sequence shown by the dotted line is VH.

[0095] The following array is provided in addition. Anti-CD16A VL SEQ ID NO:38 SYVLTQPSSVVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL Anti-CD16A VH SEQ ID NO:39 QVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS Anti-CD38 VL SEQ ID NO:40 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK Anti-CD38 VH SEQ ID NO:41 EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS Anti-CS1 VL SEQ ID NO:42 DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIK Anti - CS1 VH SEQ ID NO:43 EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS Anti - CD33 VL SEQ ID NO:44 DIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSTNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSPQPEDSATYYCQQSAHFPITFGQGTRLEIK Anti - CD33 VH SEQ ID NO:45 QVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYADKFQGRVTMTTDTSTSTAYMEIRNLGGDDTAVYYCARWSWSDGYYVYFDYWGQGTSVTVSS IL - 15 SEQ ID NO:46 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS IL - 15(L52C)SEQ ID NO:47 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS IL - 15Ra ECDSEQ ID NO:48 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT IL-15Ra Sushi 65 SEQ ID NO:49 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR IL-15Ra Sushi 73 SEQ ID NO:50 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQR IL-15Ra Sushi 77 SEQ ID NO:51 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP IL-15Ra Sushi 86 SEQ ID NO:52 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVT IL-15Ra Sushi 102 SEQ ID NO:53 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAAS IL-15Ra-sushi(S40C): SEQ ID NO:54 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIR。

[0096] All of the above IL-15 or IL-15Ra sequences can be used to construct the fusion protein of the present invention. As shown in Table 3, control antibodies such as anti-CD16a antibody, anti-CD33 antibody, elotuzumab, daratumumab, and TAA / CD16A biantibody were designed and constructed, and molecules such as the CD33 / CD16A / IL-15 control molecule GTB-3550 were constructed based on patent document US20200148737A1.

[0097] [Table 9] JPEG0007880160000020.jpg239170JPEG0007880160000021.jpg246170JPEG0007880160000022.jpg173170

[0098] Transient expression of target molecules: ExpiCHO-S cells are inoculated into FortiCHO medium (Gibco, A1148301), 8 mM GlutaMax is added, and the cells are cultured at 37°C, 120 rpm, and 8% CO2. The day before transfection, the ExpiCHO-S cell density is adjusted to 3 × 10⁶ E⁶ / mL, placed in a shaker, and cultured at 37°C, 120 rpm, and 8% CO2. On the day of transfection, samples are collected, counted, and the cell density is diluted to 6 × 10⁶ E⁶ / mL. Each bottle is 40 mL and placed in a 125 mL shaking flask. 20 μg of each co-transfected plasmid is mixed with 4.8 mL of Opti MEM, and 120 μl of Polyplus-FectoPRO transfection reagent is added. After homogeneous mixing of the DNA and transfection reagent, the mixture is left at room temperature for 10 minutes. The mixture is then slowly added to the cells, homogeneous mixing is performed, and the cells are cultured in a shaker. During incubation, add 2 mL of Feed PFF05 (OPM, F81279-001) and 1 mL of 30% glucose solution to the bottle on days 1, 4, 6, and 8, respectively. On the first day of transfection, cool the temperature to 32°C and reduce the CO2 concentration to 5%. On day 13, collect the sample, centrifuge at 8000 rpm for 20 minutes, take the supernatant, and prepare for purification.

[0099] Purification of fusion proteins: Purification by Protein A affinity chromatography The equilibrium solution is passed through the column to a volume of at least 3 CV (actual volume 20 ml), ensuring that the pH and conductivity of the solution effluent from the final instrument match those of the equilibrium solution, at a flow rate of 1 ml / min. The supernatant of the culture medium after centrifugation is passed through the column, loading 40 ml of sample at a flow rate of 0.33 ml / min. The equilibrium solution is passed through the column to a volume of at least 3 CV (actual volume 20 ml), ensuring that the pH and conductivity of the solution effluent from the final instrument match those of the equilibrium solution, at a flow rate of 0.33 ml / min. The eluent is passed through the column, and collection of the elution peak (PAC-EP) is started when UV280 rises to 15 mAU, and stopped when UV280 falls to 15 mAU, at a flow rate of 1 ml / min. After sample collection is complete, the PAC-EP is neutralized with pH adjusting solution.

[0100] CH1-XL Affinity Chromatography The sample treated with Protein A was centrifuged at 8000 rpm for 15 minutes, the supernatant was collected, and the equilibrium solution was passed through the column to an amount of at least 3 CV (actual volume 20 ml), ensuring that the pH and conductivity of the solution effluent from the final instrument matched that of the equilibrium solution, at a flow rate of 1 ml / min. The centrifuged supernatant was passed through the sample loop and then through the column at a flow rate of 0.33 ml / min. The equilibrium solution was passed through the column to an amount of at least 3 CV (actual volume 20 ml), ensuring that the pH and conductivity of the solution effluent from the final instrument matched that of the equilibrium solution, at a flow rate of 0.33 ml / min. The eluent was passed through the column, and collection of the elution peak (PAC-EP) began when UV280 rose to 10 mAU, and collection was stopped when UV280 fell to 10 mAU, at a flow rate of 1 ml / min. After sample collection was complete, CH1-EP was neutralized with pH adjusting solution.

[0101] Example 2: FACS detection of CD33 / CD16A / IL-15 tripfunctional fusion protein that binds to HL60 cells naturally expressing CD33. Experimental Objective: The human promyelocytic leukemia cell line HL60 naturally expresses CD33. This invention aims to detect the binding of the CD33 / CD16A / IL-15 trifunctional fusion protein to HL60 cells that naturally express CD33 using FACS.

[0102] Experimental method: HL60 cells were cultured at 37°C in a 5% CO2 incubator, cells were collected by trypsin digestion, 100,000 cells per well were inoculated into a 96-well plate, blocked on ice for 1 hour with 2% FBS / PBS, incubated with different concentrations of trifunctional protein and control protein, held on ice for 1 hour, washed three times with PBS, diluted with PE-anti-human FC (1:200), washed three times with PBS, resuspended in 200 μL of PBS, mean fluorescence values ​​were read by FACS, and results were analyzed using GraphPad Prism software. The results are shown in Figure 10. The CD33 antibody IgG1 wt subtype QP41154116, the CD33 / CD16A / IL-15 trifunctional fusion protein QP743745 (FC wild-type), QP43394340 (FC performs L234A / L235A mutagenesis function), QP42914285 (FC performs L234A / L235A mutagenesis function), the CD33 / CD16A bispecific antibody QP4115777778 (FC wild-type), the CD33 / CD16A bispecific antibody QP411543674368 (FC performs L234A / L235A mutagenesis function), and the CD33 antibody IgG1 wt subtype QP41154116 all bind to HL60 cells that naturally express CD33.

[0103] Example 3: FACS detection of CD38 / CD16A / IL-15 tripfunctional fusion protein that binds to Daudi cells that naturally express CD38. Experimental Objective: Human Burkitt's lymphoma cells, Daudi cells, naturally express CD38. This invention aims to detect the CD38 / CD16A / IL-15 trifunctional fusion protein that binds to Daudi cells naturally expressing CD38 using FACS.

[0104] Experimental method: Daudi cells were cultured at 37°C in a 5% CO2 incubator, cells were collected by trypsin digestion, 100,000 cells per well were inoculated into a 96-well plate, blocked on ice for 1 hour with 2% FBS / PBS, incubated with different concentrations of trifunctional protein and control protein, held on ice for 1 hour, washed three times with PBS, diluted with PE-anti-human FC (1:200), washed three times with PBS, resuspended in 200 μL of PBS, mean fluorescence values ​​were read by FACS, and results were analyzed using GraphPad Prism software. The results, as shown in Figure 11, show that the CD38 antibody IgG1 wt subtype QP34503451 and the CD38 / CD16A / IL-15 trifunctional fusion protein QP43394341 (FC performs L234A / L235A mutation removal function) bind to Daudi cells that naturally express CD38 in a concentration-dependent manner.

[0105] Example 4: ELISA detection of CS1 / CD16A / IL-15 trifunctional fusion protein binding activity to CS1 Detection reagents: Milk (BD, 232100), PBS (Sangon, B548117-0500), HRP-anti-human IgG(H+L) (Jackson, 109-035-088), TMB (Luoyang Baiaotong Experimental Materials Center, C060201), Elisa plate (Costa, 9018). Weigh 8.00g NaCl, 0.20g KCl, 2.9g Na2HPO4·12H2O, and 0.2g KH2PO4 into 1× PBS buffer, dissolve in 800mL ddH2O, and after complete dissolution, adjust to 1L volume, adjust pH to 7.4, sterilize at high temperature, and store. Alternatively, purchase commercially available 10× or 20× PBS solutions and dilute them in 1× PBS buffer before use. Blocking solution: Weigh 5g of milk into PBS, prepare a blocking solution, and use immediately. Stop solution (1 mol / L H2SO4): Weigh 109 mL of 98% concentrated H2SO4 and slowly add it dropwise to 2000 mL of ddH2O. Allow TMB to develop color at 37°C for 10 minutes, then add 100 μl / well to a shaker (120 rpm).

[0106] Experimental steps: Plate the CS1-FC fusion protein at 1 μg / ml overnight at 4°C and wash three times with PBS. Add 5% milk blocking solution according to 200 μL / well and incubate at 37°C for 1 hour. After blocking is complete, wash three times with PBS, incubate the sample, dilute five-fold with 20 μg / ml, dilute in a total of seven gradients, dilute the last well 100-fold, mix thoroughly with 100 μl / well, incubate for 1 hour, wash three times with PBST, add enzyme-labeled antibody: Incubate HRP-anti-human Fab antibody at a dilution ratio of 1:5000 with 100 μl / well, mix thoroughly, incubate for 1 hour, and wash six times with PBST. Add substrate chromogenic solution: Add substrate chromogenic solution TMB at a dose of 100 μL / well, place in a shaker, and allow to develop color at 200 rpm in the dark at 35°C for 10 minutes. Termination: After color development is complete, the reaction is quickly terminated with a stop solution at a dose of 100 μL / well. Detection: The OD value at A450 nm is measured on a microplate reader, and the results are analyzed using GraphPad Prism software. The results are shown in Figure 12, indicating that the CS1 / CD16A / IL-15 trifunctional fusion proteins QP7434262 (FC wild type) and QP43394342 (FC performs L234A / L235A mutagenesis function) all bind to the CS1 protein in a concentration-dependent manner.

[0107] Example 5: Mo7e cell proliferation experiment Mo7e (human giant cell leukemia cell line) cells express IL-15Rβγ and are cytokine-dependent growth cells. Studies have shown that quiescent NK and naive T cells express a moderate affinity IL-15Rβγ phenotype, and the cytokine IL-15 / IL-15Ra results in Mo7e(IL-15Rβγ) cell proliferation experiments are consistent with proliferation experiments in unstimulated PBMCs (Mol Cancer Ther, 11(6) June 2012). This invention utilizes Mo7e cell proliferation experiments to evaluate the activity of the multifunctional fusion protein IL-15, and the methods and results are as follows.

[0108] Experimental reagents: Mo7e cells (human giant cell leukemia cell line) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medicine; the cell proliferation and toxicity detection kit (CCK-8) was purchased from MeilunBio, product number MA0218; recombinant human GM-CSF was purchased from perprotech, product number 300-03; human IgG was purchased from Sigma, product number I4506; and other antibodies were prepared in-house.

[0109] Experimental method: Mo7e cells were cultured in RPMI1640 medium containing 10% FBS, 2 mM L-glutamine, and 8 ng / ml GM-CSF in a 37°C, 5% CO2 incubator. Mo7e cells were collected, centrifuged at 800 rpm for 5 minutes, the supernatant was discarded, the cells were washed twice with RPMI1640 medium without GM-CSF, the cells were resuspended and counted in RPMI1640 medium without GM-CSF, and 2 × 10⁴ cells were divided into 96 wells at 80 μl / well. Inoculate the cells into a plate and incubate at 37°C in a 5% CO2 incubator for 1 hour. Dilute each medicinal medium to be tested fourfold, then uniformly mix 20 μl per well with the cell suspension and incubate at 37°C in a 5% CO2 incubator for 3 days. Add 10 μl of CCK-8 reagent per well to the 96-well plate to be tested and incubate at 37°C in a 5% CO2 incubator for 4 hours. Remove the 96-well plate and detect the absorbance at a wavelength of 450 nm using a microplate reader.

[0110] The results shown in Figures 13, 14, and 15 demonstrate that the CD33 / CD16A / IL-15, CD38 / CD16A / IL-15, and CS1 / CD16A / IL-15 triplicate fusion proteins can all promote the proliferation of MO7E cells, proving that all of the triplicate fusion protein molecular forms of the present invention possess IL-15 biological activity.

[0111] Example 6: Effect of CD33 / CD16A / IL-15 trifunctional fusion protein-mediated ADCC Experimental Objective: To detect the CD33 / CD16A / IL-15 trifunctional fusion protein through ADCC experiments.

[0112] Experimental materials: PBMCs were purchased from SailyBio, HL60 cells from the Cell Bank of the Chinese Academy of Sciences, and the Cytotox96 non-radioactive cytotoxicity detection kit was purchased from Promega (G1780).

[0113] Experimental phase: Revive PBMCs and collect cells for use the following day. Target cell preparation: Digeste HL60 with trypsin at 1000 rpm for 5 minutes. After washing twice with PBS, plate 20,000 cells / well, 50 μl / well in a 96-well plate and incubate at 37°C, 5% CO2 for 2 hours. Antibody preparation: Gradient dilution of antibodies to eight concentrations in culture medium (RPMI1640 containing 10% low IgG FBS). Add 50 μl of each diluted antibody concentration to each well and incubate at 37°C for 15 minutes. PBMC preparation: Centrifuge the PBMCs revived on the first day, resuspend in culture medium (RPMI1640 containing 10% low IgG FBS), and count. PBMC: Add 50 μl per well to the above well plate according to Target cell = 20:1 or 10:1 and incubate at 37°C for 4 hours. LDH Detection: For the maximum release group and volume-corrected group, add 10 μl of lysis solution 45 minutes prior and continue culturing in the incubator. After 4 hours of culturing, pipette 50 μl of supernatant into a microplate and add 50 μl of reagent according to the instructions for the Cytotox96 Non-Radioactive Cytotoxicity Experiment Detection Kit (Promega, G1780). After reacting for 30 minutes at room temperature in the dark, add 50 μl of stop solution. Read the absorbance at 490 nm (complete the reading within 1 hour after the stop solution has been added). Calculation: Killing rate = (Sample release - Spontaneous release of target cells - Spontaneous release of effector cells) / (Maximum release of target cells - Spontaneous release of target cells) × 100. Spontaneous release (corresponding to target cells incubated with effector cells in the absence of antibody) is defined as 0% cytotoxicity, and maximum release (target cells lysed with 1% Triton X-100) is defined as 100% cytotoxicity.

[0114] Figures 16 and 17 show the results when the effector:target ratio is 10:1, and Figure 18 shows the results when the effector:target ratio is 20:1. Results: The CD33 antibody IgG1 wt subtype QP41154116 caused concentration-dependent NK cell killing in HL60 cells that naturally express CD33, and the CD33 / CD16A / IL-15 trifunctional fusion proteins QP743745 (FC wild type), QP43394340 (FC performs L234A / L235A mutagenesis function), QP42914285 (FC performs L234A / L235A mutagenesis function), C The D33 / CD16A bispecific antibody QP4115777778 (FC wild-type) and the CD33 / CD16A bispecific antibody QP411543674368 (FC performs L234A / L235A mutagenesis function) enhance HL60 cell killing by NK cells more than the CD33 / CD16A / IL-15 trifunctional fusion protein control molecule GT-3550 (QP877) and the CD33 antibody IgG1 wt subtype QP41154116. Neither the CD16A monoclonal antibody QP43354336 nor the IL-15 / IL-15Ra-FC fusion protein QP33123313 exhibit ADCC effects.

[0115] Example 7: Effect of CD38 / CD16A / IL-15 trifunctional fusion protein-mediated ADCC Experimental Objective: To detect the CD38 / CD16A / IL-15 trifunctional fusion protein through ADCC experiments.

[0116] Experimental materials: PBMCs were purchased from SailyBio, Daudi cells from the Cell Bank of the Chinese Academy of Sciences, and the Cytotox96 non-radioactive cytotoxicity experimental detection kit was purchased from Promega (G1780).

[0117] Experimental phase: Revive PBMCs and collect cells for use the following day. Target cell preparation: Digeste Daudi cells with trypsin at 1000 rpm for 5 minutes. After washing twice with PBS, plate 20,000 cells / well, 50 μl / well in a 96-well plate and incubate at 37°C, 5% CO2 for 2 hours. Antibody preparation: Gradient dilution of antibodies to eight concentrations in culture medium (RPMI1640 containing 10% low IgG FBS). Add 50 μl of each diluted antibody concentration to each well and incubate at 37°C for 15 minutes. PBMC preparation: Centrifuge the PBMCs revived on the first day, resuspend in culture medium (RPMI1640 containing 10% low IgG FBS), and count. PBMC: Add 50 μl per well to the above well plate according to Target cell = 10:1 or 5:1 and incubate at 37°C for 4 hours. LDH detection: For the maximum release group and volume-corrected group, add 10 μl of lysis solution 45 minutes prior and continue culturing in the incubator. After 4 hours of culturing, pipette 50 μl of supernatant into a microplate and add 50 μl of reagent according to the instructions of the Cytotox96 non-radioactive cytotoxicity detection kit (Promega, G1780). After reacting for 30 minutes at room temperature in the dark, add 50 μl of stop solution. Read the absorbance at 490 nm (complete the reading within 1 hour after adding the stop solution). Calculation: Killing rate = (Sample release - Spontaneous release of target cells - Spontaneous release of effector cells) / (Maximum release of target cells - Spontaneous release of target cells) × 100. Spontaneous release (corresponding to target cells incubated with effector cells in the absence of antibodies) is defined as 0% cytotoxicity, and maximum release (target cells lysed with 1% Triton X-100) is defined as 100% cytotoxicity.

[0118] Figure 19 shows the results when the effector:target ratio is 10:1, and Figure 20 shows the results when the effector:target ratio is 5:1. As shown in the figures, at different effector:target ratios, the CD38 antibody IgG1 wt subtype QP34503451 induces concentration-dependent NK cell killing in Daudi cells that naturally express CD38, while the CD38 / CD16A / IL-15 trifunctional fusion protein QP43394341 (FC performs L234A / L235A mutagenesis function) enhances NK cell killing of Daudi cells more than the CD38 antibody IgG1 wt subtype QP34503451. Neither the CD16A monoclonal antibody QP43354336 nor the IL-15 / IL-15Ra-FC fusion protein QP33123313 exhibits an ADCC effect.

[0119] Example 8: CD33 / CD16A / IL-15 trifunctional fusion protein-mediated ADCP Monocytes are isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals and differentiated into macrophages by adding 50 ng / mL of human recombinant M-CSF. HL60 cells are labeled with green fluorescent CFSE, and HL60 cells and macrophages are inoculated into 96-well plates in a 2:1 ratio. Different concentrations of the target protein are added. After incubation at 37°C for 2 hours, the reaction is stopped, and APC anti-human CD11b antibody is incubated. The cells are read by FACS to obtain the percentage of APC / FITC double-positive cells and the percentage of phagocytic macrophages at each antibody concentration.

[0120] The results, as shown in Figure 21, show that both the CD33 / CD16A / IL-15 trifunctional fusion protein QP743745 (FC wild-type) and QP43394340 (FC performs L234A / L235A mutation removal function) induce macrophages to phagocytose HL60 cells in a concentration-dependent manner. The CD33 / CD16A / IL-15 trifunctional fusion protein control molecule GT-3550 (QP877) does not possess the ADCP effector.

[0121] Although the present invention has been described in detail through the preferred embodiments described above, it should be understood that the present invention is not limited to the above description. Those skilled in the art will see that various modifications and changes to the present invention are possible upon reading the above description. Accordingly, the scope of protection of the present invention should be limited by the appended claims.

Claims

1. A triplicate fusion protein with antigen targeting, anti-CD16A, and immune effector cell activation properties, The antigen comprises a tumor-associated antigen (TAA), the trifunctional fusion protein comprises a CD16A-binding region that specifically binds to CD16A, the CD16A-binding region comprises an anti-CD16A antibody or its antigen-binding fragment, the CD16A-binding region is a Fab fragment or Fv fragment that targets CD16A, the TAA-binding region that specifically binds to a tumor-associated antigen (TAA), the TAA-binding region comprises an anti-TAA antibody or its antigen-binding fragment, the TAA-binding region is a Fab fragment or Fv fragment that targets TAA, an IL-15 / IL-15Rα complex formed by the binding of IL-15 and IL-15Rα, and an Fc domain. The fusion protein comprises a first monomer and a second monomer. The first monomer comprises the TAA binding region and the first Fc chain, the second monomer comprises the CD16A binding region, the IL-15 / IL-15Rα complex and the second Fc chain, the first Fc chain and the second Fc chain polymerize to form the Fc domain, the TAA binding region is a Fab fragment targeting TAA, the CD16A binding region is an Fv fragment targeting CD16A, or, The first monomer comprises the CD16A binding region and the first Fc chain, the second monomer comprises the TAA binding region, the IL-15 / IL-15Rα complex and the second Fc chain, the first Fc chain and the second Fc chain polymerize to form the Fc domain, the CD16A binding region is a Fab fragment targeting CD16A, and the TAA binding region is an Fv fragment targeting TAA. The first monomer comprises a first peptide chain and a second peptide chain, and the second monomer comprises a third peptide chain and a fourth peptide chain. The first peptide chain includes a light chain formed by the fusion of a VL domain and a CL domain. The second peptide chain comprises a VH domain and a heavy chain formed by the fusion of a CH1 domain and a first Fc chain. The third peptide chain comprises a VH domain, IL-15Rα, and a second Fc chain. The fourth peptide chain comprises a VL domain and IL-15, The VL domain and CL domain of the first peptide chain, and the VH domain and CH1 domain of the second peptide chain, pair up to form a Fab fragment. The VH domain of the third peptide chain and the VL domain of the fourth peptide chain pair up to form an Fv fragment. The IL-15Rα of the third peptide chain and the IL-15 of the fourth peptide chain bind to form the IL-15 / IL-15Rα complex. The Fab fragment targets TAA and the Fv fragment targets CD16A, or the Fab fragment targets CD16A and the Fv fragment targets TAA. From the N-terminus to the C-terminus of the peptide chain, the fusion sequence of the third peptide chain is VH domain ~ IL-15Rα ~ second Fc chain. The triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation is characterized in that the fusion sequence of the fourth peptide chain from the N-terminus to the C-terminus of the peptide chain is VL domain to IL-15, where "~" represents a linking fragment.

2. The amino acid sequence of the aforementioned linked fragment is characterized by having a few GS repeat sequences. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 1.

3. The amino acid sequence of the linked fragment is (GGGGS)n, where n is 1, 2, 3, 4, or 5. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 1.

4. The TAA binding region is a Fab fragment targeting TAA, and the CD16A binding region is an Fv fragment targeting CD16A. The first monomer comprises the TAA binding region and the first Fc chain, the second monomer comprises the CD16A binding region, the IL-15 / IL-15Rα complex and the second Fc chain, and the first Fc chain and the second Fc chain polymerize to form the Fc domain. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 1.

5. The VH domain and VL domain, which together form the Fv fragment, are accompanied by one or more pairs of disulfide bonds, and the molecule contains one or more of the following mutation combinations, which are counted according to the EU. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 1. Table 1

6. The IL-15 comprises IL-15, as well as mutations, cleavages, and various derivatives that can bind to IL-15Rα, and the IL-15Rα comprises IL-15Rα, as well as mutations, cleavages, and various derivatives that can bind to IL-15. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 1.

7. The IL-15 in question includes, but is not limited to, any of the following mutation combinations, and the counting method is to count the first amino acid of the IL-15 amino acid sequence as the first position. Table 2 Alternatively, the IL-15 / IL-15Rα complex may include, but is not limited to, any of the following mutation combinations, and the counting method is characterized in that the first amino acid of the amino acid sequence of IL-15 or IL-15Rα is counted as the first position. The triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 6. Table 3

8. The aforementioned TAAs are CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD217, CD239, CD274, CD276, CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, EGFR, EGFR Characterized by being selected from VIII, C-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, CCR4, VEGFR-2, CD6, Integrin α4, PDGFRα, NeuGcGM3, Integrin αVβ3, CD51, CTAA16.88, CD22, ROR1, CSPG4, SS1, or IGFR1. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 1.

9. The TAA is characterized in that it is selected from CD33, CS1, or CD38. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 1.

10. The Fc domain is selected from human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc or its variants, and the Fc domain is characterized in that it is in the form of an Fc heterodimer. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 1.

11. The Fc heterodimer includes, but is not limited to, the following mutation combinations, and the following mutations are counted according to the EU. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation, as described in claim 10. Table 4

12. The Fc domain is characterized by being selected to eliminate immunoeffector function. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 1.

13. The Fc domain comprises one of the following mutation forms, and the following mutations are counted according to the EU. The triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 12. Table 5

14. The first Fc chain does not bind to protein A, while the second Fc chain can bind to protein A. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 1.

15. The first Fc chain is characterized by containing the mutation H435R or H435R / Y436F and being counted according to the EU. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation, as described in claim 14.

16. The CD16A binding region is characterized in that it includes the VH domain shown in SEQ ID NO: 39 and the VL domain shown in SEQ ID NO: 38, or the sequence of IL-15 is as shown in SEQ ID NO: 46 or 47, or the sequence of IL-15Rα is as shown in any of SEQ ID NO: 48 to 54. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 1.

17. The CD16A binding region is characterized by having at least one pair of disulfide bonds between VH and VL, and / or between IL-15 and IL-15Rα. The triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 16.

18. (1) SEQ ID NO: 01, 02, 03, 04; (2) SEQ ID NO: 01, 05, 03, 06; (3) SEQ ID NO: 07, 08, 09, 10; (4) SEQ ID NO: 01, 02, 11, 12; (5) SEQ ID NO: 01, 02, 13, 14; (6) SEQ ID NO: 01, 05, 11, 15; (7) Characterized by being obtained by fusing with an amino acid fragment that is 90% or more identical to any set of sequences of SEQ ID NO: 01, 05, 13, 16. A triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in claim 1.

19. nucleic acid molecules, The nucleic acid molecule characterized by encoding a triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in any one of claims 1 to 18.

20. A pharmaceutical composition, The pharmaceutical composition is characterized by comprising a triplicate fusion protein for antigen targeting, anti-CD16A, and immune effector cell activation as described in any one of claims 1 to 18, and a pharmaceutically acceptable carrier.

21. Use of a tripfunctional fusion protein of antigen targeting, anti-CD16A, and immune effector cell activation according to any one of claims 1 to 18 in the preparation of a drug for inhibiting or treating cancer, infection, and immunomodulatory disorders.

22. The aforementioned cancers are characterized by including prostate cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, melanoma, kidney cancer, oral cancer, pharyngeal cancer, pancreatic cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, or hematological cancers. The use described in claim 21.

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