CD16 cleavage-resistant mutants enhance cellular functions

Abstract

The present invention provides CD16 cleavage-resistant mutants that enhance cell functions. Specifically, the CD16 mutants of the present invention have amino acid residue mutations at the following sites corresponding to wild-type CD16: one or more proline insertions between the V196 site and the S197 site and / or between the S197 site and the T198 site of CD16-F176V. The CD16 mutants of the present invention can resist ADAM17 cleavage and enhance the ADCC ability of the NK92 cell line expressing them.

Description

[Technical field]

[0001] The present invention relates to the field of immunology, specifically to CD16 cleavage-resistant mutants that enhance cell function. [Background technology]

[0002] FcγRIII (CD16) is a glycoprotein with a molecular weight of 50-70 kDa, a member of the Ig superfamily, with two C2 structures, and its gene chromosome is located at 1q23-24. FcγRIII binds to human IgG and IgG3 and is a low affinity receptor for antibodies. In normal individuals, wild-type CD16 is polymorphic, where the change in the F176V site can enhance the affinity of binding to IgG and IgG3.

[0003] ADCC is a process involving the activation of NK cells, the release of cytokines and cytolytic granules, and the induction of apoptosis of target cells, and is mainly induced and generated by the binding of low affinity mediated Fc receptor CD16 (FcγRIIIA or CD16a) and antibodies. It directly activates NK cells via CD16, promotes the formation of immune synapses between NK cells and tumor cells, and exhibits killing activity against various tumor cells.

[0004] In this process, CD16 is the receptor that activates NK cells and through signal transduction induces immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation, leading to the release of lytic granules and cytokines, such as IFN-γ and TNF-α.

[0005] Furthermore, when NK cells interact with target cells, the membrane expression level of CD16 in NK cells is downregulated while transmitting activation signals. This is the result of shedding of CD16 extracellular fragments during NK cell activation. Shedding of CD16 by activated NK cells is mediated by metalloproteinase 17 (ADAM17). This indicates the central importance of CD16 in inducing NK cell-mediated ADCC.

[0006] Considering the fact that CD16 shedding negatively regulates the ADCC function of cells expressing it, such as NK cells, it may weaken the therapeutic effect during long-term antibody drug treatment. To overcome the effects of such negative regulatory signals, there is a need in the art to develop engineered CD16 mutants that are resistant to ADAM17 cleavage and can enhance the ADCC ability of NK cells. Summary of the Invention [Problem to be solved by the invention]

[0007] It is an object of the present invention to provide modified CD16 mutants. It is also an object of the present invention to provide CD16 cleavage-resistant mutants that enhance cellular function. [Means for solving the problem]

[0008] A first aspect of the present invention provides a CD16 mutant, wherein the CD16 mutant has amino acid residue mutations at the following sites relative to wild-type CD16: an insertion of one or more amino acids between the V196 and S197 sites and / or between the S197 and T198 sites of wild-type CD16, wherein the insertion of one or more amino acids comprises the insertion of one or more prolines.

[0009] In another preferred embodiment, the CD16 mutant further comprises a mutation in which F at position 176 of wild-type CD16 is replaced with V (F176V). In another preferred embodiment, the mutation further comprises a deletion of one or more amino acids or a substitution of one or more amino acids. In another preferred embodiment, the insertion of one or more amino acids is the insertion of one, two or three prolines.

[0010] In another preferred embodiment, the insertion of one or more amino acids comprises: (1) one or two P residues are inserted between the V196 and S197 sites of wild-type CD16; (2) one or two Ps are inserted between the S197 and T198 sites of wild-type CD16, or (3) Any combination of the above.

[0011] In another preferred embodiment, the insertion of one or more amino acids comprises: (1) A single P is inserted between the V196 and S197 sites of wild-type CD16. (2) a single P is inserted between the S197 and T198 sites of wild-type CD16; or (3) One P is inserted between the V196 and S197 sites of wild-type CD16, and one P is inserted between the S197 and T198 sites of wild-type CD16.

[0012] In another preferred embodiment, the CD16 mutant further comprises a mutation in which F at position 176 of wild-type CD16 is replaced with V (F176V).

[0013] In another preferred embodiment, the insertion of one or more amino acids comprises: (1) CD16-F176V has one or two P inserted between the V196 and S197 sites; (2) one or two Ps are inserted between the S197 and T198 sites of CD16-F176V; or (3) Any combination of the above.

[0014] In another preferred embodiment, the insertion of one or more amino acids comprises: (1) CD16-F176V has one P inserted between the V196 and S197 sites; (2) a single P is inserted between the S197 and T198 sites of CD16-F176V; or (3) One P is inserted between the V196 and S197 sites of CD16-F176V, and one P is inserted between the S197 and T198 sites of CD16-F176V.

[0015] In another preferred embodiment, the CD16 mutant is selected from the group consisting of: (1) CD16-F176V-V196_S197insP, the amino acid sequence of which is as set forth in SEQ ID NO.3; (2) CD16-F176V-S197_T198insP, the amino acid sequence of which is as set forth in SEQ ID NO.4; and (3) CD16-F176V-V196_S197insP-V197_T198insP, the amino acid sequence is as shown in SEQ ID NO.5.

[0016] In another preferred embodiment, the amino acid sequence of the wild-type CD16 is as shown in SEQ ID NO.1. In another preferred embodiment, the amino acid sequence of said CD16-F176V is as set forth in SEQ ID NO. 6. In another preferred embodiment, compared to the wild-type CD16 polypeptide, the CD16 mutant exhibits reduced susceptibility to ADAM17-mediated shedding. In another preferred embodiment, the CD16 mutant is resistant to ADAM17 cleavage, as compared to a wild-type CD16 polypeptide.

[0017] A second aspect of the invention provides a fusion protein, said fusion protein comprising a functional portion of a CD16 mutant and a non-CD16 mutant according to the first aspect of the invention.

[0018] In another preferred embodiment, the non-CD16 functional moiety is Fc fragments, including but not limited to Fc fragments of human IgG1, IgG2, IgG3, IgG4, and Fc fragment mutants having 90% or more homology thereto; Human serum albumin (HSA) and 6His tag.

[0019] In a preferred embodiment, the CD16 mutant and the non-CD16 functional portion of the fusion protein can be linked directly or via a linker, which can be AAA or a GS repeat sequence, including but not limited to, a G3S repeat sequence or a G4S repeat sequence, such as (G3S)4.

[0020] A third aspect of the invention provides a polynucleotide, said polynucleotide encoding a CD16 mutant according to the first aspect of the invention or a fusion protein according to the second aspect of the invention.

[0021] A fourth aspect of the invention provides an expression vector, said expression vector comprising a polynucleotide according to the third aspect of the invention.

[0022] A fifth aspect of the invention provides a cell, said cell comprising an expression vector according to the fourth aspect of the invention or having integrated in its genome a polynucleotide according to the third aspect of the invention. In another preferred embodiment, the cells express a CD16 mutant as described above.

[0023] In another preferred embodiment, the cell is a eukaryotic cell, preferably a yeast, an insect cell, an animal cell, and may be a mammalian cell. In another preferred embodiment, the cell is a natural killer (NK) cell, a neutrophil, a monocyte, a T cell, a macrophage, a pluripotent stem cell or a differentiated cell generated from the pluripotent stem cell.

[0024] In another preferred embodiment, the pluripotent stem cells are human induced pluripotent stem cells (iPSCs) or human embryonic stem cells (ESCs), or the differentiated cells generated from the pluripotent stem cells are hematopoietic cells. In another preferred embodiment, the NK cells are NK cells derived from human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs), or NK cells induced or expanded from peripheral blood or umbilical cord blood.

[0025] In another preferred embodiment, the cells are natural killer (NK) cells, and compared to NK cells that express a CD16 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO.1, SEQ ID NO.6, or SEQ ID NO.2, the NK cells: (a) increased antitumor potential; (b) increased antiviral capacity; (c) improved antibody-dependent cellular cytotoxicity; (d) increased production of IFNγ or TNFα; (e) increased CD16-mediated activity; (f) higher CD16 surface levels; (g) enhanced cell stimulation, and (h) exhibiting one or more characteristics selected from increased in vivo anti-cancer activity.

[0026] In another preferred embodiment, the ADCC ability of the NK cells is improved by at least ≧20%, ≧30%, ≧40%, ≧50% or more, for example 20-90% or 30-80%, compared to NK cells expressing a CD16 polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.6 or SEQ ID NO.2.

[0027] A sixth aspect of the invention provides a pharmaceutical composition comprising a CD16 mutant according to the first aspect of the invention, a fusion protein according to the second aspect of the invention, or a cell according to the fifth aspect of the invention and a pharma- ceutical acceptable carrier.

[0028] In another preferred embodiment, the pharmaceutical composition is a formulation. In another preferred embodiment, the pharmaceutical composition is used for treating tumors or enhancing ADCC activity in mammalian cells. In another preferred embodiment, the pharmaceutical composition further comprises another drug used in the treatment of tumors.

[0029] In another preferred embodiment, the other drug used to treat the tumor (i) specifically recognizes a tumor antigen, or (ii) comprises an antibody or antibody fragment that specifically recognizes a tumor antigen, or (iii) specifically recognizes a viral target protein. In another preferred embodiment, the other drug used in the treatment of said tumor comprises a therapeutic NK cell drug.

[0030] In another preferred embodiment, the tumor antigen comprises HER2, CD20, EGFR, CD38, CD3, CD4, CD8, CD19, CD22, CD34, CD40, CD52, HER1, HER3, HER4, VCAM, CD11a, CD18, CD11b, VEGF, Claudin18.2, or the antibody comprises trastuzumab, rituximab, pertuzumab, ranibizumab, ublituximab, omalizumab, or the like. These include: lizumab, isatuximab, ofatumumab, inebilizumab, cetuximab, panitumumab, nimotuzumab, obinutuzumab, alemtuzumab, futuximab, imgatuzumab, matuzumab, and ertumaxomab.

[0031] In another preferred embodiment, the antibody or antibody fragment that specifically recognizes the tumor antigen is an antibody having a clear ADCC activity, and is preferably an antibody selected from the group consisting of trastuzumab, cetuximab, rituximab, pertuzumab, ranibizumab, ublituximab, omalizumab, isatuximab, ofatumumab, and the like. ), inebilizumab, panitumumab, nimotuzumab, obinutuzumab, alemtuzumab, futuximab, imgatuzumab, matuzumab, ertumaxomab, and more preferably, trastuzumab, cetuximab, and rituximab.

[0032] A seventh aspect of the invention provides an in vitro method for enhancing ADCC activity in a mammalian cell comprising introducing into the cell a polynucleotide according to the third aspect of the invention.

[0033] An eighth aspect of the present invention provides the use of a CD16 mutant according to the first aspect of the invention, a fusion protein according to the second aspect of the invention or a cell according to the fifth aspect of the invention in the preparation of a medicament for expanding T lymphocytes, natural killer (NK) cells in vitro or for treating a disease in an individual.

[0034] In another preferred embodiment, the disease is a disease in which NK cells are used for immunotherapy. In another preferred embodiment, the disease is one that is treated by stimulating the immune system or by proliferating immune cells, e.g., stimulating the immune system to kill tumor cells by mediating ADCC.

[0035] In another preferred embodiment, the disease is a cancer or tumor, e.g., lymphoma, colorectal cancer, breast cancer, lung cancer, gastric cancer, liver cancer, multiple myeloma, kidney cancer, pancreatic cancer, melanoma, thyroid cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, cervical cancer, head and neck cancer, bladder cancer, or a combination thereof.

[0036] A ninth aspect of the present invention provides a method for treating a tumour comprising administering to a subject in need thereof a CD16 mutant according to the first aspect of the invention, a fusion protein according to the second aspect of the invention, a cell according to the fifth aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention.

[0037] In another preferred embodiment, the method comprises administering to the subject a therapeutic NK cell drug. In another preferred example, the NK cell drug (i) specifically recognizes a tumor antigen, or (ii) comprises an antibody or antibody fragment that specifically recognizes a tumor antigen, or (iii) specifically recognizes a viral target protein.

[0038] In another preferred embodiment, the tumor antigen comprises HER2, CD20, EGFR, CD38, CD3, CD4, CD8, CD19, CD22, CD34, CD40, CD52, HER1, HER3, HER4, VCAM, CD11a, CD18, CD11b, VEGF, Claudin18.2, or the antibody comprises trastuzumab, rituximab, pertuzumab, ranibizumab, ublituximab, omatase inhibitor, cytomegalovirus ... These include omalizumab, isatuximab, ofatumumab, inebilizumab, cetuximab, panitumumab, nimotuzumab, obinutuzumab, alemtuzumab, futuximab, imgatuzumab, matuzumab, and ertumaxomab.

[0039] In another preferred embodiment, the antibody or antibody fragment that specifically recognizes the tumor antigen is an antibody that has significant ADCC activity.

[0040] A tenth aspect of the present invention provides a combination of active ingredients, said combination of active ingredients comprising a first active ingredient: a CD16 mutant as defined in the first aspect of the invention, a fusion protein as defined in the second aspect of the invention or a cell as defined in the fifth aspect of the invention, and a second active ingredient: an antibody or antibody fragment which specifically recognises a tumor antigen.

[0041] In another preferred embodiment, the combination of active ingredients is used to enhance ADCC activity in mammalian cells. In another preferred embodiment, the antibody or antibody fragment that specifically recognizes the tumor antigen is an antibody that has significant ADCC activity.

[0042] An eleventh aspect of the present invention provides the use of a combination of active ingredients, said combination of active ingredients comprising a first active ingredient: a CD16 mutant according to the first aspect of the invention, a fusion protein according to the second aspect of the invention or a cell according to the fifth aspect of the invention; and a second active ingredient: an antibody or antibody fragment that specifically recognizes a tumor antigen, and said combination is used for enhancing ADCC activity in mammalian cells and / or for preparing a pharmaceutical composition or kit for treating tumors.

[0043] In another preferred embodiment, said combination is used for the preparation of a pharmaceutical composition or kit for the joint treatment of tumors.

[0044] In another preferred embodiment, the antibody or antibody fragment that specifically recognizes the tumor antigen is an antibody having a clear ADCC activity, and is preferably trastuzumab, cetuximab, rituximab, pertuzumab, ranibizumab, ublituximab, omalizumab, isatuximab, or ofatumumab. , inebilizumab, panitumumab, nimotuzumab, obinutuzumab, alemtuzumab, futuximab, imgatuzumab, matuzumab, ertumaxomab, and more preferably trastuzumab, cetuximab, and rituximab.

[0045] In another preferred embodiment, the tumor antigen comprises HER2, CD20, EGFR, CD38, CD3, CD4, CD8, CD19, CD22, CD34, CD40, CD52, HER1, HER3, HER4, VCAM, CD11a, CD18, CD11b, VEGF, Claudin18.2. Effect of the Invention

[0046] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (for example, in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief description of the drawings]

[0047] [Figure 1] This shows a diagram illustrating the principle of CD16 cleavage by ADAM17. [Diagram 2] An X-ray model of the ADAM17 protein is shown. [Diagram 3] It is shown that the CD16 mutants of the present invention are able to resist cleavage by ADAM17. [Figure 4] 1 shows that the CD16 mutants of the present invention mediate more potent NK92 cell activation. [Figure 5A] 1 shows that the CD16 mutants of the present invention mediate stronger ADCC effects. [Figure 5B] 1 shows that the CD16 mutants of the present invention mediate stronger ADCC effects. [Figure 6] We show that iNK expressing CD16-mut3 exhibit resistance to cleavage (FIG. 6A) and stronger ADCC capacity (FIG. 6B). [Figure 7A] Figure 2 shows that iNK expressing CD16-mut3 exhibits potent ADCC effect against A549 tumor cells. [Figure 7B] Figure 2 shows that iNK expressing CD16-mut3 exhibits potent ADCC effect against Cal27 tumor cells. [Figure 7C] FIG. 1 shows that iNK expressing CD16-mut3 exhibits potent ADCC effect against Cal33 tumor cells. [Figure 7D] We show that iNK expressing CD16-mut3 exhibits potent ADCC effect against FADU tumor cells. [Figure 7E] Figure 2 shows that iNK expressing CD16-mut3 exhibits potent ADCC effect against SKOV3 tumor cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Through extensive and detailed research, the present inventors have unexpectedly discovered three conformational mutants of CD16 designed based on the binding method of the enzyme active pocket region of CD16 and ADAM17, which can resist ADAM17 cleavage and enhance the ADCC ability of NK cells or NK92 cell lines expressing them, thereby preventing the shedding of CD16 in NK cells, thereby enhancing the stimulation of NK cells and the killing of cancer cells. Based on this, the present invention has been completed.

[0049] term FcγRIII and NK cells FcγRIII (CD16) is a glycoprotein with a molecular weight of 50-70 kDa, a member of the Ig superfamily, has two C2 structures, and its gene chromosome is located at 1q23-24. FcγRIII binds to human IgG and IgG3 and is a low affinity receptor for antibodies.

[0050] FcγRIII has two isotypes, FcγRIIIA and FcγRIIIB. <1> The transmembrane structure of FcγRIIIA is distributed mainly in macrophages, NK cells, and eosinophils; macrophages express high levels of FcγRIIIA, whereas monocytes express lower levels. FcγRIIIA is associated with disulfide-linked CD3ζ or FcεRIγ chain dimers; FcγRIIIA on macrophages is associated with the γ chain of the CD3 complex, whereas FcγRIIIA on NK / LGL is associated with the ζ chain. TGF-β promotes the expression of FcγRIIIA in cultured monocytes. <2> FcγRIIIB is "anchored" to the surface of neutrophils via GPI, and it is mainly from this form that each individual's neutrophils express 100,000 to 200,000 soluble FcγRIII in the blood. After short-term treatment with neutrophil activators, the expression level of FcγRIIIB is significantly reduced, which may be related to the shedding of GPI-linked molecules by the activation of endogenous proteases.

[0051] FcγRIII function: FcγR function is exerted primarily by myeloid and NK cells. (1) Monocyte-macrophage: FcγRI, II and III can all mediate human monocyte ADCC to kill target cells such as tumors, and such ADCC effects are Mg2+-dependent and require the participation of adhesion molecules such as LFA-1. IFN-γ promotes the monocyte FcγRI-mediated killing effect. Monocyte-phagocytes can regulate phagocytosis through FcγRI, II and III, and exert the function of removing immune complexes.

[0052] (2) Neutrophils: GPI-linked FcγRIIIB cannot mediate the tumor-killing effect of neutrophils. Activated neutrophils can mediate phagocytosis and remove immune complexes via FcγRI and II.

[0053] (3) NK cells: ADCC mediates the killing of target cells, such as tumor cells, through FcγRIIIA. IL-2 and IFN-γ can significantly enhance the killing activity of NK cells, but there is no significant change in the expression level of FcγRIIIA.

[0054] NK cells, also known as natural killer cells, are important immune cells in the body that can non-specifically and directly kill tumor cells, and such natural killing activity is not restricted by MHC, does not depend on antibodies, and does not require antigen sensitization. In addition to their strong killing function, NK cells also have very strong immune regulation function, and interact with various other immune cells in the body to regulate the body's immune status and immune function.

[0055] The "dynamic balance" of NK cells is mainly controlled by various receptor proteins on the cell surface, which are classified into two categories: NK cell-activating receptors (KARs) and inhibitory receptors (KIRs). The activation state, degranulation response, cytokine release and cell killing function of NK cells are the result of the integration of inhibitory and activating signals.

[0056] NKG2D (also known as CD314 and KLRK1), the natural cytotoxicity receptors (NCRs), DNAM1 (also known as CD226) and CD16 are the best characterized activating NK cell receptors involved in anti-cancer immune responses.

[0057] NK cells also express the low-affinity receptor FcγRIII (CD16) for IgG1 and IgG3, which binds to the Fc section of tumor antigen-specific antibodies and can mediate NK cell-mediated identification and killing of antibody-encapsulated tumor cells, also known as antibody-dependent cell-mediated cytotoxicity (ADCC).

[0058] ADCC is a process involving the activation of NK cells, the release of cytokines and cytolytic granules, and the induction of apoptosis of target cells, and is mainly induced by the binding of low affinity mediated Fc receptor CD16 (FcγRIIIA or CD16a) and antibodies. It directly activates NK cells via CD16, promotes the formation of immune synapses between NK cells and tumor cells, and exhibits killing activity against various tumor cells.

[0059] In this process, CD16 is the receptor that activates NK cell effect and through signal transduction induces phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs), resulting in the release of lytic granules and cytokines such as IFN-γ and TNF-α.

[0060] Furthermore, when NK cells interact with target cells in the presence of specific antibodies, the membrane expression level of CD16 on NK cells is downregulated while transmitting activation signals, as a result of shedding of CD16 extracellular fragments during NK cell activation.

[0061] Shedding of CD16 by activated NK cells is mediated by metalloproteinase 17 (ADAM17), indicating the central importance of CD16 in triggering NK cell-mediated ADCC.

[0062] CD16 mutants of the present invention In comparison to the point mutation strategy used in the CD16 mutant CD16-F176V-S197P (PC) reported in the art (F176V is a naturally occurring high affinity mutant of CD16), the present invention employs a design in which an amino acid P, opposite the polarity characteristic required for the pocket region of ADAM17, is inserted on one or both sides of S197 where CD16 binds to ADAM17.

[0063] As used herein, the terms "CD16 cleavage resistant mutant of the invention" and "CD16 mutant of the invention" are used interchangeably and refer to a CD16 mutant according to the first aspect of the invention.

[0064] Preferably, said CD16 mutant comprises a mutation in an amino acid residue at the following site relative to wild-type CD16: an insertion of one or more amino acids, for example an insertion of one, two or three prolines, between the V196 and S197 sites and / or between the S197 and T198 sites of wild-type CD16. Preferably, said CD16 mutant further comprises an F176V mutation (CD16-F176V).

[0065] In another preferred embodiment, the mutation further comprises a deletion of one or more amino acids or a substitution of one or more amino acids. In another preferred embodiment, the insertion of one or more amino acids is the insertion of one, two or three prolines.

[0066] In another preferred embodiment, the insertion of one or more amino acids comprises: (1) CD16-F176V has one or two P inserted between the V196 and S197 sites; (2) one or two Ps are inserted between the S197 and T198 sites of CD16-F176V; or (3) Any combination of the above.

[0067] In another preferred embodiment, the insertion of one or more amino acids comprises: (1) CD16-F176V has one P inserted between the V196 and S197 sites; (2) a single P is inserted between the S197 and T198 sites of CD16-F176V; or (3) One P is inserted between the V196 and S197 sites of CD16-F176V, and one P is inserted between the S197 and T198 sites of CD16-F176V.

[0068] Specifically, the sequences of wild-type CD16 (WT), CD16(F176V), a reported CD16 mutant, and the three CD16 mutants of the present invention are as follows:

[0069] CD16-WT (SEQ ID NO.1) MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLR CHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETTVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

[0070] CD16-F176V-S197P(PC)(SEQ ID NO.2) MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAV P TISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSTRDWKDHKFKWRKDPQDK

[0071] CD16-F176V-V196_S197insP Mut1(SEQ ID NO.3) MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAV P.S. TISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

[0072] CD16-F176V-S197_T198insP Mut2(SEQ ID NO.4) MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAV SP TISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

[0073] CD16-F176V-V196_S197insP-V197_T198insP Mut3(SEQ ID NO.5) MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAV PSP TISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

[0074] CD16-F176V (SEQ ID NO. 6) MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAV S TISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSTRDWKDHKFKWRKDPQDK

[0075] Fusion proteins Based on the CD16 mutant of the present invention, it is well known to those skilled in the art that the CD16 mutant of the present invention and other non-CD16 functional parts can be prepared into fusion proteins. In a specific embodiment, the non-CD16 functional part includes, but is not limited to, Fc fragment, human serum albumin (HSA), anti-HSA antibody or antibody fragment, transferrin, human chorionic gonadotropin β subunit carboxyl terminal peptide (CTP), elastin-like peptide (ELP) and antigen-binding part. In a preferred embodiment, the antigen-binding part can be an antibody or its active antibody fragment, a Fab molecule, a scFv molecule and a VHH molecule, an immunoglobulin molecule, a receptor protein molecule or a ligand protein molecule, and the immunoglobulin molecule can be an IgG molecule.

[0076] Based on routine operations in the art, those skilled in the art are well aware of how to obtain fusion proteins containing the CD16 mutants of the present invention.For example, the CD16 mutants of the present invention can be directly linked to other non-CD16 functional parts or linked via a linker.The linker can be a repeat sequence of AAA or GS, including but not limited to a repeat sequence of G3S or a repeat sequence of G4S, such as (G3S)4.

[0077] A preferred type of active derivative refers to a polypeptide in which up to three, preferably up to two, more preferably up to one amino acid is replaced by an amino acid having similar or similar properties, compared to the amino acid sequence of the invention. These conservative variant polypeptides are preferably generated by replacing the amino acids according to Table A.

[0078] [Table A]

[0079] The present invention further provides analogs of the fusion proteins of the present invention. The difference between these analogs and the polypeptides of the present invention can be differences in amino acid sequence, differences in modified forms that do not affect the sequence, or both. Analogs further include analogs with residues different from natural L-amino acids (e.g., D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0080] Furthermore, the CD16 mutant or fusion protein of the present invention can also be modified. Modified forms (which do not usually change the primary structure) include polypeptides that are chemically derivatized, such as acetylation or carboxylation in vivo or in vitro. Modifications further include glycosylation, such as purified polypeptides, for example, by glycosylation modification of the polypeptide during synthesis and processing, or in a further processing step. Such modification can be achieved by exposing the polypeptide to a glycosylating enzyme (e.g., mammalian glycosylase or deglycosylase). Modified forms further include sequences with phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Further included are polypeptides that are modified to enhance their proteolytic performance or optimize their solubility performance.

[0081] The term "polynucleotide of the invention" can include polynucleotides that encode a CD16 mutant or fusion protein of the invention, and can further include polynucleotides that add coding and / or non-coding sequences.

[0082] The present invention also relates to variants of the above polynucleotides that code for fragments, analogs and derivatives of the polypeptides or fusion proteins having the same amino acid sequences as those of the present invention. These nucleotide variants include substitution, deletion and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the CD16 mutant or fusion protein it encodes.

[0083] The present invention further relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the above polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refers to (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 degrees, or (2) addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 degrees, or (3) hybridization that occurs only when the identity between the two sequences is at least 90% or more, more preferably 95% or more.

[0084] The CD16 mutant or fusion proteins and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, are provided in a homogeneously produced form.

[0085] The full-length polynucleotide of the present invention can be obtained by PCR amplification, recombinant methods, or artificial synthesis. In the case of PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and the relevant sequences are obtained by amplifying a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, it is often necessary to carry out two or more rounds of PCR amplification, and then splice the amplified fragments of each round in the correct order.

[0086] Once the relevant sequence is obtained, recombinant methods can be used to obtain large quantities of the relevant sequence, typically by cloning it into a vector, then transforming it into a cell, and then isolating the relevant sequence from the host cells that have been propagated by conventional methods.

[0087] Additionally, related sequences can be synthesized by synthetic methods, especially when the fragments are relatively short in length, usually by first synthesizing several smaller fragments and then ligating them together to obtain fragments of much longer sequences.

[0088] Currently, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) can be obtained entirely by chemical synthesis, which can then be introduced into a variety of existing DNA molecules (or vectors, etc.) and cells known in the art.

[0089] The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotide of the present invention. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-rapid amplification of cDNA ends) can be preferably used, and the primers used in PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.

[0090] Expression vector The present invention also relates to vectors comprising a polynucleotide of the invention, host cells genetically engineered with a vector of the invention or a CD16 mutein or fusion protein coding sequence of the invention, as well as methods for purifying a polypeptide according to the invention by recombinant techniques.

[0091] Conventional recombinant DNA techniques can be used to express or produce recombinant fusion proteins using the polynucleotide sequences of the present invention, which generally include the following steps: (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a fusion protein of the invention, or with a recombinant expression vector containing said polynucleotide; (2) culturing the host cells in an appropriate medium; (3) Isolating and purifying the protein from the medium or cells.

[0092] In the present invention, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmids and vectors can be used as long as they are replicable and stable in the host. The important features of an expression vector are that it usually contains an origin of replication, a promoter, a marker gene and a translation control element.

[0093] In the method for preparing the CD16 mutein or its fusion protein of the present invention, any suitable vector can be used, which can be selected from one of pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), and pDHFR, and the expression vector comprises a fusion DNA sequence linked to suitable transcriptional and translational regulatory sequences.

[0094] cell In the present invention, the cell expressing a CD16 mutant may be a natural killer (NK) cell, a neutrophil, a monocyte, a T cell, a macrophage, or a pluripotent stem cell (e.g., human induced pluripotent stem cell (iPSC) and human embryonic stem cell (ESC)) or an immune cell differentiated from said pluripotent stem cell. Preferably, said cell is a NK cell, and said NK cell may be a NK cell derived from human induced pluripotent stem cell (iPSC) and human embryonic stem cell (ESC), or a NK cell induced and expanded from peripheral blood or umbilical cord blood, and more preferably is a NK cell population.

[0095] Preferably, said NK cell population can be used as a therapeutic agent.In addition, compared with the NK cell or NK cell population expressing CD16 polypeptide comprising the amino acid sequence as shown in SEQ ID NO.1 (wild type) or SEQ ID NO.6 (naturally occurring CD16 mutant CD16-F176V), the NK cell or NK cell population expressing CD16 mutant comprising the amino acid sequence as shown in SEQ ID NO.3, 4 or 5 of the present invention has higher ADCC-mediated function, which is equivalent to the ADCC function mediated by SEQ ID NO.2 (CD16-F176V-S197P).

[0096] Both eukaryotic / prokaryotic host cells can be used for expressing the CD16 mutant of the present invention or its fusion protein, the eukaryotic host cells are preferably mammalian or insect host cell culture systems, preferably cells such as COS, CHO, NS0, sf9 and sf21, and the prokaryotic host cells are preferably one of DH5a, BL21(DE3), TG1.

[0097] Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding the fusion protein of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The DNA sequence can be operatively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include E. coli lac or trp promoters; lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and several other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector further includes a ribosome binding site for translation initiation and a transcription terminator.

[0098] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for use in E. coli.

[0099] The vector containing the appropriate DNA sequence as described above and an appropriate promoter or control sequence can be used to transform an appropriate host cell to express the protein.

[0100] The cell of the present invention may be a host cell, for example a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells (e.g., carrot cells), etc.

[0101] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, the transcription is enhanced by inserting an enhancer sequence into the vector. Enhancers are cis-acting DNA elements usually about 10 to 300 base pairs in length, which act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

[0102] A person skilled in the art knows how to select appropriate vectors, promoters, enhancers and host cells.

[0103] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of absorbing DNA can be obtained by treating with CaCl2 method after exponential growth phase, the steps used being well known in the art. Another method is to use MgCl2. Transformation can also be carried out by electroporation method if necessary. When the host is a eukaryotic organism, DNA transfection methods such as calcium phosphate co-precipitation method; conventional mechanical methods such as microinjection; electroporation; liposome packaging, etc. can be selected.

[0104] The obtained transformant can be cultured in a conventional manner to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used for the culture can be selected from a variety of conventional media. The culture is performed under conditions suitable for the growth of the host cell. After the host cell has grown to an appropriate cell density, the selected promoter is induced using an appropriate method (e.g., temperature change or chemical induction), and the cell is further cultured for a certain period of time.

[0105] The recombinant polypeptide in the method can be expressed intracellularly or in the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various isolation methods using physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (salting out), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid phase chromatography (HPLC) and various other liquid chromatography techniques, as well as combinations of these methods.

[0106] Affinity chromatography can be used to separate and purify the CD16 mutant or its fusion protein disclosed in the present invention, and depending on the characteristics of the affinity column used, conventional methods such as high salt buffers, changes in pH, etc. can be used to elute the CD16 mutant or its fusion protein bound to the affinity column.

[0107] Using the above method, the CD16 mutant or fusion protein thereof can be purified to substantial homogeneity, such as a single band on SDS-PAGE electrophoresis.

[0108] Pharmaceutical Compositions The present invention further provides a pharmaceutical composition comprising the above-mentioned CD16 mutant or fusion protein.

[0109] The CD16 mutant or fusion protein exerts a more stable therapeutic effect by combining with a pharma- ceutical acceptable excipient to form a drug formulation, and these formulations can ensure the structural integrity of the amino acid core sequence of the CD16 mutant or fusion protein of the present invention while protecting the polyfunctional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation). The formulations can take various forms, and generally, in the case of liquid formulations, they can be stably stored for at least one year under conditions of 2°C to 8°C, and in the case of lyophilized formulations, they can be stably stored for at least six months at 30°C. The formulations herein may be formulations such as suspensions, aqueous injections, and lyophilized formulations commonly used in the pharmaceutical field, preferably aqueous injections or lyophilized formulations.

[0110] In the case of a water injection or lyophilized formulation of the CD16 mutant or its fusion protein of the present invention, the pharma- ceutically acceptable excipient comprises one or a combination of a surfactant, a solution stabilizer, an isotonicity adjusting agent and a buffer, wherein the surfactant is a non-ionic surfactant such as polyoxyethylene sorbitol fatty acid ester (Tween 20 or 80), a poloxamer (e.g., poloxamer 188), Triton, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, a tetradecyl group, a linoleyl group or an octadecyl sarcosine, Pluronics, MONAQUA, or the like. TTM, etc., the amount of which should be added should minimize the tendency of the protein to granulate, the solution stabilizer can be a sugar including reducing sugar and non-reducing sugar, the amino acid can include monosodium glutamate or histidine, the alcohol can include one or a combination of a triol, a higher sugar alcohol, propylene glycol, polyethylene glycol, the amount of solution stabilizer added should be such that the final formulation formed remains stable over a period of time according to one skilled in the art, the isotonicity adjuster can be one of sodium chloride, mannitol, and the buffer can be one of TRIS, a histidine buffer, or a phosphate buffer.

[0111] When using the above CD16 mutant or its fusion protein and its drug formulation to be administered to animals including humans, the dosage varies according to the age and weight of the patient, the characteristics and severity of the disease, and the administration route, and can refer to the results of animal experiments and various situations, and the total dosage cannot exceed a certain range.Specifically, the dosage in the case of intravenous injection is 0.1-3000mg / day.

[0112] The CD16 mutant or its fusion protein of the present invention and drug formulations containing the same can be used as anti-tumor drugs for tumor treatment, and the anti-tumor drug referred to in the present invention refers to a drug that inhibits and / or treats tumors, and can include delaying the progression of symptoms associated with tumor growth and / or reducing the severity of these symptoms, which further includes reducing existing tumor growth accompanied by symptoms, and preventing the occurrence of other symptoms, reducing or preventing the transcription.

[0113] The above CD16 mutant or its fusion protein and its drug formulations can also be administered in combination with other antitumor drugs used to treat tumors, and these antitumor drugs for combination administration include: 1. Cytotoxic drugs (1) Drugs acting on the chemical structure of DNA: alkylating agents such as nitrogen mustard, nitrosourea, methanesulfonic acid esters, etc.; platinum compounds such as cisplatin, carboplatin and platinum oxalate; mitomycin (MMC); (2) Drugs affecting nucleic acid synthesis: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta; thymidine synthetase inhibitors such as fluorouracil (5FU, FT-207, capecitabine); purine nucleoside synthase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG; nucleotide reductase inhibitors such as hydroxyurea (HU); DNA polymerase inhibitors such as cytarabine (Ara-C) and Gemz; (3) Drugs acting on nucleic acid transcription: drugs that act selectively on DNA templates, inhibit DNA-dependent RNA polymerase, and thereby inhibit RNA synthesis: actinomycin D, daunorubicin, doxorubicin, epirubicin, aclarithromycin, rajimicin, etc.; (4) drugs that act mainly on tubulin synthesis: paclitaxel, taxotere, vinblastine, vinorelbine, podophylline alkaloids, homoharringtonine; (5) other cytotoxic drugs: asparaginase, mainly inhibiting protein synthesis; 2. Hormonal antiestrogens: tamoxifen, droxifene, exemestane, etc.; aromatase inhibitors: aminoglutethimide, lantrone, letrozole, arimidex, etc.; antiandrogens: flutamide RH-LH agonists / antagonists: noreid, enastron, etc.; 3. Biological response regulators: mainly inhibit tumor interferon through the body's immune function; interleukin-2; thymosin; 4.Monoclonal antibodies: MabThera; Cetuximab (C225); Trastuzumab; Bevacizumab (Avastin); Yervoy (Ipilimumab); Nivolumab (OPDIVO); Pembrolizumab (Keytruda); Atezolizumab (Tecentriq); 5. Other drugs for which some mechanisms are currently unknown and require further study; Cell differentiation inducers such as retinoids; Cell apoptosis inducers, including but not limited to.

[0114] Preferably, when the CD16 mutant of the present invention is used in combination with an antibody having a clear or observable ADCC effect (preferably an antibody such as trastuzumab, cetuximab, rituximab, etc.), the ADCC effect in NK cells expressing the CD16 mutant is unexpectedly and significantly improved, thereby improving the anti-tumor effect.

[0115] The main advantages of the present invention are: (1) The CD16 mutants of the present invention can resist ADAM17 cleavage and enhance the ADCC ability of NK cells expressing them. (2) The CD16 mutant of the present invention can be used in immunotherapy to increase the anti-cancer activity of immune cells, particularly NK cells, NK92 cells, etc. (3) The CD16 mutant nucleotide fragment of the present invention can be inserted into a specific site (such as AAVS1) of pluripotent stem cells (iPSCs, ESCs, etc.) by gene editing, and the differentiated immune cells, such as NK cells, have stronger ADCC-mediating ability. (4) The CD16 mutant nucleotide fragment of the present invention can be expressed on the surface of various immune cells by various genetic engineering techniques (e.g., viral vector transfection, plasmid transfection, transposon transfection, etc.), and immune cells genetically engineered by such methods have stronger cleavage resistance ability and ADCC efficiency, as well as better discrimination and killing ability against target cells.

[0116] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. In the following examples, the experimental methods that do not show detailed conditions usually follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those suggested by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0117] Experimental Method The experimental method referred to in this invention is as follows. 1. Design of CD16 mutants In this study, three CD16 mutants were designed, based on CD16-F176V, proline was inserted near the S197 site either individually or simultaneously, and the mutants were linked to EGFP (as a reporter gene) via the P2A sequence, and the corresponding fragments were inserted into pMSCV retroviral vector to pMSCV-CD16-F176V-V196_S197insP(Mut1), pMSCV-CD16-F176V-S197_T198insP(Mut2), To form pMSCV-CD16-F176V-V196_S197insP-V197_T198insP (Mut3), In addition, wild type pMSCV-CD16-WT (WT), pMSCV-CD16-WT (F176V) and positive control pMSCV-CD16-S197P are constructed (all vectors are synthesized and extracted by GENEWIZ, and the sequencing results are all accurate).

[0118] 2.Cell culture HEK293T and NK92 are from species stored in this laboratory, and Raji cells are purchased from ATCC. HEK293T are cultured in DMEM + 10% FBS medium, Raji are cultured in RPMI-1640 + 10% FBS medium, and the components of NK92 cell medium include α-MEM (Shanghai Culture Co., Ltd., L560KJ), 0.2 mM inositol (Sigma-Aldrich, I7508), 55 mM β-mercaptoethanol (MESGEN, MG1004), 20 mM folic acid (Sigma-Aldrich, F8758-5G), 1000 U / ml IL-2 (Nearshore Proteins, GMP-CD66), 12.5% ​​FBS (CAPRICORN), and 12.5% ​​horse serum (Gibco, 26050088). Other tumor cells are grown according to culture methods recommended by the ATCC.

[0119] 3. Virus Packaging and Infection The retrovirus packaging vectors are BaEV-TR, pCMV-gag-pol, respectively, which are designed by this laboratory and then sent to GENEWIZ for synthesis and extraction. The virus packaging process is as follows: a) After digesting HEK-293T cells in good condition, resuspend them in the corresponding medium and inoculate them into 10cm culture dishes at a density of 7-8E5 / ml; b) After culturing in an incubator for 16 hours, observe the cell density, and start transfection of plasmid when the density is about 80%; c) Prepare two 1.5ml centrifuge tubes, add 500ul Opti-MEM™ I serum reduced medium (Gibco, 31985062) to each, and in one of the centrifuge tubes, 7.5ug BaEV-TR, 10ug pCMV-gag-pol and CD16 mutant expressing vector are added. Add 20ug of the corresponding pCMV vector to the centrifuge tube and add 40ul of PEIpro solution (Polyplus, 115-010) to another one of the centrifuge tubes; d) Mix the solutions in the two centrifuge tubes gently and evenly, and then leave them at room temperature for 15-20 minutes to form the transfection complex; e) Gently drop the above transfection complex into the HEK-293T culture supernatant, place it in the medium and continue to culture for 4-6 hours; f) Replace the pre-warmed fresh medium, and place the cells in the incubator and continue to culture for 48 hours; g) Collect the cell culture supernatant and incubate with Lenti-X. Concentrate the virus using a Concentrator (Takara, 631232); h) resuspend the virus using 100ul of medium, place 5E5 NK92 cells in a 12-well plate, add 50ul of virus concentrate each, and add polybrene to a final concentration of 5ug / ml; i) centrifuge the cells under the conditions of 32℃ and 800g for 1 hour, then place in an incubator and culture overnight; j) replace the cells with new medium, continue to culture for 3 to 5 days, and then perform flow cytometry detection.

[0120] 4. Flow Cytometry Detection Flow cytometry detection is performed according to standard operating procedures. Taking the detection of CD16 as an example, the operating procedures are as follows: a) Collect 1E5 corresponding cells from each well, centrifuge at 300g, 4℃ for 5 minutes, discard the supernatant; b) Add 200ul pre-chilled PBS buffer to each well, mix gently and homogeneously, then centrifuge at 300g, 4℃ for 5 minutes, discard the supernatant; c) Add CD16 antibody (Biolegend, 302012) or Isotype (Biolegend, 400122), add PBS to the remaining wells, the volume of each well is 100ul, mix gently and homogeneously, place at 4℃, and incubate for 30 minutes; d) Centrifuge at 300g, 4℃ for 5 minutes, discard the supernatant; e) Add 200ul pre-chilled PBS buffer to each well, mix gently and homogeneously, then centrifuge at 300g, 4℃ for 5 minutes, discard the supernatant; f) Add 200ul pre-chilled 7-AAD (BD PBS buffer containing Pharmingen™ 559925) was added and mixed gently to homogenize, followed by machine detection (Attune) and data analysis.

[0121] 5. Experiments to test for resistance to ADAM17 cleavage Four groups were set up as follows, in which a stimulatory cocktail (Invitrogen, 00-4970-93) was used to activate NK92 cells, and an ADAM17 inhibitor (Selleck, S7434, 20uM) was used to specifically inhibit the activity of ADAM17. The reaction system was 100ul, and the cells were collected after 4 hours, and the expression status of CD16 on the surface of NK92 in each group was detected by flow cytometry. [Table B]

[0122] 6. ADCC Activity Detection Take 3E4 individual NK92-CD16-WT, NK92-CD16-PC, NK92-CD16-Mut1, NK92-CD16-Mut2, and NK92-CD16-Mut3, place them in one well of a 96-well plate (each group has three replicates), and set up four stimulation conditions for each cell type: blank, Raji (3E4), Rituximab (Selleck, A2009, 100ng / ml), and Raji + Rituximab, and place them in an incubator for 1 hour. Then Brefeldin A (Invitrogen, 00-4506-51, 1x) and Monensin (Invitrogen, 00-4505-51, 1x) and anti-human CD107a (Biolegend, 328620) were added, and the cells were continuously placed in an incubator for 3-5 hours, during which the reaction system of each group was kept constant. Then anti-human CD56 (Biolegend, 362508) and 7-AAD were added for staining according to the standard flow cytometry staining protocol. The machine detected and analyzed the data.

[0123] Example 1. Design of CD16 mutants Considering the fact that CD16 shedding negatively regulates the ADCC function of cells expressing CD16, such as NK cells, the therapeutic effect during long-term cell and antibody drug treatment may be weakened. To overcome the influence of such negative regulatory signals, the applicant analyzes the molecular structure of ADAM17 (Figure 2) and the molecular mechanism by which ADAM17 cleaves peptide chains. Based on the binding method of CD16 and the enzyme active pocket region of ADAM17, three conformational mutants of CD16 are designed. As expected, none of these three mutants are cleaved by ADAM17.

[0124] Through experiments, Applicant confirms that these three CD16 mutants are indeed able to resist ADAM17 cleavage and enhance the ADCC ability of NK92 cell lines expressing them. In comparison with the point mutation strategy adopted by the art-reported CD16 mutant CD16-F176V-S197P (positive control, PC) (F176V is a naturally occurring CD16 high affinity mutant), the three mutants designed by the applicant are as follows: CD16-F176V-V196_S197insP(Mut1), CD16-F176V-S197_T198insP(Mut2), and CD16-F176V-V196_S197insP-V197_T198insP(Mut3), The design used is one in which the amino acid P, opposite the polar feature required for the ADAM17 pocket region, is inserted on either or both sides of S197 where CD16 binds to ADAM17.

[0125] Experimental results prove that such an idea actually works with presets.

[0126] Example 2. Resistance of CD16 mutants to ADAM17 cleavage Wild-type CD16 (WT), a reported positive control CD16 mutant (PC) and three applicant-designed CD16 mutants are overexpressed in the NK92 cell line via a gamma-retroviral vector. To verify whether ADAM17 has a cleavage effect on these mutants, four experimental conditions G1 to G4 are set up (Table 1), where a PMA / ionomycin mixture is used to activate NK92 cells and TAPI-1 is a small molecule inhibitor of ADAM17.

[0127] [Table 1]

[0128] For wild-type CD16 (WT), it can be seen that under non-activated conditions (G1, G2), NK92 cells express higher levels of CD16; under the action of PMA / ionomycin, NK cells are activated and at the same time the expression level of CD16 is significantly reduced (G3); after the addition of TAPI-1, the activity of ADAM17 is inhibited and the expression of CD16 returns to the normal level (G4) (Figure 3, A).

[0129] The above experimental results indicate that wild-type CD16 (WT) can be specifically cleaved by ADAM17 in activated NK92 cells. Compared with wild-type CD16 (WT), the positive control (PC) and the three mutants of the present invention (Mut1, Mut2, Mut3) can fully achieve resistance to ADAM17 cleavage (Figure 3, B-E).

[0130] Example 3. CD16 mutants mediate stronger NK92 cell activation To further verify whether the CD16 mutants of the present invention have the function of mediating ADCC, the expression status of CD107a (a marker of NK92 activation) in cells of each group under different stimulation conditions is detected.

[0131] It can be seen that in the presence of Raji cells or Rituximab (a monoclonal antibody targeting CD20) alone, the NK92 cells of each group cannot be effectively activated, whereas in the simultaneous presence of Raji cells and Rituximab, the NK92 cells are activated and express CD107a (Figure 4). Similar to the positive control (PC), the three CD16 mutants of the present invention can resist ADAM17 cleavage, thereby better mediating ADCC and more potently activating NK92 cells.

[0132] The above experimental results indicate that the three CD16 mutations of the present invention can all mediate stronger ADCC effects.

[0133] Example 4. CD16 mutants that mediate stronger ADCC effects To prove that NK92 cells expressing CD16 mutants have a stronger ability to mediate ADCC, trastuzumab and cetuximab are used to detect ADCC against SKOV3 (HER2 expression 90%) and A549 (EGFR expression 100%), respectively. The results show that NK92 expressing CD16 mutants can mediate stronger ADCC effect than NK92 expressing wild-type CD16 (including CD16WT and CD16WT(F176V)) (Figure 5A, 5B). The calculation method of ADCC is as follows: ADCC%=(Experimental cytotoxicity - Ab-independent cytotoxicity) / Maximum cytotoxicity×100%.

[0134] Example 5. iNK expressing CD16-mut3 exhibits resistance to cleavage and stronger ADCC capacity To further verify the function of CD16 mutants in NK (iNK) cells differentiated from induced pluripotent stem cells (iPSCs), CD16-mut3 was knocked into the iPSC genome via CRISPR / Cas9 and screened to obtain iPSC monoclones expressing CD16-mut3, which were then expanded and differentiated into iNKs, and finally iNKs (iNK-mut3) with CD16-mut3 expression rates of over 90% were obtained. After differentiation of unedited iPSCs, they expressed about 45% wild-type CD16 (iNK-wt). After adding Raji cells and rituximab to the two groups of cells respectively, the iNK cells were activated and produced an ADCC effect. The expression of wild-type CD16 was reduced by 40%, while the expression of CD16-mut3 was reduced by only 9%, indicating an obvious cleavage resistance effect (Figure 6A).

[0135] The effects of iNK-wt and iNK-mut3 on Raji cells mediating ADCC were simultaneously detected, and it was found that iNK-mut3 exhibited a stronger ADCC effect (FIG. 6B).

[0136] Example 6. iNK expressing CD16-mut3 exhibits strong ADCC effect against various tumor cells Then, ADCC detection was performed against cells such as A549 (lung cancer), Cal27 (head and neck cancer), Cal33 (head and neck cancer), FADU (head and neck cancer), and HCC-1954 (breast cancer), and under the condition of E:T of 3:1, iNK expressing CD16-mut3 showed stronger ADCC killing ability than iNK (Figure 7A). At the same time, iNK-mut3 (iNK-vCD16) showed strong ADCC effect against cells such as Cal27 (Figure 7B), Cal33 (Figure 7C), FADU (Figure 7D), and SKOV3 (Figure 7E).

[0137] The above results indicate that when combined with antibodies, iNKs edited with CD16-mut3 and differentiated into iPSCs have stronger ADCC function, and have potential for clinical development and application.

[0138] All documents mentioned in the present invention are incorporated by reference in this application as if each document was incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalents are also included in the scope defined by the appended claims of this application.

Claims

1. A CD16 mutant, The CD16 mutant is characterized in that an amino acid residue mutation occurs at the following sites corresponding to wild-type CD16: one or more amino acid insertions occur between the V196 site and the S197 site and / or between the S197 site and the T198 site of wild-type CD16, wherein the one or more amino acid insertions include one or more proline insertions.

2. The CD16 mutant further comprises a mutation in which F at position 176 of wild-type CD16 is replaced with V (F176V). A CD16 mutant according to claim 1.

3. The one or more amino acid insertions may be: (1) one P is inserted between the V196 site and the S197 site of CD16-F176V; (2) one P is inserted between the S197 site and the T198 site of CD16-F176V, or (3) A CD16-F176V vector comprising one P inserted between the V196 site and the S197 site, and one P inserted between the S197 site and the T198 site, A CD16 mutant according to claim 1.

4. A fusion protein comprising: The fusion protein is characterized in that it contains functional portions of a CD16 mutant and a non-CD16 mutant described in claim 1.

5. The non-CD16 functional part comprises Fc fragments, including, but not limited to, human IgG1, IgG2, IgG3, IgG4 Fc fragments, and Fc fragment mutants having 90% or more homology thereto; Human serum albumin (HSA), and 6His tag. The fusion protein of claim 4.

6. A polynucleotide comprising: The polynucleotide, characterized in that it encodes a CD16 mutant as described in claim 1 or a fusion protein as described in claim 4.

7. An expression vector comprising: The expression vector, characterized in that it comprises the polynucleotide according to claim 6.

8. A cell, The cell, characterized in that it contains the expression vector described in claim 7 or that the genome of the cell has integrated therein the polynucleotide described in claim 6.

9. The cell is a natural killer (NK) cell, a neutrophil, a monocyte, a T cell, a macrophage, a pluripotent stem cell, or a cell differentiated from the pluripotent stem cell. The cell described in claim 8.

10. The pluripotent stem cells include human induced pluripotent stem cells (iPSCs) or human embryonic stem cells (ESCs), or the cells differentiated from the pluripotent stem cells are immune cells. The cell described in claim 9.

11. The NK cells are characterized in that they are NK cells derived from human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs), or NK cells induced or expanded from peripheral blood or umbilical cord blood. The cell described in claim 9.

12. 1. A pharmaceutical composition comprising: The pharmaceutical composition is characterized in that it comprises a CD16 mutant described in claim 1, a fusion protein described in claim 4, or a cell described in claim 8 and a pharma- ceutically acceptable carrier.

13. The pharmaceutical composition further comprises another drug used in the treatment of a tumor, the other drug used in the treatment of the tumor being characterized in that (i) it specifically recognizes a tumor antigen, or (ii) it comprises an antibody or an antibody fragment that specifically recognizes a tumor antigen, or (iii) it specifically recognizes a viral target protein. The pharmaceutical composition according to claim 4.

14. 1. An in vitro method for enhancing ADCC activity in a mammalian cell, comprising:

7. The method, comprising introducing the polynucleotide of claim 6 into a cell.

15. Use of a combination of active ingredients, Use of a combination of active ingredients, characterized in that the combination of active ingredients comprises a first active ingredient: a CD16 mutant described in claim 1, a fusion protein described in claim 4, or a cell described in claim 8, and a second active ingredient: an antibody or antibody fragment that specifically recognizes a tumor antigen, and the combination is used for enhancing ADCC activity in mammalian cells and / or for preparing a pharmaceutical composition or kit for treating tumors.

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