CD16A targeted binding protein as well as derivative and application thereof

Abstract

The invention specifically discloses a CD16A targeted binding protein as well as a derivative and application thereof, and relates to the technical field of biological medicines. The CD16A targeting binding protein provided by the invention has high affinity to CD16A, the molecular weight of the CD16A targeting binding protein is only about 18kDa and is about one tenth of that of an antibody, and the CD16A targeting binding protein has good tissue penetrability, is simple in production process and high in yield, has good targeting property to NK cells with positive CD16 expression, has an efficient immune activation effect, and can be used for preparing the immunopotentiator. The compound can promote the killing effect of an organism immune system on tumor cells, and can be used for tumor targeted drug delivery or NK cell targeted drug delivery and tumor immune cell therapy. The invention provides a theoretical basis and an experimental basis for developing a new treatment strategy, and has good clinical transformation and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a CD16A targeting binding protein and an application thereof. Background Art

[0002] Natural killer cells (NK cells) are a type of lymphocyte in the innate immune system that has characteristics such as cytotoxicity that is not restricted by MHC, the ability to secrete cytokines, and the formation of immune memory. An important feature of NK cells is that they can recognize a variety of damaged cells, especially tumor cells and virus-infected cells. They can not only directly kill target cells, but also participate in the occurrence, regulation, and maintenance of a variety of cellular immune responses.

[0003] NK cells can recognize target cells through the low-affinity IgG Fc region receptor CD16A on their surface in the presence of antibodies, triggering antibody-dependent cell cytotoxicity (ADCC). NK cells then eliminate target cells through two main mechanisms: direct cytotoxicity and cytokine secretion. The immune synapse formed between NK cells and target cells enables the cytotoxic granules released by NK cells to act accurately on target cells without affecting the surrounding bystander cells. The ADCC effect mediated by CD16A on the surface of NK cells is believed to help improve the efficacy of therapeutic antibodies (such as the anti-CD20 antibody rituximab).

[0004] Therefore, CD16A target is preferred in NK cell-based anti-tumor therapy. It is an important target for the development of anti-tumor drugs and has a wide range of uses. However, the current antibody drugs targeting CD16A have the characteristics of large molecular weight, poor tumor penetration, complex process, and high production cost. Therefore, it is of great significance to develop low molecular weight antibody drugs with high affinity and high permeability. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a CD16A targeting binding protein, wherein the protein is selected from:

[0006] (1) a targeted binding protein having an amino acid sequence as shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3; or

[0007] (2) a targeted binding protein that is homologous to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID No. 2 or SEQ ID No. 3 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity; or

[0008] (3) A targeting binding protein derived from (1) or (2) in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (1) or (2).

[0009] In one embodiment, the amino acid sequence comprises amino acid changes at positions 30, 32, 35, 41, 42, 63, 65, 66, 68, 74, 75, 96, 98, 101, 107 and 108 of SEQ ID NO: 1.

[0010] In one embodiment, the amino acid change is any amino acid substitution.

[0011] In another aspect, the present invention further provides a fusion protein, which comprises the above-mentioned targeting binding protein, a connecting peptide, and a tumor penetrating peptide.

[0012] In one embodiment, the connecting peptide includes one or a combination of G, GGGSG, GGGGS, GGSGG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGGGSGGGGSGGGGS, GGSGGGGSGGGGSG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGG, (G4S)n, wherein n is selected from a natural number within 1-10.

[0013] In one embodiment, the connecting peptide is GGGGSGGGGSGGGGS.

[0014] In one embodiment, the tumor penetrating peptide comprises one or a combination of Tat peptide, Penetratin, R8 peptide, iRGD, RGD, iNGR, NGR, LyP-1, CGKRK, ANGiopep-2, pHLIP, K237.

[0015] In one embodiment, the tumor penetrating peptide is iRGD.

[0016] In one embodiment, the fusion protein is a fusion protein selected from the amino acid sequence shown in SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7.

[0017] In one embodiment, the amino acid change of the fusion protein is any amino acid substitution.

[0018] On the other hand, the present invention also provides a chimeric antigen receptor, which comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain comprises a CD16A antigen binding domain that specifically binds to CD16A, and wherein the antigen binding domain is selected from the protein according to any one of claims 1-2 or the fusion protein according to any one of claims 3-6.

[0019] In one embodiment, the intracellular domain comprises at least one co-stimulatory domain.

[0020] In one embodiment, the costimulatory domain is a signaling region of the following substances: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death factor-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10 , Fcγ receptors, class I MHC molecules, TNF receptor proteins, immunoglobulins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α., CD8β, IL-2Rβ, IL -2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM , CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI(CD226), SLAMF4(CD244, 2B 4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

[0021] In one embodiment, the costimulatory domain comprises the signaling region of 4-1BB / CD137.

[0022] In one embodiment, the intracellular domain comprises at least one activation domain.

[0023] In one embodiment, the activation domain comprises CD3.

[0024] In another aspect, the present invention also provides a nucleic acid molecule, wherein the nucleic acid encodes the protein, fusion protein or chimeric antigen receptor as claimed in claim 1.

[0025] In one embodiment, the nucleic acid molecule is selected from mRNA.

[0026] The present invention also provides a vector in another aspect, wherein the vector contains the above-mentioned nucleic acid molecule.

[0027] In one embodiment, the vector comprises one or a combination of a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-associated vector, and a lentiviral vector.

[0028] In another aspect, the present invention further provides an engineered immune cell, characterized in that the engineered immune cell expresses the above-mentioned chimeric antigen receptor, nucleic acid molecule or vector.

[0029] In one embodiment, the immune cell is one or a combination of T cells, tumor infiltrating lymphocytes (TIL), NK cells, cells expressing TCR, dendritic cells or NK-T cells.

[0030] In one embodiment, the cell is a NK cell.

[0031] In another aspect, the present invention also provides a use of the above-mentioned protein, fusion protein, chimeric antigen receptor, nucleic acid molecule, vector or engineered immune cell in the preparation of a drug for treating tumors and / or tumor-related diseases and / or symptoms.

[0032] In one embodiment, the tumor includes breast cancer, gastric cancer, liver cancer, lymphoma, intestinal cancer, lung cancer, and cervical cancer.

[0033] In another aspect, the present invention also provides a pharmaceutical composition, comprising:

[0034] (1) a therapeutically effective amount of the above-mentioned protein, fusion protein, chimeric antigen receptor, nucleic acid molecule, vector or engineered immune cell;

[0035] (2) A pharmaceutically or immunologically acceptable carrier or excipient.

[0036] In another aspect, the present invention further provides a pharmaceutical preparation, which comprises the above-mentioned pharmaceutical composition.

[0037] In one embodiment, the pharmaceutical composition is formulated for administration via an administration route selected from the group consisting of intranasal, intralesional, intrathecal, intravenous, intramuscular, subcutaneous, sublingual, oral, and intracerebral administration routes, and combinations thereof.

[0038] In one embodiment, the pharmaceutical composition is formulated for administration via an intravenous route of administration.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. The CD16A targeting binding protein provided by the present invention has a high affinity for CD16A, and its molecular weight is only about 18 kDa, which is about one tenth of that of an antibody. It has good tissue penetrability, and its production process is simple and its yield is high.

[0041] 2. The targeted binding protein of the present invention has good targeting to CD16-positive NK cells and has a highly efficient immune activation effect, which can promote the killing effect of the body's immune system on tumor cells. It can be used for tumor-targeted drug delivery or NK cell-targeted drug delivery, as well as tumor immune cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 : Binding of targeted binding proteins to CD16A protein molecules. Targeted binding proteins #1, #2, and #3 have significant binding to CD16A, among which targeted binding proteins #1 and #2 have the strongest binding to CD16A, while the non-targeted targeted binding protein #4 in the control group has no significant binding.

[0044] Figure 2 :Numerical detection of affinity between superior CD16A targeting binding protein and CD16A protein molecule.

[0045] Figure 3 :The binding status of dominant CD16A targeting binding protein and NK cells. Figure 3 A. After the targeted binding protein #1 was co-incubated with NK cells, the fluorescence intensity was detected by flow cytometry. Its average fluorescence intensity was significantly different from that of the targeted binding protein #4 in the control group; Figure 3 B. Fluorescence images of cell slides observed under a confocal microscope show that targeted binding protein #1 can bind to NK cells, while targeted binding protein #4 in the control group cannot bind to NK cells.

[0046] Figure 4 :The fusion protein anti-CD16A-iRGD can upregulate the CD137 activation marker on the surface of NK cells.

[0047] Figure 5 : The fusion protein anti-CD16A-iRGD can promote the killing function of NK cells against MKN45 tumor cells.

[0048] Figure 6 :Analysis of the effect of fusion protein anti-CD16A-iRGD on the survival of MKN45 subcutaneous tumor nude mouse model. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] As used herein, “containing,” “having,” or “including” include “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of;” “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0051] definition

[0052] As used herein, the term "specific binding" means that an antibody or polypeptide can preferentially bind to a binding partner, e.g., CD16A, in a competitive binding assay, such as assessed using a recombinant form of the protein, an epitope therein, or the native protein present on the surface of an isolated target cell. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.

[0053] As used herein, the term "affinity" means the strength of binding of an antibody or protein to an epitope. The affinity of an antibody is given by the dissociation constant KD, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant KA is defined by 1 / KD. Preferred methods for determining protein affinity can be found in Harlow et al., "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988); Coligan et al., eds., "Current Protocols in Immunology", Greene Publishing Assoc and Wiley Interscience, New York, (1992, 1993); and Muller, Meth. Enzymol., Vol. 92: 589-601 (1983). A preferred standard method for determining protein affinity, well known in the art, is to use surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore™ SPR analysis instrument).

[0054] As used herein, the term "identity" or "same" when used in the context of a relationship between sequences of two or more polypeptides refers to the degree of sequence relatedness between the polypeptides, as determined by the number of matches between chains of two or more amino acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences with gapped alignments (if any) solved by a particular mathematical model or computer program (i.e., an "algorithm"). The identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in: "Computational Molecular Biology", Lesk, AM, ed., Oxford University Press, New York, 1988; "Biocomputing: Informatics and Genome Projects", Smith, DW, ed., Academic Press, New York, 1993; "Computer Analysis of Sequence Data, Part 1", Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; "Sequence Analysis in Molecular Biology", von Heinje, G., Academic Press, 1987; "Sequence Analysis Primer", Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1993. Press, New York, 1991; and Carillo et al., SIAM J. Applied Math., 48, 1073 (1988).

[0055] Preferred methods for determining identity are designed to give the maximum match between the sequences tested. The method for determining identity is described in publicly available computer programs. Preferred computer program methods for determining the identity between two sequences include GCG program packages, including GAP (Devereux et al., Nucl. Acid. Res.), Vol. 12, p. 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol., Vol. 215, p. 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources ("BLAST Manual", Altschul et al., NCB / NLM / NIH Bethesda, Maryland (Md.), 20894; Altschul et al., supra). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0056] As used herein, "NK cell" refers to a lymphocyte subgroup involved in unconventional immunity. NK cells can be identified by certain characteristics and biological properties, such as the expression of specific surface antigens (including CD56 and / or NKp46) of human NK cells, the absence of α / β or γ / δTCR complexes on the cell surface, the ability to bind and kill cells that cannot express "self" MHC / HLA antigens by activating specific lytic cell mechanisms, the ability to kill tumor cells or other diseased cells expressing ligands of NK activation receptors, and the ability to release protein molecules (called cytokines) that stimulate or inhibit immune responses. Using methods well known in the art, any of these characteristics and activities can be used to identify NK cells. Any subgroup of NK cells is also covered in the term NK cells. In the context of this article, "active" NK cells refer to biologically active NK cells, including NK cells with the ability to lyse target cells or enhance the immune function of other cells. NK cells can be obtained by various techniques known in the art, such as separation from blood samples, cell apheresis, tissue or cell collection, etc. Useful experimental protocols involving NK cells can be found in "Natural Killer Cells Protocols" (Edited by Campbell KS and Colonna M). Humana Press, pp. 219-238 (2000).

[0057] As used herein, the terms "peptide connector" or "amino acid linker" are used interchangeably and refer to a short peptide that acts as a link between the amino acid sequence of a protein and the amino acid sequence of a tumor-penetrating peptide. The length of the peptide connector is generally 1-20 amino acids, preferably 3-10 amino acids, and most preferably 4-6 amino acids. A skilled person can design a peptide connector according to conventional methods in the art (e.g., see PNAS 1998; 95: 5929-5934; Protein Eng, 2000; 13 (5): 309-312; Protein Eng, 2003; 15 (11): 871-879, etc.). Generally, the peptide connector does not affect or seriously affects the correct folding and spatial conformation of the amino acid sequence of the protein and the amino acid sequence of the tumor-penetrating peptide.

[0058] Preferred examples of connecting peptides include (but are not limited to): in order to facilitate the protein to fold into independent domains with sufficient biological activity in space, it is appropriate to use sequences such as GG, AAAGGGS, SGGGSGGG and GGGGSGGGGSGGGGS as connecting arms; in order to facilitate purification, 6His can be used as a connecting arm to purify the CD16A targeting binding protein-iRGD fusion protein by metal affinity chromatography. In one embodiment, the connecting peptide is GGGGSGGGGSGGGGS.

[0059] As used herein, "chimeric antigen receptor (CAR)" is a protein that specifically recognizes a target antigen (e.g., a target antigen on a cancer cell). When bound to a target antigen, CAR can activate immune cells to attack and destroy cells (e.g., cancer cells) bearing the antigen. CAR can also incorporate a co-stimulatory or signaling domain to increase its effectiveness.

[0060] The chimeric antigen receptor disclosed in the present invention comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain comprises a CD16A antigen binding domain that specifically binds to CD16A. In some embodiments, the CD16A-specific CAR comprises the following elements from 5' to 3': a signal sequence, a CD16A antigen binding domain, a hinge and a transmembrane region, and one or more continuous signaling domains.

[0061] As used herein, "antigen binding domain" refers to any polypeptide that binds to a specified target antigen, for example, a specified target antigen may be a CD16A protein or a fragment thereof (interchangeably referred to herein as "CD16A antigen", "CD16A target antigen" or "CD16A target"). In some embodiments, the antigen binding domain binds to the CD16A antigen on tumor cells. In some embodiments, the antigen binding domain binds to the CD16A antigen on cells involved in a hyperproliferative disease.

[0062] As used herein, the term "transmembrane domain" has the ability to: (a) be expressed on a surface immune cell, such as, but not limited to, a lymphocyte, such as a T helper (Th) cell, a cytotoxic T (Tc) cell, a T regulatory (Treg) cell, or a natural killer (NK) cell, and / or (b) interact with an extracellular antigen binding domain and an intracellular signaling domain to direct the cellular response of the immune cell to a target cell.

[0063] The transmembrane domain may be derived from natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0064] The transmembrane region used in the present disclosure may be derived from (contain or correspond to) CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death factor 1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fc gamma receptors, class 1 MHC molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2 Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITG AM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2 B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

[0065] As used herein, the term "intracellular (cytoplasmic) domain" can activate at least one normal effector function of an immune cell comprising CAR, such as signal 1 / activation and / or signal 2 / co-stimulation. For example, the effector function of a T cell can refer to cytolytic activity or auxiliary activity, including the secretion of cytokines.

[0066] It is understood that suitable (e.g., activating) intracellular domains include, but are not limited to, signaling domains derived from (or corresponding to) the following: CD3ζ, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death factor 1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, class 1 MHC molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, L FA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(C D244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

[0067] The intracellular domain of the CAR disclosed in the present invention may also be incorporated into a costimulatory signaling domain (hereinafter interchangeably referred to as a costimulatory molecule) in addition to the above-mentioned activation domain to increase its effectiveness. In addition to the main signal provided by the activation molecule described herein, the costimulatory domain may also provide a signal.

[0068] It will be appreciated that suitable costimulatory domains within the scope of the present disclosure may be derived from (or correspond to) for example CD28, OX40, 4-1BB / CD137, CD2, CD3 (α, β, δ, ε, γ, ζ), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, class I MHC molecule, TNFR, integrin, signaling lymphocyte activation molecule, BT LA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), N Kp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA 6. VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29 , ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tact ile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, or fragments or combinations thereof.

[0069] It should be understood that other co-stimulatory molecules or fragments thereof not listed above are also within the scope of the present disclosure.

[0070] As used herein, an "immune cell" is a T cell (e.g., inflammatory T lymphocytes, cytotoxic T lymphocytes,

[0071] In some embodiments, the engineered immune cells are T cells. In some embodiments, the engineered immune cells are γδT cells. In some embodiments, the engineered immune cells are macrophages. In some embodiments, the engineered immune cells are natural killer (NK) cells.

[0072] As used herein, "tumor" includes tumors including breast cancer, gastric cancer, liver cancer, lymphoma, intestinal cancer, lung cancer, cervical cancer. In some embodiments, the tumor is gastric cancer.

[0073] As used herein, the term "pharmaceutical composition" refers to a composition comprising extracellular vesicles formulated together with one or more pharmaceutically acceptable carriers.

[0074] The formulation of the pharmaceutical composition can be adjusted according to the application. In particular, the pharmaceutical composition can be formulated using methods known in the art to provide rapid, continuous or delayed release of the active ingredient after administration to a mammal. For example, the formulation can be selected from any of the following: plasters, granules, lotions, liniments, lemonades, perfumed waters, powders, syrups, eye ointments, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, eye drops, tablets, suppositories, injections, alcoholic agents, capsules, creams, lozenges, tinctures, pastes, pills and soft or hard gelatin capsules.

[0075] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH regulators, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc. that are compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide supplementary, additional or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch or talc, or liquid carriers, such as water, fatty oils or liquid paraffin. Other examples of the carrier include culture media, such as DMEM or RPMI; and low temperature storage media, comprising components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates and ion concentrations, which can balance the intracellular state at low temperatures; and mixtures of organic solvents and water.

[0076] The pharmaceutical composition is formulated for administration by an administration route selected from intranasal, intralesional, intrathecal, intravenous, intramuscular, subcutaneous, sublingual, oral and intracerebral administration routes. According to one embodiment, the pharmaceutical composition is formulated for intranasal administration. According to some embodiments, this pharmaceutical composition is in the form of a liquid solution, nasal drops, spray, or metered spray. According to other embodiments, the pharmaceutical composition is formulated into a preparation for injection, such as intralesional, intrathecal or intravenous injection. According to this embodiment, the pharmaceutical composition is in the form of a sterile injection solution.

[0077] Any known method can be used to administer the pharmaceutical composition of the present invention. One of the various methods known to those skilled in the art can be used to implement the term "giving" or "administering" a substance, compound or medicament to a subject. For example, a compound or medicament can be administered intranasally (e.g., by inhalation), intrathecal (entering the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., by a skin catheter). Compounds or agents can also be appropriately introduced by rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or preparations), which provide extended, slowed, or controlled release for compounds and agents. It can also be administered, for example, once, multiple times, and / or over one or more extended periods.

[0078] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level includes the type and severity of the subject, age, sex, drug activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant medications, and other factors well known in the medical field.

[0079] As used herein, the term "treating" a condition or patient means taking steps to obtain a beneficial or desired result, including a clinical result. Beneficial or desired clinical results include, but are not limited to, or eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, condition, or disorder, substantially improving or alleviating the clinical or aesthetic symptoms of a condition, substantially preventing the clinical or aesthetic symptoms of a disease, condition, or disorder, and avoiding harmful or annoying symptoms. Treatment also refers to accomplishing one or more of the following: (a) lessening the severity of the condition; (b) limiting the development of symptoms characteristic of the condition being treated; (c) limiting the worsening of symptoms characteristic of the condition being treated; (d) limiting the recurrence of the condition in patients who previously had the condition; and / or (e) limiting the recurrence of symptoms in patients who were previously free of symptoms of the condition.

[0080] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0081] Example 1

[0082] Enzyme-linked immunosorbent assay (ELISA) was used to detect the affinity of the target binding protein to the CD16A molecule:

[0083] 1. Preparation and purification of targeted binding proteins:

[0084] The following targeted binding proteins #1, #2, and #3 were synthesized using the E. coli expression system, and the negative control targeted binding protein #4 was expressed. The proteins were purified using a nickel column, and the endotoxin removal kit was used to remove endotoxin to <0.1EU / μg:

[0085] Targeted binding protein #1 (SEQ ID No. 1 with a histidine tag at the N-terminus)

[0086] Targeted binding protein #2 (SEQ ID No. 2 with a histidine tag at the N-terminus)

[0087] Targeted binding protein #3 (SEQ ID No. 3 with a histidine tag at the N-terminus)

[0088] Targeted binding protein #4 (SEQ ID No. 4 with a histidine tag at the N-terminus)

[0089] 2. Co-incubation of targeted binding protein with CD16A

[0090] CD16A protein was coated on a flat-bottom 96-well plate, blocked with 1% BSA, and incubated with the same concentration of target binding proteins #1, 2, 3, and 4 for 2 hours. After washing away each group of target binding proteins with PBS containing 0.5% Tween 20 (0.5% PBST), diluted anti-his secondary antibody was added and incubated for another 1 hour. After washing away the anti-his secondary antibody with 0.5% PBST, TMB solution was added; reacted at room temperature in the dark for 30-120 seconds, the solution changed from colorless to blue; 1M dilute hydrochloric acid was added to terminate the reaction, and the solution changed from blue to yellow.

[0091] 3. Use a microplate reader to measure the absorbance at OD450:

[0092] like Figure 1 As shown, compared with the negative control targeted binding protein #4, the targeted binding proteins #1, #2, and #3 all have obvious binding with the CD16A protein, among which the targeted binding protein #1 has the strongest binding with the CD16A protein.

[0093] The specific protein sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 are as follows:

[0094] SEQ ID No.1:

[0095] HHHHHHDLGKKLLEAARAGQDDEVRILLKAGADVNAHDKQGSTPLHLAAEEGHLEIVEVLLKYGADVNATDVSGWTPLHLAAFYGHLEIVEVLLKYGADVNAHDHLGTTPLHLAAAVGHLEIVEVLLKYGADVNAQDKSGKTPADLAADAGHEDIAEVLQKAA

[0096] SEQ ID No.2:

[0097] HHHHHHDLGKKLLEAARAGQDDEVRILLKAGADVNAADFNGSTPLHLAAREGHLEIVEVLLKYGADVNATDVSGHTNLHLAAQYGHLEIVEVLLKYGADVNAHDHNGETPWHLAAAHNHLEIVEVLLKYGADVNAQDKSGKTPADLAADAGHEDIAEVLQKAA

[0098] SEQ ID No.3:

[0099] HHHHHHDLGKKLLEAARAGQDDEVRILLKAGADVNAYDKAGTTPLHLAALAGHLEIVEVLLKYGADVNATDVSGTTDLHLAAFWGHLEIVEVLLKYGADVNAHDHSGDTPIHLAAALWHLEIVEVLLKYGADVNAQDKSGKTPADLAADAGHEDIAEVLQKAA

[0100] SEQ ID No.4:

[0101] HHHHHHDLGKKLLEAASAGQDDEVRILLKAGADVNAADAAGATPLHLAAAAGHLEIVEVLLKYGADVNAADAAGATPLHLAAAAGHLEIVEVLLKYGADVNAADAAGATPLHLAAAAGHLEIVEVLLKYGADVNAQDKSGKTSADLAADAGHEDIAEVLQKAA

[0102] Example 2

[0103] The affinity value of the superior CD16A targeting binding protein (CD16A targeting binding protein #1) to the CD16A molecule was detected by the surface plasmon resonance (SPR) method:

[0104] SPR detection using CM5 chip and Biacore T200 instrument

[0105] 1. Coupling CD16A protein:

[0106] 10 μg / mL anti-His antibody was prepared and flowed through the first and second channels of the CM5 chip at a flow rate of 10 μL / min for 420 seconds; then the residual activated carboxyl groups were blocked with ethanolamine at a flow rate of 10 μL / min for 420 seconds. The human CD16A recombinant protein molecule with a His tag was flowed through the surface of the CM5 chip at a flow rate of 10 μg / mL, and the first channel was set as the reference channel.

[0107] 2. Determine the affinity of the targeted binding protein #1 to the human CD16A recombinant protein:

[0108] After the protein was fixed on the chip surface, multi-cycle kinetics was used to detect the affinity of the analyte to the CD16A protein. The analyte concentrations were 0.425, 0.85, 2.215, 4.25, 21.25, 42.5, 63.75, 85, 148.75, and 212.5 nM, the flow rate was 30 μL / min, the association time was 180 s, the dissociation time was 320 s, and it was regenerated with 0.75 mM NaOH.

[0109] 3. The affinity data were fitted using Biacore Evaluation Software of Biacore T200 instrument.

[0110] like Figure 2 As shown, the affinity of targeted binding protein #1 to human CD16A recombinant protein is 12 nM.

[0111] Example 3

[0112] Advantages of targeted binding proteins binding to CD16A on the surface of natural killer cells (NK cells):

[0113] 1. Flow cytometry detection of cells

[0114] (1) Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation, and CD16+ cells in PBMCs were obtained by Miltenyi Biotec's Human CD16 MicroBeads magnetic bead sorting.

[0115] (2) Interleukin-12 (IL12), interleukin-15 (IL15), and interleukin-18 (IL18) were used for overnight culture to induce the memory NK cell phenotype, and then interleukin-15 (IL15) was used for 7 days to promote NK cell activation.

[0116] (3) Targeted binding proteins #1 and #4 were added to NK cells respectively, and co-cultured in a cell incubator at 37°C for 1 h. Dylight 650 anti-His secondary antibody was then added for staining. After washing, the cell surface fluorescence intensity was detected by flow cytometry.

[0117] The results are as follows Figure 3As shown in A, compared with the non-targeted targeted binding protein #4, the average fluorescence intensity of the targeted binding protein #1 was significantly enhanced, indicating that the dominant CD16A targeted binding protein (CD16A targeted binding protein #1) has a stronger ability to bind to CD16-positive NK cells.

[0118] 2. Observe cell fluorescence under confocal microscope

[0119] NK cells of a certain density were attached to the cell slide by centrifugation, and the target binding proteins #1 and 4 were added to the NK cells respectively, and co-cultured in a 37°C cell incubator for 1 hour; unbound proteins were washed away, and Dylight 650 anti-His secondary antibody was added for staining, and incubation was continued for 1 hour. After washing, DAPI dye was used for cell nucleus staining, and finally the slide was taken to observe the cell surface fluorescence under a confocal microscope.

[0120] The results are as follows Figure 3 As shown in B, compared with the non-targeted targeted binding protein #4, the fluorescence intensity of the targeted binding protein #1 is significantly different, further indicating that the superior CD16A targeted binding protein (CD16A targeted binding protein #1) has a stronger ability to bind to CD16-positive NK cells.

[0121] Example 4

[0122] Preparation of CD16A targeting binding protein-iRGD fusion protein and its activation effect on NK cells 1. Preparation and purification of CD16A targeting binding protein-iRGD fusion protein:

[0123] The CD16A targeting binding protein-iRGD fusion protein (anti-CD16A-iRGD) was synthesized and expressed using an E. coli expression system. The fusion proteins #5, #6, and #7 were composed of one of the CD16A targeting binding proteins #1, #2, and #3 and the tumor penetrating peptide iRGD connected via a connecting peptide GGGGSGGGGSGGGGS. The protein was purified using a nickel column, and the endotoxin removal kit of GenScript was used to remove endotoxin to <0.1EU / μg.

[0124] 2. Flow cytometry detection of NK cell surface activation marker CD137:

[0125] (1) PBMCs were isolated and NK cells were sorted according to the method described in Example 2, and cytokine culture was used to promote NK cell differentiation.

[0126] (2) MKN45 cells and NK cells were co-incubated at an E:T ratio of 5:1, and different concentrations of fusion protein #5 (0, 1, 10, 100 μg / ml) were added. After 24 h, the expression of CD137 on the surface of NK cells was detected by flow cytometry, and its average fluorescence intensity was statistically analyzed.

[0127] like Figure 4 As shown, CD137 is one of the surface markers of NK cell activation. The activation effect of fusion protein #5 on NK cells is concentration-dependent. As the dose increases, the proportion of CD137-positive NK cells also increases, indicating that fusion protein #5 has a very strong immune activation effect.

[0128] The specific protein sequences of SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7 are as follows:

[0129] SEQ ID No.5:

[0130] HHHHHHDLGKKLLEAARAGQDDEVRILLKAGADVNAHDKQGSTPLHLAAEEGHLEIVEVLLKYGADVNATDVSGWTPLHLAAFYGHLEIVEVLLKYGADVNAHDHLGTTPLHLAAAVGHLEIVEVLLKYGADVNAQDKSGKTPADLAADAGHEDIAEVLQKAAGGGGSGGGGSGGGGSGGGGSCRGDKGPDC

[0131] SEQ ID No.6:

[0132] HHHHHHDLGKKLLEAARAGQDDEVRILLKAGADVNAADFNGSTPLHLAAREGHLEIVEVLLKYGADVNATDVSGHTNLHLAAQYGHLEIVEVLLKYGADVNAHDHNGETPWHLAAAHNHLEIVEVLLKYGADVNAQDKSGKTPADLAADAGHEDIAEVLQKAAGGGGSGGGGSGGGGSGGGGSCRGDKGPDC

[0133] SEQ ID No.7:

[0134] HHHHHHDLGKKLLEAARAGQDDEVRILLKAGADVNAYDKAGTTPLHLAALAGHLEIVEVLLKYGADVNATDVSGTTDLHLAAFWGHLEIVEVLLKYGADVNAHDHSGDTPIHLAAALWHLEIVEVLLKYGADVNAQDKSGKTPADLAADAGHEDIAEVLQKAAGGGGSGGGGSGGGGSGGGGSCRGDKGPDC

[0135] Example 5

[0136] Advantages: anti-CD16A-iRGD promotes NK cell killing of MKN45:

[0137] The NK cells obtained according to the method described in Example 2 were mixed with MKN45 cells transfected with luciferase at an effector-target ratio of E:T=5:1, and different concentrations of fusion protein #5 (0, 1, 5, 10 μg / ml) were added. After co-culture at 37°C for 24 h, the fluorescent substrate D-luciferin potassium salt was added, and the bioluminescence intensity was counted using the luminescence detection function of the microplate reader.

[0138] like Figure 5 As shown, compared with the control group without fusion protein #5, the experimental groups with different concentrations of fusion protein #5 protein showed better NK cell killing effect, and a low concentration of 1 μg / ml could play a significant role in promoting NK cell killing.

[0139] Example 6

[0140] In vivo detection advantages Anti-tumor activity test of anti-CD16A-iRGD in MKN45 mouse subcutaneous tumor model:

[0141] (1) MKN45 cells were cultured until adherent, and 4-week-old nude mice were selected and inoculated subcutaneously in the groin of the nude mice for 10 6 The cells were grown and observed for several days until the tumor grew to 100 mm 3 about.

[0142] (2) The tumor-bearing mice were randomly divided into three groups: blank group (PBS), control group (NK), and experimental group (NK+fusion protein #5), with 6 mice in each group. The blank group was injected with 100 μL of PBS per tail vein once a week for two consecutive weeks; the control group was injected with 1×10 NK cells per tail vein. 7 1×10 NK cells were injected into the tail vein of the experimental group each time, once a week for two consecutive weeks. 7 Each mouse was treated with 2 mg / kg of fusion protein #5 once a week for two consecutive weeks. During the treatment period, each mouse was intraperitoneally injected with 50,000 IU rIL-2 three times a week to maintain NK cell activity.

[0143] (3) Observe and count the survival status of each mouse in the blank group (PBS), control group (NK), and experimental group (NK+fusion protein #5), and the observation and statistics time length is 60 days. The mouse survival curve was drawn using the Kaplan-Meier method, and the differences in the survival period of mice in each group were statistically compared using the log-rank (Mantel-Cox) test.

[0144] like Figure 6As shown, the survival time of the fusion protein #5 group was significantly longer than that of the blank PBS group and the control group, indicating that fusion protein #5 effectively inhibited the proliferation and growth of MKN45 cells and significantly prolonged the survival time of mice.

[0145] The above experiments collectively illustrate that the CD16A targeting binding protein #1 screened by the present invention can specifically bind to the CD16A target with high affinity, can be widely used in the prevention and treatment of tumors, and provides a new option for the prevention or treatment of tumors.

[0146] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0147] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A CD16A targeting binding protein, characterized in that: The targeted binding protein is selected from: (1) a targeted binding protein having an amino acid sequence as shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3; or (2) a protein that is homologous to or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 1, SEQ ID No. 2 or SEQ ID No. 3; or (3) A targeted binding protein derived from (1) or (2) in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (1) or (2).

2. The targeted binding protein according to claim 1, characterized in that The amino acid sequence SEQ ID NO:1 comprises amino acid changes at positions 30, 32, 35, 41, 42, 63, 65, 66, 68, 74, 75, 96, 98, 101, 107 and 108.

3. A fusion protein, characterized in that The fusion protein comprises the targeting binding protein according to any one of claims 1 to 2, a connecting peptide, and a tumor penetrating peptide.

4. The fusion protein according to claim 3, characterized in that The connecting peptide includes one or a combination of G, GGGSG, GGGGS, GGSGG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGGGSGGGGSGGGGS, GGSGGGGSGGGGSG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGG, (G4S)n; wherein n is selected from a natural number within 1-10.

5. The fusion protein according to claim 4, characterized in that The tumor penetrating peptide includes one or a combination of Tat peptide, Penetratin, R8 peptide, iRGD, RGD, iNGR, NGR, LyP-1, CGKRK, ANGiopep-2, pHLIP, K237.

6. The fusion protein according to claim 5, characterized in that The fusion protein is a fusion protein selected from the amino acid sequence shown in SEQ ID No.5, SEQ ID No.6 or SEQ ID No.

7.

7. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain comprises a CD16A antigen binding domain that specifically binds to CD16A, and wherein the antigen binding domain is selected from the targeted binding protein of any one of claims 1-2 or the fusion protein of any one of claims 3-6.

8. A nucleic acid molecule, characterized in that The nucleic acid encodes the targeted binding protein according to any one of claims 1 to 2, the fusion protein according to any one of claims 3 to 6, or the chimeric antigen receptor according to claim 7.

9. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 8.

10. The carrier according to claim 9, characterized in that The vector includes one or a combination of a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-associated vector, and a lentiviral vector.

11. An engineered immune cell, characterized in that: The engineered immune cell expresses the chimeric antigen receptor of claim 7, the nucleic acid molecule of claim 8, or the vector of claim 9 or 10.

12. The engineered immune cell according to claim 11, characterized in that: The immune cell is one or a combination of T cells, tumor infiltrating lymphocytes (TIL), NK cells, cells expressing TCR, dendritic cells or NK-T cells.

13. Use of the protein according to any one of claims 1 to 6, the chimeric antigen receptor according to claim 7, the nucleic acid molecule according to claim 8, the vector according to claim 9 or 10, or the engineered immune cell according to claim 11 or 12 in the preparation of a medicament for treating tumors and / or tumor-related diseases and / or symptoms.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (1) a therapeutically effective amount of the protein of any one of claims 1 to 6, the chimeric antigen receptor of claim 7, the nucleic acid molecule of claim 8, the vector of claim 9 or 10, or the engineered immune cell of claim 11 or 12; (2) A pharmaceutically or immunologically acceptable carrier or excipient.

15. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the pharmaceutical composition according to claim 14.

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