Anti-CD33 / CLL1 bispecific antibody-natural killer cell conjugates and uses thereof

Abstract

The present invention provides anti-CD33 / CLL1 bispecific antibody-natural killer cell conjugates, pharmaceutical compositions comprising the conjugates, and pharmaceutical uses of the conjugates, as well as methods for their preparation.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority from Chinese Patent Application No. 202211728567.1, filed on December 30, 2022, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates generally to the field of biopharmaceuticals, and specifically to an anti-CD33 / CLL1 bispecific antibody-natural killer cell conjugate, a pharmaceutical composition comprising the conjugate, as well as pharmaceutical uses of the conjugate, methods for producing the same, etc. [Background technology]

[0003] Acute myeloid leukemia (AML) is a highly heterogeneous group of malignant clonal proliferative myeloid primitive cell disorders of the hematopoietic system. It results from the malignant transformation of myeloid hematopoietic progenitor cells at different stages of differentiation and development. In recent years, the incidence of AML has continued to increase with the increasing proportion of aging populations, and the proportion of relapsed and refractory AML has subsequently gradually increased. While 60%–80% of adult AML patients achieve complete remission (CR) after initial induction chemotherapy, approximately 30% subsequently undergo consolidation therapy and allogeneic bone marrow transplantation in an effort to achieve complete cure. However, 10%–30% of patients still fail to achieve CR after induction therapy and develop primary refractory AML. Approximately half of younger patients and 80%–90% of older patients ultimately relapse.

[0004] The basic treatment principles for relapsed / refractory acute myeloid leukemia are as follows: 1) For patients who can tolerate intensive chemotherapy, salvage therapy with combination chemotherapy such as the FLAG regimen (fludarabine, cytarabine, and granulocyte colony-stimulating factor G-CSF), the CLAG regimen (cladribine, cytarabine, and G-CSF), or intermediate-dose (high-dose) cytarabine (IDAC / HiDAC) is recommended. If the patient achieves complete remission, allogeneic bone marrow transplantation can be performed. 2) For patients who cannot tolerate intensive chemotherapy, combination therapy with venetoclax (VEN) and azacitidine (AZA) or low-dose cytarabine (LDAC) is performed. Depending on the presence or absence of gene mutations such as FMS-like tyrosine kinase 3 (FLT3) or isocitrate dehydrogenase 1 / 2 (IDH1 / 2), corresponding targeted drugs such as gilteritinib or ivosidenib can also be administered. Mutations in genes such as FLT3 and IDH1 / 2 typically occur at a low incidence in AML, occurring in approximately 5% to 15% of cases. The above treatment regimens can be adjusted accordingly based on the patient's karyotype status. For example, allogeneic bone marrow transplantation is recommended for the intermediate-high risk group.

[0005] The prognosis for relapsed / refractory acute myeloid leukemia is poor. According to literature, the complete remission rate after chemotherapy in relapsed / refractory acute myeloid leukemia patients (those who can tolerate intensive chemotherapy) is 20–30%, with a median overall survival (OS) of 5–8 months. To increase the remission rate and extend patient survival, numerous clinical trials are currently underway worldwide. For example, a multicenter clinical trial of uproleselan (GMI-1271, APL-106) combination chemotherapy is currently being conducted globally, and a bridging study was also conducted for Chinese patients. According to published phase II clinical trial data, the combined complete remission rate (including complete remission or complete remission with incomplete hematologic remission, CR / CRi) of this combination treatment regimen was 41%, with an OS of 8.8 months.

[0006] In addition to conventional chemotherapy and hematopoietic stem cell transplantation, several cellular immunotherapies have been reported, including a) autologous tumor-infiltrating lymphocyte (TIL) therapy, b) NK cell immunotherapy, c) dendritic cell-cytokine-induced killer cell (DC-CIK) therapy, d) macrophage activation therapy, and e) chimeric antigen receptor T cell (CAR-T) immunotherapy. Although several clinical studies on CAR-T and NK cell therapy for AML have been conducted, no approved products have been approved yet.

[0007] The field urgently needs more treatments for acute myeloid leukemia (AML). Summary of the Invention

[0008] According to a first aspect, the present invention provides an antibody-natural killer cell (NK cell) conjugate, wherein the antibody is a bispecific antibody comprising a CD33 antigen-binding fragment and a CLL1 antigen-binding fragment, and the bispecific antibody is conjugated to the NK cell via a linker.

[0009] According to a second aspect, the present invention provides a cell population comprising an antibody-natural killer cell (NK cell) conjugate according to the first aspect.

[0010] According to a third aspect, the present invention provides a pharmaceutical composition comprising an antibody-natural killer cell (NK cell) conjugate according to the first aspect, or a cell population according to the second aspect, and a pharmaceutically acceptable carrier.

[0011] According to a fourth aspect, the present invention provides the use of an antibody-natural killer cell (NK cell) conjugate according to the first aspect or a cell population according to the second aspect in the manufacture of a medicament for the treatment of acute myeloid leukemia (AML) in an individual.

[0012] According to a fifth aspect, the present invention provides a method of treating acute myeloid leukemia (AML) in an individual, the method comprising administering to said individual an effective amount of an antibody-natural killer cell (NK cell) conjugate according to the first aspect, or a cell population according to the second aspect, or a pharmaceutical composition according to the third aspect.

[0013] By way of non-limiting example, the present invention provides the following embodiments. [1] An antibody-natural killer cell (NK cell) conjugate, wherein the antibody is a bispecific antibody comprising a CD33 antigen-binding fragment and a CLL1 antigen-binding fragment, and the bispecific antibody is conjugated to the NK cell via a linker.

[0014] [2] The CD33 antigen-binding fragment comprises: HCDR1 as set forth in SEQ ID NO: 9 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 9; an HCDR2 as set forth in SEQ ID NO: 10 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 10; an HCDR3 as set forth in SEQ ID NO: 11 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 11; LCDR1 as set forth in SEQ ID NO: 12 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 12; An LCDR2 as set forth in SEQ ID NO: 13 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 13, and comprising an LCDR3 as set forth in SEQ ID NO: 14 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 14; wherein the amino acid sequences of the HCDR and LCDR are as defined by Kabat: 2. The antibody-natural killer cell (NK cell) conjugate of embodiment 1.

[0015] [3] 3. The antibody-natural killer cell (NK cell) conjugate of embodiment 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the CD33 antigen-binding fragment is set forth in SEQ ID NO: 5 or has at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 7 or has at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 7.

[0016] [4] The CLL1 antigen-binding fragment comprises: HCDR1 as set forth in SEQ ID NO: 15 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 15; HCDR2 as set forth in SEQ ID NO: 16 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 16; an HCDR3 as set forth in SEQ ID NO: 17 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 17; LCDR1 as set forth in SEQ ID NO: 18 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 18; An LCDR2 as set forth in SEQ ID NO: 19 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 19, and comprising an LCDR3 as set forth in SEQ ID NO:20 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO:20; 4. The antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 3, wherein the amino acid sequences of the HCDR and LCDR are as defined by Kabat.

[0017] [5] 5. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1 to 4, wherein the amino acid sequence of the heavy chain variable region of the CLL1 antigen-binding fragment is set forth in SEQ ID NO: 1 or has at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 3 or has at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 3.

[0018] [6] 6. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1 to 5, wherein the CD33 antigen-binding fragment and the CLL1 antigen-binding fragment are in the form of a single chain antibody (scFv) or a Fab fragment.

[0019] [7] the bispecific antibody comprises a CD33 antibody arm comprising a CD33 antigen-binding fragment and a CLL1 antibody arm comprising a CLL1 antigen-binding fragment; the CD33 antibody arm comprises a heavy chain variable region set forth in SEQ ID NO:5, a heavy chain constant region set forth in SEQ ID NO:6, a light chain variable region set forth in SEQ ID NO:7, and a light chain constant region set forth in SEQ ID NO:8; and / or the CLL1 antibody arm comprises a heavy chain variable region set forth in SEQ ID NO: 1, a heavy chain constant region set forth in SEQ ID NO: 2, a light chain variable region set forth in SEQ ID NO: 3, and a light chain constant region set forth in SEQ ID NO: 4; An antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 6.

[0020] [8] the bispecific antibody is a complete antibody, and / or the bispecific antibody is a humanized antibody or a fully human antibody, and / or the bispecific antibody is a monoclonal antibody, and / or the bispecific antibody is of the IgG1, IgG2 or IgG4 isotype, and / or the bispecific antibody comprises a light chain constant region of the κ subtype; An antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 7.

[0021] [9] The NK cells are CD16 + and / or NKG2D + and preferably CD16 + NKG2D + The antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 8, wherein

[0022]

[10] The conjugate CD16 + NKG2D + The proportion of NK cells is at least 90%, preferably CD56 + The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1 to 9, wherein the proportion of NK cells is at least 95%.

[0023]

[11] The NK cells are obtained from in vitro culture and expansion of NK cells derived from peripheral blood mononuclear cells (PBMCs), or The NK cells are obtained from the in vitro culture and expansion of NK cells derived from umbilical cord blood, or The NK cells are obtained from in vitro induction, culture, and expansion of induced pluripotent stem cells (iPSCs / iPS cells) or mesenchymal stem cells (ESCs); The antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 10.

[0024]

[12] 12. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1 to 11, wherein the bispecific antibody and the NK cell are conjugated via a click chemistry reaction of a linker.

[0025]

[13] 13. The antibody-natural killer cell (NK cell) conjugate of embodiment 12, wherein the bispecific antibody and the NK cell are conjugated via a first linker and a second linker, wherein the first linker is conjugated to the bispecific antibody, the second linker is conjugated to the NK cell, and the first linker and the second linker are conjugated to each other to form the antibody-NK cell conjugate.

[0026]

[14] 14. The antibody-natural killer cell (NK cell) conjugate of embodiment 13, wherein the first linker is an active ester capable of forming conjugation to a lysine residue of the antibody via an ester bond to amide bond reaction, and the active ester is, for example, a pentafluorophenyl ester, for example, piperidine acid pentafluorophenyl ester.

[0027]

[15] 15. The antibody-natural killer cell (NK cell) conjugate of embodiment 14, wherein the first linker further comprises a carbon-carbon triple bond structure capable of undergoing a cyclization reaction with the azide group to form a five-membered triazazole ring, e.g., the carbon-carbon triple bond structure is an octyne group.

[0028]

[16] The first linker has the following structure: [ka] [In the formula, n is an integer of 0 to 8.] dibenzoazacyclooctynyl-glutaryl-aminopolyethylene glycol-acetylpiperidine acid pentafluorophenyl ester having the formula 16. The antibody-natural killer cell (NK cell) conjugate of embodiment 15.

[0029]

[17] The first linker has the following structure: [ka] dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester having the formula 17. The antibody-natural killer cell (NK cell) conjugate of embodiment 16.

[0030]

[18] 14. The antibody-natural killer cell (NK cell) conjugate of embodiment 13, wherein said second linker is an azidoacetylcyclohexosamine, e.g., azidoacetylcyclogalactosamine, or azidoacetylglucosamine.

[0031]

[19] The second linker has the following structure: [ka] 1,3,4,6-oxo-tetraacetyl-2-azidoacetamido-2-deoxy-a,bD-galactose having the formula 19. The antibody-natural killer cell (NK cell) conjugate of embodiment 18.

[0032]

[20] 20. The antibody-natural killer cell (NK cell) conjugate of embodiment 19, wherein the proportion of said second linker in a single α or β configuration is at least 90%.

[0033] 〔twenty one〕 A cell population comprising the antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1 to 20.

[0034] 〔twenty two〕 CD3 - CD56 + CD16 + and / or CD3 - CD56 + NKG2D + 22. The cell population of embodiment 21, wherein the number of cells represents at least 95%, preferably at least 98%, of the total number of cells in said cell population.

[0035] 〔twenty three〕 CD3 + CD56 + the number of cells comprises 5% or less of the total number of cells in said cell population; and / or CD3 - CD19 + the number of cells accounts for 2% or less of the total number of cells in said cell population; and / or CD3 + CD4 + and CD3 + CD8 + The number of cells accounts for 2% or less of the total number of cells in the cell population, 22. The cell population of embodiment 21.

[0036] 〔twenty four〕 24. The cell population of any one of embodiments 21 to 23, wherein the antibody-natural killer cell (NK cell) conjugate accounts for at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably at least 99% of the total cells in the cell population.

[0037] 〔twenty five〕 A pharmaceutical composition comprising the antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1 to 20 or the cell population of any one of embodiments 21 to 24, and a pharmaceutically acceptable carrier.

[0038]

[26] 26. The pharmaceutical composition of embodiment 25, comprising sodium chloride and / or human serum albumin.

[0039]

[27] 27. The pharmaceutical composition of embodiment 25 or 26, comprising trehalose, sucrose, dextran, DMSO, or any combination thereof.

[0040]

[28] 28. The pharmaceutical composition according to any one of embodiments 25 to 27, for use in the treatment of acute myeloid leukemia (AML) in an individual.

[0041]

[29] 29. The pharmaceutical composition of embodiment 28 for use in the treatment of initially diagnosed acute myeloid leukemia.

[0042]

[30] 29. The pharmaceutical composition of embodiment 28 for use in treating relapsed or refractory acute myeloid leukemia.

[0043]

[31] The pharmaceutical composition of embodiment 30, wherein the individual has undergone chemotherapy and / or targeted drug therapy.

[0044]

[32] The pharmaceutical composition of embodiment 31, wherein the targeted drug is gilteritinib and / or venetoclax.

[0045]

[33] for use in combination with venetoclax and / or azacitidine for the combination treatment of relapsed or refractory acute myeloid leukemia; The pharmaceutical composition according to any one of embodiments 25 to 32.

[0046]

[34] Use of an antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 20 or a cell population according to any one of embodiments 21 to 24 in the manufacture of a medicament for the treatment of acute myeloid leukemia (AML).

[0047]

[35] The use according to embodiment 34, wherein the medicament is for use in the treatment of initially diagnosed acute myeloid leukemia.

[0048]

[36] The use of embodiment 34, wherein the medicament is for use in treating relapsed or refractory acute myeloid leukemia.

[0049]

[37] The use of embodiment 36, wherein the individual has undergone chemotherapy and / or targeted drug therapy.

[0050]

[38] The use of embodiment 37, wherein the targeted drug is gilteritinib and / or venetoclax.

[0051]

[39] The use according to any one of embodiments 34 to 38, wherein the medicament is for use in combination with venetoclax and / or azacitidine for the combination treatment of relapsed or refractory acute myeloid leukemia.

[0052]

[40] 32. A method of treating acute myeloid leukemia (AML) in an individual, comprising administering to said individual an effective amount of the antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1 to 20, the cell population of any one of embodiments 21 to 24, or the pharmaceutical composition of any one of embodiments 25 to 33.

[0053]

[41] 41. The method of embodiment 40, wherein the individual has initially diagnosed acute myeloid leukemia.

[0054]

[42] 41. The method of embodiment 40, wherein the individual has relapsed or refractory acute myeloid leukemia.

[0055]

[43] The method of embodiment 42, wherein the individual has undergone chemotherapy and / or targeted drug therapy.

[0056]

[44] The method of embodiment 43, wherein the targeted drug is gilteritinib and / or venetoclax.

[0057]

[45] The method of any one of embodiments 40-44, further comprising administering to the individual venetoclax and / or azacitidine for combination treatment of relapsed or refractory acute myeloid leukemia. [Brief explanation of the drawings]

[0058] [Figure 1] FIG. 1 shows a structural schematic of an exemplary anti-CD33 / CLL1 bispecific antibody-natural killer cell conjugate of the invention. [Figure 2] FIG. 1 shows a schematic representation of the mechanism of action of an exemplary anti-CD33 / CLL1 bispecific antibody-natural killer cell conjugate of the invention. [Figure 3] FIG. 1 shows the sequence and structural analysis of exemplary anti-CD33 / CLL1 bispecific antibodies of the invention. [Figure 4] FIG. 1 shows a schematic diagram of the in vitro synthesis of an anti-CD33 / CLL1 bispecific antibody in an exemplary embodiment of the invention. [Figure 5] FIG. 1 shows flow cytometry analysis of IBR733 stock solution (Lot No. DSE20220407): CD56+, CD3-, CD16+, NKG2D+, CD19-. [Figure 6]FIG. 1 shows the flow cytometry analysis results of IBR733 formulation (Lot No. FE20220407): CD56+, CD3-, CD16+, NKG2D+, CD19-. [Figure 7] FIG. 1 shows the results of flow cytometry analysis of the conjugation positivity rate and geometric mean value for UNK, IBR733 stock solution, and IBR733 formulations. [Figure 8] FIG. 1 shows a schematic diagram of the production process flow of an L2 linker. [Figure 9] FIG. 1 shows the structural formula of the L2 linker (Japanese name: (Z)-19-(1′-aza-2′-one-dibenzo[b,f]cyclo-7′-octynyl)-3,6,9,12-tetraoxa-15-aza-16,20-dioxodecanoylpiperidine-4-carboxylic acid pentafluorophenyl ester, English name: pentafluorophenyl (Z)-20-(1′-aza-2′-oxo-dibenzo[b,f]cyclo-7′-octynyl)-3,6,9,12-tetraoxa-15-aza-16,20-dioxodecanoyl piperidine-4-carboxylate). [Figure 10] FIG. 1 shows a schematic flow diagram of the manufacturing process of the N1 linker. [Figure 11] FIG. 1 shows the structural formula of the N1 linker (Japanese name: 1,3,4,6-tetra-O-acetyl-2-azidoacetylamido-2-deoxy-a,bD-mannopyranose, English name: 1,3,4,6-tetra-O-acetyl-2-azidoacetylamido-2-deoxy-a,bD-mannopyanose). [Figure 12] FIG. 1 shows a schematic diagram of the conjugation mechanism between the L2 linker and a bispecific antibody. [Figure 13] FIG. 1 shows a schematic diagram of the binding mechanism between the N1 linker and NK cells. [Figure 14] FIG. 1 shows a schematic diagram of the structure of UNK connected with an N1 linker. [Figure 15]FIG. 1 shows a schematic diagram of the binding mechanism between UNK and an L2-conjugated antibody. [Figure 16] FIG. 1 shows a comparison of the cytotoxic effects of IBR733, UNK cells, and NK cells on U937 cells. [Figure 17] FIG. 1 shows a comparison of the cytotoxic effects of IBR733, UNK cells, and NK cells on THP-1 cells. [Figure 18] FIG. 1 shows a comparison of the cytotoxic effects of IBR733, UNK cells, and NK cells on HL60 cells. [Figure 19] 1 shows the levels of TraiL following the action of IBR733 on U937 tumor cells, where panels A and B show the time course at an effector to target ratio of 10:1, and panel C shows the time course at different effector to target ratios. [Figure 20] Figure 1 shows the levels of FasL following the action of IBR733 on U937 tumor cells, where panels A and B show the time course at an effector to target ratio of 10:1, and panel C shows the time course at different effector to target ratios. [Figure 21] 1 shows the survival curves for each group during the animal experiment in a tumor model test of U937 tumor-implanted mice, where the vertical axis represents the percentage of surviving animals and the horizontal axis represents the number of days. [Figure 22] 1 shows the survival curves of each group during the animal experiment in a tumor model test of THP-1 tumor-implanted mice, where the vertical axis represents the percentage of surviving animals and the horizontal axis represents the number of days. [Figure 23] Figure 1 shows a comparison of the cytotoxic effects of anti-CD33 / CLL1 bispecific antibody-natural killer cell conjugates with different L linkers, UNK cells, and NK cells on U937 cells, where the bars for each effector-to-target ratio are L1, L2, L3, NK, and UNK, from left to right. DETAILED DESCRIPTION OF THE INVENTION

[0059] [Definition] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as understood by those skilled in the art. For definitions and terms in the art, those skilled in the art can refer, in particular, to "Current Protocols in Molecular Biology" (Ausubel). Abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0060] Notwithstanding the numerical ranges and approximations of parameters set forth in the broad scope of the invention, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, all numerical values ​​inherently contain a certain degree of error due to the standard deviation that exists in each measurement. Furthermore, all ranges disclosed herein should be understood to cover all subranges contained within that range. For example, a recited range of "1 to 10" should be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10 (including the endpoints), i.e., all subranges beginning with a minimum value of 1 or greater, e.g., 1 to 6.1, and all subranges ending with a maximum value of 10 or less, e.g., 5.5 to 10. Furthermore, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0061] As used herein, the term "individual" or "subject" refers to a mammal such as a human, but may also refer to other animals such as wild, domestic, or laboratory animals (e.g., orangutans, monkeys, rats, mice, rabbits, guinea pigs, marmots, round squirrels, etc.).

[0062] As used herein, the term "antigen" refers to a predetermined target to which an antibody can selectively bind. Examples of antigens include, but are not limited to, polypeptides, sugars, nucleic acids, lipids, haptens, or other natural or synthetic compounds.

[0063] In a broad sense, an "antibody" can refer to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule, and thus encompasses a complete antibody / full-length antibody, a single antibody chain, or any antigen-binding fragment of an antibody (also called an "antigen-binding portion"). When "antibody" and "antigen-binding fragment / antigen-binding portion" appear in the same context, "antibody" can be understood as being complete with respect to "antigen-binding fragment / antigen-binding portion," and both commonly correspond to the broad concept of an antibody.

[0064] The term "agonist antibody" refers to an antibody that elicits a response, e.g., an antibody that mimics at least one functional activity of a target polypeptide. Agonist antibodies include antibodies that are ligand mimetics, e.g., a ligand binds to a cell surface receptor, the binding of which induces cell signaling or activity through an intracellular cell signaling pathway, and an antibody induces similar cell signaling or activation.

[0065] A "full-length antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains linked together via disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region comprises a CL domain. The VH and VL regions may be further subdivided into multiple regions of hypervariability called complementarity-determining regions (CDRs), interspersed with multiple more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains comprise the binding domains that interact with antigens. The constant region of an antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. A full-length antibody (intact antibody) can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or a subclass thereof), but antibodies need not belong to any particular class. Immunoglobulins can be assigned to different classes based on the amino acid sequence of the constant domain of their heavy chains. There are generally five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are known. The antibodies of the present invention also include chimeric or humanized antibodies. Those skilled in the art know that the complementarity-determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest influence on the affinity and specificity of an antibody.There are many common definition methods for the CDR amino acid sequences of VH or VL, such as the Kabat definition, the IMGT definition, and the Chothia definition. For a given antibody variable region amino acid sequence, the CDR amino acid sequences in the VH and VL amino acid sequences can generally be determined by various definition methods. In an embodiment of the present invention, Kabat is used to define the CDR amino acid sequences. For a given antibody variable region amino acid sequence, the CDR amino acid sequences of the variable region amino acid sequence can be analyzed by various methods.

[0066] The term "humanized antibody" refers to an antibody obtained by grafting CDR sequences from other mammalian species, such as mice, onto human framework sequences. Some residues in the framework (referred to as FR) section can be modified to maintain binding affinity. The humanized antibody or fragment thereof according to the present invention can be produced by techniques well known to those skilled in the art.

[0067] The term "chimeric antibody" refers to an antibody whose variable region sequences are derived from one species and whose constant region sequences are derived from another species, e.g., whose variable region sequences are derived from a mouse antibody and whose constant region sequences are derived from a human antibody. The chimeric antibody or fragment thereof of the present invention can be produced using genetic engineering techniques. For example, the chimeric antibody can be produced by cloning recombinant DNA containing a promoter, a sequence encoding the variable region of a non-human (especially mouse) monoclonal antibody described in the present invention, and a sequence encoding the constant region of a human antibody. The chimeric antibody of the present invention encoded by such a recombinant gene is, for example, a mouse-human chimera, and the antibody's specificity is determined by the variable region derived from the mouse DNA and its allotype is determined by the constant region derived from the human DNA.

[0068] The term "partially humanized antibody" refers to an antibody that comprises a constant region of human origin and a variable region (including CDRs) of non-human (such as murine) origin. The term "semi-humanized antibody" refers to a type of humanized antibody, in which one antibody chain contains a mouse variable region and the other antibody chain contains a humanized variable region, i.e., a semi-humanized antibody. The term "monoclonal antibody" refers to an antibody obtained from a nearly homogeneous antibody population, in which the individual antibodies comprising the population are identical except for the possible presence of naturally occurring mutations in a few individuals.

[0069] As used herein, the terms "antigen-binding fragment" or "antigen-binding portion" or "antigen-binding region" are used interchangeably and refer to the portion of an antibody that contains the amino acid residues that interact with an antigen and confer specificity and affinity to the binder for the antigen, particularly antibody fragments such as Fv, Fab, F(ab')2, and Fab', or any fragment whose half-life can be extended by chemical modification or incorporation into liposomes. Such chemical modifications include, for example, the addition of poly(alkylene) glycols, such as polyethylene glycol ("pegylated, PEGylated") (referred to as "pegylated fragments" of Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, and Fab'-PEG, where "PEG" is polyethylene glycol). Preferably, the functional fragment consists of or comprises a subsequence of the heavy or light variable chain of the antibody from which it is derived, the subsequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, and the functional fragment comprises at least 5 consecutive amino acids, preferably 10, 15, 25, 50, or 100 consecutive amino acids of the antibody sequence from which it is derived. Examples of antigen-binding fragments include, but are not limited to, (1) a Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain, (2) an F(ab')2 fragment, which may be a bivalent fragment having two Fab' fragments linked by a disulfide bridge in the hinge region (i.e., an Fab' dimer), and (3) an Fv fragment of the VL and VH domains comprising a single arm of an antibody.

[0070] The term "single-chain antibody (scFv)" refers to a single polypeptide chain in which a VH domain and a VL domain are linked via a peptide linker. (scFv)2 contains two VH domains linked via a peptide linker and two VL domains connected to the two VH domains via disulfide bridges. The terms "Fc fragment," "Fc region," "Fc domain," "Fc portion," or similar terms refer to a portion of the antibody heavy chain constant region, including the hinge region, CH2 fragment, and CH3 fragment of the constant region. The Fc region of an antibody can be engineered or modified, including modifications related to effector function, for example, to reduce or eliminate antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), which can be achieved by introducing one or more amino acid substitutions / mutations into the Fc region of the antibody. As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0071] The term "bispecific antibody" refers to an antibody capable of simultaneously binding to two different antigenic epitopes. The two epitopes may be on different antigens or on the same antigen. Bispecific antibodies may have multiple structural configurations. For example, a bispecific antibody may be composed of two Fc fragments fused to two antigen-binding moieties (similar to natural antibodies, except that the two arms bind to different antigenic targets or epitopes). The antigen-binding moieties may be in the form of a single-chain antibody (scfv) or Fab fragments. The two different binding moieties of a bispecific antibody are each attached to the N-terminus of a single Fc fragment, and the antigen-binding moieties of the two arms can be configured in four combinations: scfv + Fab fragment, Fab fragment + scfv, scfv + scfv, or Fab fragment + Fab fragment. The Fc fragment can contain mutations that ensure heavy chain heteromerization, and knob-in-hole (KIH) technology is a strategy to address heavy chain heteropolymerization. Generally, KIH technology refers to a technique that alters the amino acid sequence of the CH3 region to form a favorable structure for heterologous incomplete antibodies to pair with each other, thereby forming a bispecific antibody while maintaining the structure of a normal antibody as much as possible.

[0072] Generally, to prepare monoclonal antibodies or functional fragments thereof, in particular murine-derived monoclonal antibodies or functional fragments thereof, reference can be made in particular to the techniques described in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988), or to the technique of preparation from hybridoma cells by Kohler and Milstein (Nature, 256: 495497, 1975).

[0073] The term "conservative mutation" or "conservative amino acid substitution" refers to a substitution that does not substantially affect or reduce the affinity of a protein. For example, an antibody may contain about 1 or less, about 2 or less, about 5 or less, about 10 or less, or about 15 or less conservative substitutions and specifically bind to a target antigen. The term "conservative mutation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, as long as the antibody specifically binds to the target antigen.

[0074] The term "isolated" biological component (e.g., nucleic acid, protein (including antibody), or organelle) refers to one that has been substantially separated or purified from other biological components (i.e., other chromosomal and additional chromosomal DNA and RNA, proteins and organelles) in the component's naturally occurring environment (e.g., a cell). Nucleic acids and proteins that have been "isolated" include those that have been purified by standard purification methods. The term also includes nucleic acids and proteins produced by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0075] As used herein, the term "pharmaceutical composition" refers to a combination of at least one drug and a pharmaceutically acceptable carrier or auxiliary substance that are combined together to achieve a specific purpose. In some embodiments, the pharmaceutical composition includes a combination of drugs separated in time and / or space, as long as they can act together to achieve the purpose of the present invention. For example, the components contained in the pharmaceutical composition (e.g., the antibody-cell conjugates of the present invention) may be administered to an individual all at once or separately. When the components contained in the pharmaceutical composition are administered to an individual separately, the components may be administered to the individual simultaneously or sequentially. The pharmaceutical composition of the present invention may contain conventional components for cell culture, particularly NK cell culture, to maintain the activity of NK cells in the conjugate. Pharmaceutically acceptable carriers may further include water, aqueous buffer solutions, isotonic salt solutions such as PBS (phosphate buffer), glucose, mannitol, dextroglucose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol and triglycerides, etc. The pharmaceutical composition or formulation according to the present invention can be administered by any suitable route, such as intravenous administration, intradermal administration, subcutaneous administration, intramuscular injection, etc. The composition according to the present invention may contain additives such as wetting agents, emulsifying agents or buffer substances.

[0076] As used herein, the term "therapeutically effective amount" or "effective amount" refers to a dose sufficient to show its benefit to the individual to whom it is administered. The actual amount administered, the rate and time course of administration depend on the condition and severity of the individual being treated. The prescription for treatment (e.g., determining the dosage) is ultimately the responsibility of specialists and other physicians, and is generally determined taking into account the disease to be treated, the individual patient's condition, delivery site, administration method, and other factors known to physicians.

[0077] EC 50The EC value primarily refers to the concentration of a corresponding drug, antibody, or toxin that can reach 50% of its maximum biological effect after a specific exposure time. In pharmacology, it is used to characterize the activation ability of agonists in in vitro experiments, and also to indicate the blood concentration required to reach half of the maximum biological effect in vivo. In some literature, the EC 50 It is also used to characterize the potency (including agonism and antagonism) of compounds at a cellular level, and EC 50 The value can be measured by methods such as ELISA.

[0078] The term "identity / homology / matching" of an amino acid or nucleic acid sequence is defined as the percentage of identical residues in amino acid or nucleotide sequence variants after sequence alignment and capping, if necessary, to reach the maximum percentage of identity. Methods and computer programs for alignment are known to those skilled in the art.

[0079] The inventors of the present application have conducted extensive research in the field of cellular immunotherapy, and in particular have developed anti-CD33 / CLL1 bispecific antibody-natural killer cell conjugates for acute myeloid leukemia (AML). Figure 1 shows a structural schematic diagram of an exemplary anti-CD33 / CLL1 bispecific antibody-natural killer cell conjugate of the present invention. Figure 2 shows a schematic diagram of the mechanism of action of an exemplary anti-CD33 / CLL1 bispecific antibody-natural killer cell conjugate of the present invention.

[0080] NK cells recognize target antigens without major histocompatibility complex (MHC) restriction, and allogeneic (allogeneic) NK cells have an extremely low risk of causing graft-versus-host disease (GVHD). Therefore, allogeneic NK cell therapy is clinically feasible. Furthermore, NK cells also have an extremely low risk of causing cytokine release syndrome (CRS). In 2020, Liu et al. published the results of a phase I / II clinical trial of CAR-NK cell therapy for lymphoma in the New England Journal of Medicine. Following CAR-NK cell infusion in 11 enrolled subjects, eight showed remission, seven of whom achieved complete remission, without any CRS, neurotoxicity, or GVHD. This indicates that CAR-NK cell therapy has a relatively high safety profile. Furthermore, NK cells can recognize target antigens without being restricted by MHC, allowing them to be manufactured into general-purpose products without being limited to autologous cells. Therefore, the NK cell sources used in this therapy can be selected from a wide range of sources, including allogeneic peripheral blood, umbilical cord blood, embryonic stem cells, human-induced pluripotent stem cells, and the NK-92 cell line.

[0081] The cytotoxic activity of NK cells is primarily mediated by: 1) Direct lysis of target cells: NK cells release cytotoxic particles such as perforin and granzymes via exocytosis, which activate the caspase pathway to induce necrosis or apoptosis of target cells. 2) Cytokine secretion: NK cells have cytokine-mediated damaging effects, and can synthesize and secrete various cytokines, such as IFN-γ, TNF-α, IL-1, IL-5, IL-8, IL-10, and G-CSF, which induce apoptosis of target cells. 3) Induction of apoptosis: Activated NK cells express Fas (CD95) ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) molecules, which induce apoptosis of CD95+ target cells and TRAIL receptor-positive target cells through a cascade of endogenous enzymes. 4)ADCC: Antibody-dependent cell-mediated cytotoxicity. 5) Immune checkpoint pathway: It exerts its effect by expressing programmed death receptor 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4), among others, and inhibiting immune checkpoints.

[0082] The multiple mechanisms of action described above, combined with the potential for commercial application and reliable safety profile, make NK cell therapy an attractive immunotherapy. Currently, NK cells used in treatment include natural NK cells and CAR-NK cells. In the field of natural NK cell therapy, CYNK-001, developed by Cellularity, Inc., has received Fast Track Designation from the FDA for the treatment of acute myeloid leukemia. Like CAR-T cells, CAR-NK cells combine the antigen-targeting ability with the natural killer capabilities of NK cells to overcome many of the limitations of other AML treatments, such as chemotherapy-induced severe bone marrow suppression, creating a new cell therapy platform. Furthermore, CAR-NK cells retain their natural receptors, such as NKp46, NKp30, NKp44, NKG2D, and CD226, which can independently recognize ligands and maintain the basic cytotoxic functions of NK cells.

[0083] In this application, one of the targets of the selected bispecific antibodies is CD33, which is expressed on more than 90% of primary cells from patients with acute myeloid leukemia, but is not expressed on the surface of hematopoietic stem cells or on mature granulocytes or other tissues, making CD33 an excellent target for the treatment of myeloid leukemia.

[0084] Another target chosen for the bispecific antibody of the present invention is C-type lectin-like molecule-1 (CLL-1), which is present on myeloid cells in peripheral blood and bone marrow and on most AML leukemia cells, and is expressed on most CD34 cells in AML patients. + CD38 -It is also expressed on stem cells, but normal human CD34 + CD38 - It is not expressed on stem cells. The CLL1 receptor targets leukemia stem cells (a small, slow-growing subset of leukemia cells that are resistant to conventional drug therapy while also being able to stably proliferate into new leukemia cell lines), and CLL1 is associated with leukemia stem cell and disease relapse. As a marker antigen for leukemia stem cells, CLL1 contributes to the differentiation of leukemia stem cells from normal stem cells and is useful for specific targeted cell immunotherapy targeting CLL1 for AML.

[0085] According to a first aspect, the present invention provides an antibody-natural killer cell (NK cell) conjugate, wherein the antibody is a bispecific antibody comprising a CD33 antigen-binding fragment and a CLL1 antigen-binding fragment, and the bispecific antibody is conjugated to the NK cell via a linker.

[0086] In some embodiments, the CD33 antigen-binding fragment comprises: HCDR1 as set forth in SEQ ID NO: 9 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 9; an HCDR2 as set forth in SEQ ID NO: 10 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 10; an HCDR3 as set forth in SEQ ID NO: 11 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 11; LCDR1 as set forth in SEQ ID NO: 12 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 12; An LCDR2 as set forth in SEQ ID NO: 13 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 13, and comprising an LCDR3 as set forth in SEQ ID NO: 14 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 14; Here, the amino acid sequences of the HCDR and LCDR are defined by Kabat.

[0087] In some embodiments, the amino acid sequence of the heavy chain variable region of the CD33 antigen-binding fragment is set forth in SEQ ID NO:5 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:7 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth in SEQ ID NO:7.

[0088] In some embodiments of the second aspect, the amino acid sequence of the heavy chain variable region of the CD33 antigen-binding fragment differs from the amino acid sequence set forth in SEQ ID NO: 5 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 5 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids and still maintain similar function as the heavy chain variable region of the antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 5, and the resulting amino acid sequence still maintains similar function as the heavy chain variable region of the antibody. In some embodiments, the modified amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids added or deleted in a region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 5, as long as the modified amino acid sequence maintains substantially similar functions to the heavy chain variable region of the antibody.

[0089] In some embodiments, the amino acid sequence of the light chain variable region of the CD33 antigen-binding fragment differs from the amino acid sequence set forth in SEQ ID NO: 7 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 7 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids and still maintain a similar function to the light chain variable region of the antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids may be further added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 7 and the resulting amino acid sequence still maintains a similar function to the light chain variable region of the antibody. In some embodiments, the modified amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids added or deleted at the C-terminus or a region other than the C-terminus of the amino acid sequence shown in SEQ ID NO: 7, as long as the modified amino acid sequence maintains substantially similar function to the light chain variable region of the antibody.

[0090] In some embodiments, the CLL1 antigen-binding fragment is HCDR1 as set forth in SEQ ID NO: 15 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 15; HCDR2 as set forth in SEQ ID NO: 16 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 16; an HCDR3 as set forth in SEQ ID NO: 17 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 17; LCDR1 as set forth in SEQ ID NO: 18 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 18; An LCDR2 as set forth in SEQ ID NO: 19 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 19, and comprising an LCDR3 as set forth in SEQ ID NO:20 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO:20; Here, the amino acid sequences of the HCDR and LCDR are defined by Kabat.

[0091] In some embodiments, the amino acid sequence of the heavy chain variable region of the CLL1 antigen-binding fragment is set forth in SEQ ID NO:1 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence set forth in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:3 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence set forth in SEQ ID NO:3.

[0092] In some embodiments, the amino acid sequence of the heavy chain variable region of the CLL1 antigen-binding fragment differs from the amino acid sequence set forth in SEQ ID NO: 1 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 1 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids and still maintain similar function as the heavy chain variable region of the antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 1 and the resulting amino acid sequence still maintains similar function as the heavy chain variable region of the antibody. In some embodiments, the modified amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids added or deleted in a region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 1, as long as the modified amino acid sequence maintains substantially similar functions to the heavy chain variable region of the antibody.

[0093] In some embodiments, the amino acid sequence of the light chain variable region of the CLL1 antigen-binding fragment differs from the amino acid sequence set forth in SEQ ID NO: 3 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 3 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids and still maintain a similar function to the light chain variable region of the antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids may be further added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 3 and still maintain a similar function to the light chain variable region of the antibody. In some embodiments, the modified amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids added or deleted in a region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 3, as long as the modified amino acid sequence maintains substantially similar function to the light chain variable region of the antibody.

[0094] In some embodiments, the forms of the CD33 antigen-binding fragment and the CLL1 antigen-binding fragment are independently selected from a single chain antibody (scFv) or a Fab fragment.

[0095] In some embodiments, the bispecific antibody comprises a CD33 antibody arm comprising a CD33 antigen-binding fragment and a CLL1 antibody arm comprising a CLL1 antigen-binding fragment; the CD33 antibody arm comprises a heavy chain variable region set forth in SEQ ID NO:5, a heavy chain constant region set forth in SEQ ID NO:6, a light chain variable region set forth in SEQ ID NO:7, and a light chain constant region set forth in SEQ ID NO:8; and / or The CLL1 antibody arms comprise a heavy chain variable region set forth in SEQ ID NO: 1, a heavy chain constant region set forth in SEQ ID NO: 2, a light chain variable region set forth in SEQ ID NO: 3, and a light chain constant region set forth in SEQ ID NO: 4. FIG. 3 shows the sequence and structural analysis of an exemplary anti-CD33 / CLL1 bispecific antibody comprising the CD33 antibody arm and the CLL1 antibody arm described above.

[0096] In some embodiments, the bispecific antibody is a whole antibody. In some embodiments, the bispecific antibody is a humanized or fully human antibody. In some embodiments, the bispecific antibody is a monoclonal antibody. In some embodiments, the bispecific antibody is an IgG1, IgG2, or IgG4 isotype. In some embodiments, the bispecific antibody is an IgG1 isotype. In some embodiments, the bispecific antibody comprises a light chain constant region of the kappa subtype.

[0097] Once the antibody structure / sequence has been identified, techniques for producing the corresponding bispecific antibody are within the skill of the art. An exemplary methodology for producing an anti-CD33 / CLL1 bispecific antibody in the Examples of this application involves first producing anti-CD33 and anti-CLL1 monoclonal antibodies, and then separating and reassembling the monoclonal antibody arms into the bispecific antibody by reducing the disulfide bond in the hinge region (see Figure 4 for a schematic diagram of the reaction).

[0098] Specifically, the sequences of anti-CD33 and anti-CLL1 monoclonal antibodies are obtained by screening a phage library, screened for binding activity to a target protein and cells expressing the target protein, confirmed by affinity testing, and then selected to obtain anti-CD33 / CLL1 antibodies. The anti-CLL1 monoclonal antibody contains a heavy chain variable region shown in SEQ ID NO: 1 (HCDR1-3 are SEQ ID NOs: 15, 16, and 17, respectively), a heavy chain constant region shown in SEQ ID NO: 2, a light chain variable region shown in SEQ ID NO: 3 (LCDR1-3 are SEQ ID NOs: 18, 19, and 20, respectively), and a light chain constant region shown in SEQ ID NO: 4. The anti-CD33 monoclonal antibody contains a heavy chain variable region shown in SEQ ID NO: 5 (HCDR1-3 are SEQ ID NOs: 9, 10, and 11, respectively), a heavy chain constant region shown in SEQ ID NO: 6, a light chain variable region shown in SEQ ID NO: 7 (LCDR1-3 are SEQ ID NOs: 12, 13, and 14, respectively), and a light chain constant region shown in SEQ ID NO: 8. The DNA sequences of the anti-CD33 and anti-CLL1 antibodies were determined, and recombinant plasmids (designated IB12-CD33 and IB12-CLL1 in this example) expressing the anti-CD33 and anti-CLL1 monoclonal antibodies, respectively, were constructed. The construction of monoclonal cell lines was based on known techniques: the IB12-CD33 and IB12-CLL1 plasmids were transfected into CHO cells by electroporation, followed by one round of minipool screening and two rounds of screening after plating the monoclonals. These were confirmed by early passage stable culture, resulting in cell lines capable of stably expressing the anti-CLL1 and anti-CD33 monoclonal antibodies. To produce anti-CD33-CLL1 bispecific antibodies, cell lines stably expressing the above-mentioned anti-CLL1 monoclonal antibody and anti-CD33 monoclonal antibody are obtained, and after expansion and large-scale culture, the unprocessed cell suspension (UPB) is collected, clarified, and filtered to obtain cell culture supernatants, which contain the anti-CD33 monoclonal antibody and anti-CLL1 monoclonal antibody, respectively.The anti-CD33 monoclonal antibody and the anti-CLL1 monoclonal antibody were then purified through a protein A affinity purification process, followed by assembly into an anti-CD33-CLL1 bispecific antibody under conditions of reducing agent 2-MEA and air oxidation. The anti-CD33-CLL1 bispecific antibody was then obtained through two-step chromatography of anion exchange and cation exchange.

[0099] In addition to the above methodologies, cells can be transfected with plasmids (e.g., three-plasmid or four-plasmid systems) expressing the antigen-binding portion (Fab or scFv) and constant regions of a bispecific antibody, and the expressed antibody fragments can be directly assembled into a bispecific antibody. "Knob-into-hole" and "CrossMab" technologies can be used in such methods.

[0100] In some embodiments, the NK cells are CD16 + and / or NKG2D + In some embodiments, the NK cells are CD16 + NKG2D + is. In some embodiments, the CD16 + NKG2D + In some embodiments, the percentage of NK cells is at least 90%, e.g., at least 95%, at least 98%, or at least 99%. + NKG2D + The proportion of NK cells is at least 90% and CD56 + The proportion of NK cells is at least 95% (eg, at least 96%, at least 97%, at least 98%, or at least 99%). There are a wide variety of techniques for culturing, expanding and obtaining NK cells in vitro, the general principles and methodologies of which are known to those skilled in the art.

[0101] In some embodiments, NK cells are obtained by in vitro culture and expansion of peripheral blood mononuclear cell (PBMC)-derived NK cells, which is also an exemplary method in the examples of this application. PBMCs are one of the major sources of NK cells, and have the advantages of being relatively easy to collect, easy to expand in vitro, and without toxicity or side effects. However, the percentage of NK cells in PBMCs is only 10% to 15%. Methods for expanding PBMC-derived NK cells include stimulating in vitro proliferation of NK cells using a combination of cytokines, feeder cells, or membrane particles; these different expansion systems exhibit different levels of NK cell expansion efficiency. In some embodiments, PBMCs are screened for HLA and KIR. In some embodiments, PBMCs are screened for CD16a variants, i.e., 176V and 176F. In some embodiments, one or more cytokines are used to maintain or activate the activity of natural killer cells in culture. In some embodiments, one or more immunoglobulins or fusion proteins are used to suppress the proliferation of B cells, macrophages, and other immune cells.

[0102] PBMCs may be derived from peripheral blood lymphocyte collection from allogeneic healthy donors. After separation of T cells and red blood cells, cells are transferred to primary cell cryopreservation solution, resulting in a total of 6.0 x 10 viable cells per vial. 7 The PBMCs are then aliquoted into 100 or more aliquots to obtain PBMCs. PBMCs are stored long-term in liquid nitrogen at temperatures below -175°C. PBMCs cryopreserved in liquid nitrogen are removed, resuscitated in an adapted medium, and expanded in culture (i.e., by increasing the medium volume). NK cell-associated cytokines, including but not limited to IL2 and IL15, are added to maintain their proliferation capacity and activity. For an exemplary production method, see Example 1 of the present application.

[0103] In some embodiments, NK cells are obtained by in vitro culture and expansion of NK cells derived from umbilical cord blood. There are generally two different methods for obtaining large amounts of NK cells from umbilical cord blood. One method is to expand NK cells in umbilical cord blood, and the other method is to extract CD34 + The goal is to induce hematopoietic stem / progenitor cells to differentiate into NK cells and then proliferate.

[0104] In some embodiments, the NK cells are obtained by in vitro culture and expansion of an NK cell line. As an example, NK-92, a homogeneous immortalized NK lymphoma cell line, is the first NK cell-based immunotherapy approved by the FDA for clinical trials. In some embodiments, NK cells are obtained by in vitro derivation, culture, and expansion of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (ESCs). In some embodiments, the bispecific antibody and the NK cell are conjugated via a click chemistry reaction of a linker.

[0105] "Click chemistry," also known as "link chemistry" or "speed-matching combined chemistry," is a synthetic concept introduced by chemist Barry Sharpless in 2001. Its main purpose is to rapidly and reliably complete the chemical synthesis of various molecules by linking small units. There are four main types of click chemistry reactions: cycloaddition reactions, nucleophilic ring-opening reactions, non-aldol carbonyl chemical reactions, and carbon-carbon multiple bond addition reactions.

[0106] In some embodiments, the bispecific antibody and the NK cell are conjugated via a first linker and a second linker, wherein the first linker is conjugated to the bispecific antibody, the second linker is conjugated to the NK cell, and the first linker and the second linker are conjugated to each other to form the antibody-NK cell conjugate.

[0107] In some embodiments, the first linker is an activated ester that can form conjugation to a lysine residue of an antibody through an ester bond-to-amide bond reaction, and the activated ester is, for example, a pentafluorophenyl ester, such as piperidine acid pentafluorophenyl ester. The activated ester may be a molecule that is stable in an aqueous phase and can specifically covalently bond to a lysine residue of an antibody. In some embodiments, the activated ester has a tetraethylene glycol chain structure and can be gradually hydrolyzed in water. In some embodiments, the activated ester reacts with an amino group at the hydrophilic interface of a protein to convert the ester bond to an amide bond, thereby establishing a binding mechanism (conjugation mechanism).

[0108] In some embodiments, the first linker further comprises a carbon-carbon triple bond structure that can undergo a cyclization reaction with the azide group to form a five-membered triazazole ring, for example, the carbon-carbon triple bond structure is an octyne group.

[0109] In some embodiments, the first linker has the following structure: [ka] [In the formula, n is an integer of 0 to 8.] Dibenzoazacyclooctynyl-glutaryl-aminopolyethylene glycol-acetylpiperidine acid pentafluorophenyl ester having the formula:

[0110] In some embodiments, the first linker has the following structure: [ka] Dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester having the formula:

[0111] Dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester is also referred to as L2 linker in the present invention. The synthesis pathway of L2 linker is shown in Figure 8, and its structural formula is shown in Figure 9. It includes three main synthesis steps: amide condensation, hydrolysis reaction, and synthesis of activated ester.

[0112] <Step 1> Amide condensation Dibenzoazacyclooctyne glutaric acid (L2-1) and aminotetraethyleneglycol acetylpiperidine methyl ester (L2-2) were obtained commercially and then subjected to a chemical condensation reaction to obtain the intermediate dibenzoazacyclooctynylglutaryl aminotetraethyleneglycol acetylpiperidine methyl ester (L2-3), which is a stable compound.

[0113] Specifically, compounds L2-1 and L2-2 (1:1.1, L2-2 is in excess) were dissolved in dichloromethane (DCM), and hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbonyldiimide (EDCI) were added, followed by triethanolamine (TEA). The mixture was stirred at room temperature for 4-12 hours, quenched with water, extracted twice with dichloromethane (DCM), and purified on a silica gel column (dichloromethane:methanol = 20:1) to obtain compound L2-3 as a yellow oil.

[0114] <Step 2> Hydrolysis reaction The intermediate L2-3 obtained in Step 1 was hydrolyzed to give the intermediate dibenzoazacyclooctynyl glutaryl aminotetraethylene glycol acetylpiperidine acid (L2-4). The hydrolysis reaction is usually quantitative, and the product is used directly in the next synthesis step without further purification.

[0115] Specifically, compound L2-3 was dissolved in a mixture of methanol and water, cooled to 0°C, and then 1 mol / L aqueous lithium hydroxide (LiOH) solution was added. The mixture was stirred at 0°C to room temperature for 4 to 12 hours, acidified to pH 2 to 3 with 1 mol / L hydrochloric acid, extracted three times with ethyl acetate (EA), and dried to obtain the intermediate dibenzoazacyclooctynyl glutarylaminotetraethylene glycol acetylpiperidine acid (L2-4) as a yellow oil.

[0116] <Step 3> Synthesis of activated ester The intermediate L2-4 (unpurified) obtained in Step 2 was condensed with pentafluorophenol and dicyclohexylcarbodiimide (DCC) to give L2 (i.e., dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester).

[0117] Specifically, compound L2-4 was dissolved in tetrahydrofuran (THF) and cooled to 0°C. Then, pentafluorophenol, hydroxybenzotriazole (HOBt), and dicyclohexylcarbodiimide (DCC) were added. The mixture was stirred at 0°C to room temperature for 4 to 12 hours, extracted three times with ethyl acetate (EA), dried, and purified on a silica gel column (dichloromethane:methanol = 20:1) to obtain dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester (L2) as a yellow oil (see Figure 9 for the structural formula).

[0118] The L2 linker and anti-CD33-CLL1 bispecific antibody can then undergo a site-directed coupling reaction in a PBS system at pH 7.2 to 7.4 (preferably HEPES at pH 7.2) (see Figure 12 for a schematic diagram). Typically, one anti-CD33-CLL1 bispecific antibody can be conjugated to one to four (preferably one to two) L2 linkers. The reaction can be stopped by adjusting the pH to approximately 5.0 to obtain an L2-conjugated antibody.

[0119] In some embodiments, the second linker is an azidoacetylated cyclohexosamine, such as azidoacetylated cyclogalactosamine or azidoacetylated glucosamine. In some embodiments, the second linker is a molecule stable in an aqueous phase that can specifically and covalently bind to a membrane protein modified with sialic acid. In some embodiments, the second linker is transferred to the membrane protein modified with sialic acid through the cell's own metabolic pathway during cell culture. In some embodiments, the azidoacetyl group of the second linker can undergo a cyclization reaction with the carbon-carbon triple bond of the first linker to form a stable five-membered triazole ring.

[0120] In some embodiments, the second linker has the following structure: [ka] and 1,3,4,6-oxo-tetraacetyl-2-azidoacetamido-2-deoxy-a,bD-galactose having the formula:

[0121] In some embodiments, the proportion of a single α or β configuration of 1,3,4,6-oxo-tetraacetyl-2-azidoacetamido-2-deoxy-α,bD-galactose is at least 90%, for example, at least 95%, at least 98%, or at least 99%.

[0122] 1,3,4,6-Oxo-tetraacetyl-2-azidoacetamido-2-deoxy-a,bD-galactose is also referred to as the N1 linker in the present invention. The synthesis pathway of the N1 linker is shown in Figure 10, and its structural formula is shown in Figure 11. The N1 linker involves two major synthesis steps: aminoazidoacetylation and hydroxyacetylation.

[0123] <Step 1> Aminoazide acetylation 1.2 times the amount of α-azidoacetic acid (compound 1), 2 times the amount of hydroxybenzotriazole (HOBt), and triethylamine (EtN) were added to a solution of D-galactosamine hydrochloride (compound 2) in N,N-dimethylformamide (DMF). A small amount of methanol (MeOH) was added to aid dissolution. The mixture was allowed to react at room temperature for 12 hours. The reaction mixture was then poured into dichloromethane (DCM) / methanol (MeOH), shaken to mix well, and ether was added to precipitate the oily product (compound 3). The ether layer was poured off and this process was repeated twice. The mixture was then dried under vacuum. The product was then used directly for the next step without further purification.

[0124] <Step 2> Hydroxyacetyl esterification Compound 3 obtained above was added to anhydrous pyridine (Pyr) and acetic anhydride (AcO) and 4-dimethylaminopyridine (DMAP) catalyst at room temperature. The reaction was allowed to proceed for 12 hours. The reaction was detected to be nearly complete by HPLC and then concentrated to yield a racemic mixture of tetraacetyl-N-azidoacetyl-a,b-D-galactosamine. The solid was precipitated in ethyl acetate / petroleum ether, which revealed the product was predominantly in the b-configuration (β-configuration) with a purity of over 85%. Further purification using a silica gel column (dichloromethane:methanol = 20:1) yielded the N1 product with a single enantiomer purity of over 90%. The two a,b-isomers can isomerize with each other intracellularly, and through multiple metabolic and synthetic steps, they are converted to N-azidoacetylsialic acid, which is ultimately expressed on the surface of NK cells. The structural formula of the N1 linker is shown in Figure 11.

[0125] After culturing the NK cells for 15 to 16 days, N1 linker is added to the culture medium and incubated for 12 to 18 hours to obtain N1 linker-modified NK cells (also referred to as "UNK" in the examples of the present application) (see Figures 13 and 14 for the binding mechanism between the N1 linker and NK cells and the structural schematic of UNK).

[0126] Finally, to obtain an antibody-NK cell conjugate, UNK and the L2 conjugated antibody are subjected to a coupling reaction in the medium (see FIG. 15 for the reaction mechanism), and the antibody-NK cell conjugate can be obtained.

[0127] Subsequent processes further include producing an antibody-NK cell conjugate formulation to effectively extend the stability of the antibody-NK cell conjugate. The formulation may contain components that maintain isotonicity, such as sodium chloride and human serum albumin, and may also contain components that maintain the low-temperature resistance of cells and the activity of proteins and enzymes, such as trehalose, sucrose, dextran, and DMSO.

[0128] According to a second aspect, the present invention provides a cell population comprising an antibody-natural killer cell (NK cell) conjugate according to the first aspect.

[0129] In some embodiments, CD3 - CD56 + CD16 + In some embodiments, the CD3 - CD56 + CD16 + In some embodiments, the CD3 - CD56 + NKG2D + In some embodiments, the CD3 - CD56 + NKG2D + The number of cells represents at least 98% of the total number of cells in said cell population.

[0130] In some embodiments, CD3 + CD56 + The number of cells accounts for 5% or less of the total number of cells in the cell population. In some embodiments, CD3 - CD19+ The number of cells accounts for 2% or less of the total number of cells in the cell population. In some embodiments, CD3 + CD4 + and CD3 + The number of CD8 cells accounts for 2% or less of the total number of cells in the cell population.

[0131] In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 90% of the total cells in the cell population. In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 95% of the total cells in the cell population. In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 98% of the total cells in the cell population. In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 99% of the total cells in the cell population.

[0132] According to a third aspect, the present invention provides a pharmaceutical composition comprising an antibody-natural killer cell (NK cell) conjugate according to the first aspect, or a cell population according to the second aspect, and a pharmaceutically acceptable carrier.

[0133] In some embodiments, the pharmaceutical composition comprises sodium chloride and / or human serum albumin. In some embodiments, the pharmaceutical composition comprises trehalose, sucrose, dextran, DMSO, or any combination thereof. In some embodiments, the pharmaceutical composition is used to treat acute myeloid leukemia (AML) in an individual.

[0134] In some embodiments, the pharmaceutical composition is used to treat newly diagnosed acute myeloid leukemia. In some embodiments, the pharmaceutical composition is used to treat relapsed or refractory acute myeloid leukemia in an individual. In some embodiments, the AML patient individual has received chemotherapy and / or targeted drug therapy. In some embodiments, the targeted drug is gilteritinib and / or venetoclax. In some embodiments, the pharmaceutical composition is used in combination with venetoclax and / or azacitidine for the combination treatment of relapsed or refractory acute myeloid leukemia.

[0135] According to a fourth aspect, the present invention provides the use of an antibody-natural killer cell (NK cell) conjugate according to the first aspect or a cell population according to the second aspect in the manufacture of a medicament for the treatment of acute myeloid leukemia (AML) in an individual.

[0136] In some embodiments, the medicament is used to treat initially diagnosed acute myeloid leukemia in an individual. In some embodiments, the medicament is used to treat relapsed or refractory acute myeloid leukemia in an individual. In some embodiments, the individual has undergone chemotherapy and / or targeted drug therapy.

[0137] In some embodiments, the targeted drug is gilteritinib and / or venetoclax. In some embodiments, the drug is used in combination with venetoclax and / or azacitidine for the combination treatment of relapsed or refractory acute myeloid leukemia.

[0138] According to a fifth aspect, the present invention provides a method of treating acute myeloid leukemia (AML) in an individual, the method comprising administering to said individual an effective amount of an antibody-natural killer cell (NK cell) conjugate according to the first aspect, or a cell population according to the second aspect, or a pharmaceutical composition according to the third aspect.

[0139] In some embodiments, the individual has initially diagnosed acute myeloid leukemia. In some embodiments, the individual has relapsed or refractory acute myeloid leukemia. In some embodiments, the individual has undergone chemotherapy and / or targeted drug therapy.

[0140] In some embodiments, the targeted drug is gilteritinib and / or venetoclax. In some embodiments, the method further comprises administering to the individual venetoclax and / or azacitidine for combination treatment of relapsed or refractory acute myeloid leukemia.

[0141] It should be understood that the above detailed description is intended only to enable those skilled in the art to more clearly understand the present invention, but is not intended to limit any aspect thereof. Those skilled in the art may make various modifications and changes to the above embodiments.

[0142] The present invention will now be further described with reference to specific examples, which are intended to illustrate the invention only and are not intended to limit the scope of the invention. [Example]

[0143] Example 1: Preparation of anti-CD33 / CLL1 bispecific antibody-NK cell conjugate The production of antibody-NK cell conjugates in this example generally involves: (1) steps for producing an anti-CD33 / CLL1 bispecific antibody; (2) NK cell manufacturing steps; (3) Preparation of the antibody linker (also referred to as the L2 linker in this example); (4) Preparation of the NK cell linker (referred to as N1 linker in this example); (5) applying an antibody linker (L2 linker) to the antibody; (6) applying an NK cell linker (N1 linker) to the NK cells; and (7) Conjugating each antibody having a linker to an NK cell. It can be divided into:

[0144] (1) Production of anti-CD33 / CLL1 bispecific antibodies Briefly, the methodology used to produce the anti-CD33 / CLL1 bispecific antibody in this example involves first producing anti-CD33 and anti-CLL1 monoclonal antibodies, and then reducing the disulfide bond in the hinge region to separate the monoclonal antibody arms and reassemble them into the bispecific antibody (see Figure 4 for a reaction schematic).

[0145] Specifically, the sequences of anti-CD33 and anti-CLL1 monoclonal antibodies are obtained by screening a phage library, screened for binding activity to a target protein and cells expressing the target protein, confirmed by affinity testing, and then selected to obtain anti-CD33 / CLL1 antibodies. The anti-CLL1 monoclonal antibody contains a heavy chain variable region shown in SEQ ID NO: 1 (HCDR1-3 are SEQ ID NOs: 15, 16, and 17, respectively), a heavy chain constant region shown in SEQ ID NO: 2, a light chain variable region shown in SEQ ID NO: 3 (LCDR1-3 are SEQ ID NOs: 18, 19, and 20, respectively), and a light chain constant region shown in SEQ ID NO: 4. The anti-CD33 monoclonal antibody contains a heavy chain variable region shown in SEQ ID NO: 5 (HCDR1-3 are SEQ ID NOs: 9, 10, and 11, respectively), a heavy chain constant region shown in SEQ ID NO: 6, a light chain variable region shown in SEQ ID NO: 7 (LCDR1-3 are SEQ ID NOs: 12, 13, and 14, respectively), and a light chain constant region shown in SEQ ID NO: 8. The DNA sequences of the anti-CD33 and anti-CLL1 antibodies were determined, and recombinant plasmids expressing the anti-CD33 and anti-CLL1 monoclonal antibodies, respectively (designated IB12-CD33 and IB12-CLL1 in this example), were constructed.

[0146] The construction of monoclonal cell lines was based on known techniques. The IB12-CD33 and IB12-CLL1 plasmids were transfected into CHO cells by electroporation (electrotransfection). After one round of screening of minipools and two rounds of screening after plating of monoclones, the cells were confirmed by early passage stable culture to obtain cell lines capable of stably expressing anti-CLL1 and anti-CD33 monoclonal antibodies.

[0147] To produce the anti-CD33-CLL1 bispecific antibody, cell lines stably expressing the anti-CLL1 and anti-CD33 monoclonal antibodies were obtained. After expansion and large-scale cultivation, the unprocessed cell suspension (UPB) was harvested, clarified, and filtered to obtain the cell culture supernatant, which contained the anti-CD33 and anti-CLL1 monoclonal antibodies, respectively. The anti-CD33 and anti-CLL1 monoclonal antibodies were then purified through a protein A affinity purification process, assembled into the anti-CD33-CLL1 bispecific antibody under conditions of reducing agent 2-MEA and air oxidation, and then purified through two-step anion exchange and cation exchange chromatography to obtain the anti-CD33-CLL1 bispecific antibody.

[0148] (2) NK cell production PBMCs are derived from peripheral blood lymphocyte collection from allogeneic healthy donors. After separation of T cells and red blood cells, cells are transferred to primary cell cryopreservation solution, resulting in a total of 6.0 × 10 viable cells per vial. 7 The cells are then aliquoted to give PBMCs, which are then stored for long periods in liquid nitrogen at temperatures below -175°C.

[0149] PBMCs cryopreserved in liquid nitrogen are removed, resuscitated in adapted medium, and culture scale expanded (i.e., expanded with increased medium), with the addition of NK cell-associated cytokines, including, but not limited to, IL2 and IL15, to maintain proliferation capacity and activity. Exemplary NK cell culture steps, process parameters, and process control indicators are shown in Table 1 below.

[0150] [Table 1]

[0151] Quality control for final cell purity of NK cells meets the requirements in Table 2 below (other requirements such as cell density and biosafety comply with industry standards).

[0152] [Table 2]

[0153] Figures 5 and 6 show the flow cytometry detection results of the above immunological markers in the final antibody-NK cell conjugate stock solution and formulation.

[0154] (3) Preparation of antibody linker (also referred to as L2 linker in this example) The synthetic pathway of the L2 linker is shown in Figure 8 and the structural formula is shown in Figure 9, which includes three main synthetic steps: amide condensation, hydrolysis reaction, and synthesis of the activated ester.

[0155] <Step 1> Amide condensation Dibenzoazacyclooctynyl glutaric acid (L2-1) and aminotetraethylene glycol acetylpiperidine methyl ester (L2-2) were obtained commercially and then subjected to a chemical condensation reaction to obtain the intermediate dibenzoazacyclooctynyl glutaryl aminotetraethylene glycol acetylpiperidine methyl ester (L2-3), which is a stable compound.

[0156] Specifically, compounds L2-1 and L2-2 (1:1.1, L2-2 is in excess) were dissolved in dichloromethane (DCM), and hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbonyldiimide (EDCI) were added, followed by triethanolamine (TEA). The mixture was stirred at room temperature for 4-12 hours, quenched with water, extracted twice with dichloromethane (DCM), and purified on a silica gel column (dichloromethane:methanol = 20:1) to obtain compound L2-3 as a yellow oil.

[0157] <Step 2> Hydrolysis reaction The intermediate L2-3 obtained in Step 1 was hydrolyzed to give the intermediate dibenzoazacyclooctynyl glutaryl aminotetraethylene glycol acetylpiperidine acid (L2-4). The hydrolysis reaction is usually quantitative, and the product is used directly in the next synthesis step without further purification.

[0158] Specifically, compound L2-3 was dissolved in a mixture of methanol and water, cooled to 0°C, and then 1 mol / L aqueous lithium hydroxide (LiOH) solution was added. The mixture was stirred at 0°C to room temperature for 4 to 12 hours, acidified to pH 2 to 3 with 1 mol / L hydrochloric acid, extracted three times with ethyl acetate (EA), and dried to obtain the intermediate dibenzoazacyclooctynyl glutarylaminotetraethylene glycol acetylpiperidine acid (L2-4) as a yellow oil.

[0159] <Step 3> Synthesis of activated ester The intermediate L2-4 (unpurified) obtained in Step 2 was condensed with pentafluorophenol and dicyclohexylcarbodiimide (DCC) to give L2 (i.e., dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester).

[0160] Specifically, compound L2-4 was dissolved in tetrahydrofuran (THF) and cooled to 0°C. Then, pentafluorophenol, hydroxybenzotriazole (HOBt), and dicyclohexylcarbodiimide (DCC) were added. The mixture was stirred at 0°C to room temperature for 4 to 12 hours, extracted three times with ethyl acetate (EA), dried, and purified on a silica gel column (dichloromethane:methanol = 20:1) to obtain dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester (L2) as a yellow oil (see Figure 9 for the structural formula).

[0161] (4) Production of NK cell linker (referred to as N1 linker in this example) The synthetic pathway of the N1 linker is shown in Figure 10 and its structural formula is shown in Figure 11, which includes two major synthetic steps: aminoazidoacetylation and hydroxyacetylation.

[0162] <Step 1> Aminoazide acetylation 1.2 times the amount of α-azidoacetic acid (compound 1), 2 times the amount of hydroxybenzotriazole (HOBt), and triethylamine (EtN) were added to a solution of D-galactosamine hydrochloride (compound 2) in N,N-dimethylformamide (DMF). A small amount of methanol (MeOH) was added to aid dissolution. The mixture was allowed to react at room temperature for 12 hours. The reaction mixture was then poured into dichloromethane (DCM) / methanol (MeOH), shaken to mix well, and ether was added to precipitate the oily product (compound 3). The ether layer was poured off and this process was repeated twice. The mixture was then dried under vacuum. The product was then used directly for the next step without further purification.

[0163] <Step 2> Hydroxyacetyl esterification Compound 3 obtained above was added to anhydrous pyridine (Pyr) and acetic anhydride (AcO) and 4-dimethylaminopyridine (DMAP) catalyst at room temperature. The reaction was allowed to proceed for 12 hours. The reaction was detected to be nearly complete by HPLC and then concentrated to yield a racemic mixture of tetraacetyl-N-azidoacetyl-a,b-D-galactosamine. The solid was precipitated in ethyl acetate / petroleum ether, which revealed the product was predominantly in the b-configuration (β-configuration) with a purity of over 85%. Further purification using a silica gel column (dichloromethane:methanol = 20:1) yielded the N1 product with a single enantiomer purity of over 90%. The two a,b-isomers can isomerize with each other intracellularly, and through multiple metabolic and synthetic steps, they are converted to N-azidoacetylsialic acid, which is ultimately expressed on the surface of NK cells. The structural formula of the N1 linker is shown in Figure 11.

[0164] (5) Applying an antibody linker (L2 linker) to the antibody The L2 linker and the anti-CD33-CLL1 bispecific antibody can undergo a site-specific coupling reaction in a PBS system at pH 7.2 to 7.4 (preferably HEPES at pH 7.2) (see Figure 12 for a schematic diagram). Typically, one anti-CD33-CLL1 bispecific antibody can be conjugated to one to four (preferably one to two) L2 linkers. The reaction can be stopped by adjusting the pH to approximately 5.0 to obtain an L2-conjugated antibody.

[0165] (6) Applying NK cell linker (N1 linker) to NK cells After culturing the NK cells for 15 to 16 days, N1 linker is added to the culture medium and incubated for 12 to 18 hours to obtain N1 linker-modified NK cells (also referred to as "UNK" in the examples of the present application) (see Figures 13 and 14 for the binding mechanism between the N1 linker and NK cells and the structural schematic of UNK).

[0166] (7) Conjugating antibodies with linkers to NK cells The UNK and L2 conjugated antibodies were coupled in the culture medium (see Figure 15 for the reaction mechanism) to obtain a stock solution (DS) of the antibody-NK cell conjugate. The antibody-NK cell conjugate was named "IBR733," and this name will be used in subsequent examples to represent the anti-CD33 / CLL1 bispecific antibody-NK cell conjugate.

[0167] Subsequent processes further include producing an antibody-NK cell conjugate formulation to effectively extend the stability of the antibody-NK cell conjugate. The formulation may contain components that maintain isotonicity, such as sodium chloride and human serum albumin, and may also contain components that maintain the low-temperature resistance of cells and the activity of proteins and enzymes, such as trehalose, sucrose, dextran, and DMSO.

[0168] Flow cytometry was used to detect the NK cell purity in the antibody-NK cell conjugate stock solution (Figure 5) and formulation (Figure 6), and the results were over 98%, exceeding the standard requirement of 95%. Furthermore, flow cytometry was used to detect the binding positivity rate of the antibody-NK cell conjugate stock solution and formulation using UNK as a control, and the results showed that the binding positivity rate of both the stock solution and formulation exceeded 99% (Figure 7), compared to the standard requirement of 90%.

[0169] Example 2: Comparison of basic antibody properties of conjugate IBR733 and anti-CD33-CLL1 bispecific antibody In this example, several basic antibody properties of the conjugate IBR733 prepared in Example 1 and the anti-CD33-CLL1 bispecific antibody prepared in step (1) of Example 1 were compared, and the results are shown in Table 3 below. The methodology for each test item follows conventional test methods in the art.

[0170] [Table 3]

[0171] As can be seen from the results in Table 3, conjugation of the anti-CD33-CLL1 bispecific antibody to NK cells does not significantly affect the properties of the antibody itself, and retains the desirable properties of the antibody.

[0172] Example 3: Evaluation of the in vitro cytotoxic activity of conjugate IBR733 For the evaluation of cytotoxic activity in this example, three tumor cell lines (U937, THP-1, and HL-60) that highly express CD33 and CLL1 were selected, and their previously measured CD33 and CLL1 expression levels (flow cytometry measurement) are shown in Table 4 below.

[0173] [Table 4]

[0174] First, a calcein AM release assay was used to detect the cytotoxic activity of the conjugate IBR733 against tumor cell lines U937, THP-1, and HL60 cells, as well as NK cells (obtained in step (2) of Example 1) and UNK cells (obtained in step (6) of Example 1) as controls. A maximum release control, a spontaneous release control, and a medium control were set up in the experiment. NK cells, UNK cells, and the conjugate IBR733 were each set up at three concentrations, with each concentration set up in three replicate wells. After staining the tumor cells with calcein AM, they were co-incubated with NK cells, UNK cells, and IBR733 for 4 hours, then centrifuged to collect the supernatant. The changes in fluorescence intensity were detected using a microplate reader, and the cytotoxicity rate (percentage) was calculated. The results are shown in Figures 16-18. The results showed that the cytotoxic activity of IBR733 against U937, THP-1, and HL60 cells was significantly higher than that against NK and UNK cells.

[0175] Next, we used calcein AM release assay to detect the dose-effect relationship of IBR733 on U937, THP-1, and HL60 cells. Nine concentrations of IBR733 were used, with six replicate wells per concentration. Changes in fluorescence intensity were detected using a microplate reader, and the cytotoxicity rate and EC 50 The values ​​were calculated. Several batches of IBR733 were produced at different times and tested for their cytotoxic effects on tumor cells U937, THP-1 and HL-60, and the results are shown in Table 5 below.

[0176] The results showed that multiple batches of IBR733 had significant cytotoxic effects on each of the three tumor cell lines, and that the effects were relatively stable. 50 The effector-to-target ratio (Effector-to-target ratio) was in the range of 0.6-0.8, and the maximum inhibitory rate was over 90%. 50 The effector-to-target ratio (Effector-to-target ratio) ranged from 0.8 to 1.6, and the maximum inhibitory rate was over 75%. 50 The effector-to-target ratio ranged from 0.7 to 4.0, and the maximum inhibition rate was over 80%.

[0177] [Table 5]

[0178] Example 4: Evaluation of the factor-releasing function of conjugate IBR733 In this example, we functionally investigated the cytokine secretion induced by the IBR733 conjugate. The cytokines studied were IL-2 (interleukin-2), IL-6 (interleukin-6), IL-15 (interleukin-15), IL-1β (interleukin-1β), TNFα (tumor necrosis factor α), IFNγ (interferon-γ), IL-8 (interleukin-8), CCL2 (chemokine 2), CCL3 (chemokine 3), and CCL5 (chemokine 5). Cytokines were measured using commercially available ELISA reagent kits in various cell culture supernatants according to the manufacturer's instructions.

[0179] (1) Conjugate IBR733 was co-incubated with two tumor effector cells (THP-1 and U937) at different effector-to-target ratios (1:1, 3:1, 10:1, and 30:1), and the secretion levels of IL-2, IL-6, IL-15, and IL-1β in the cell culture supernatant were measured by ELISA. The results are shown in Tables 6 and 7. The secretion levels of IL-2, IL-6, IL-15, and IL-1β were all lower than the average levels in healthy subjects. The IL-2 factor levels were all less than 120 pg / mL, showing a dose-dependent increase, and were lower than the IL-2 level in healthy subjects (9.40 ± 2.31 ng / mL). The IL-6 factor levels were all less than 2.5 pg / mL, lower than the IL-6 level in healthy subjects (60.32 ± 3.24 pg / mL). The IL-15 factor levels were all less than 0.2 pg / mL, lower than the IL-15 level in healthy subjects (13.38 ± 4.41 pg / mL). The IL-1β secretion levels were all less than 0.5 pg / mL.

[0180] [Table 6]

[0181] [Table 7]

[0182] (2) Conjugate IBR733 was co-incubated with U937 and THP-1 tumor cells at effector-to-target ratios of 30:1, 10:1, 3:1, and 1:1, and the secretion levels of TNFα and IFNγ in the cell culture supernatants were measured by ELISA. As shown in Table 8, after co-incubation of IBR733 with tumor cells, the secretion level of TNFα did not change significantly by several orders of magnitude and was lower than the average level of 22.75 ± 6.28 μg / mL in healthy subjects. The secretion level of IFNγ was positively correlated with the effector-to-target ratio. After co-incubation of IBR733 with tumor cells, the secretion level of IFNγ significantly increased, indicating a certain correlation with the tumor-damaging effect.

[0183] [Table 8]

[0184] (3) Conjugate IBR733 was co-incubated with U937 and THP-1 tumor cells at different effector-to-target ratios (1:1, 3:1, 10:1, and 30:1), and the secretion levels of CCL2, CCL3, CCL5, and IL-8 (CXCL8) in the cell culture supernatants were measured by ELISA. As shown in Tables 8 and 9, the levels of CCL3, CCL5, and IL-8 were positively correlated with the IBR733 exposure dose. CCL3, CCL5, and IL-8 may contribute to the cytotoxic effect of IBR733 conjugates on tumor cells, which is achieved by chemotaxis of related immune cells such as macrophages and leukocytes. The factor secretion level of CCL2 was negatively correlated with the exposure dose of IBR733, which may be because tumor cells could achieve migration and invasion by secreting CCL2, while NK cells suppressed the chemotactic ability of tumor cells, as evidenced by the decrease in CCL2 concentration with the increase in the effector-to-target ratio of NK cells.

[0185] [Table 9]

[0186] [Table 10]

[0187] (4) The conjugate IBR733 was co-incubated with U937 and THP-1 tumor cells at different effector-to-target ratios of 10:1, 3:1, and 1:1 (a control containing the conjugate IBR733 at the same effector-to-target ratio but without tumor cells was also established). The cell culture supernatant was collected and the secretion levels of granzyme B and perforin were detected by ELISA. As shown in Tables 11 and 12, IBR733 alone secreted a certain amount of granzyme B and perforin in a dose-dependent manner. When IBR733 was co-incubated with U937 and THP-1 cells, the secretion of granzyme B and perforin did not change significantly before and after tumor stimulation.

[0188] [Table 11]

[0189] [Table 12]

[0190] (5) Flow cytometry was used to detect changes in FasL / TRAIL secretion levels after co-incubation of IBR733 and U937 tumor cells. After co-incubation of IBR733 and U937 tumor cells for 1, 2, 3, and 4 hours, respectively, flow cytometry was used to analyze changes in TRAIL and FasL expression on the IBR733 cell surface. When IBR733 injures U937 tumor cells, FasL and TRAIL expression on the NK cell surface changed over time, initially increasing and then decreasing, but no significant correlation with dose was observed. The results are shown in Figures 19 and 20.

[0191] Example 5: Efficacy study of conjugate IBR733 on U937 tumor-bearing mouse model Pharmacodynamics (PD) To establish an in vivo efficacy model, IBR733 was used to inoculate the U973 tumor cell line, which showed good tumor suppression in vitro, into NPG mice lacking NK cell function after gene knockout. Given the lack of cytokines such as IL-15 in NPG mice, exogenous IL-15 was administered to mimic the maintenance of NK cells in vivo, thereby extending the duration of IBR733 treatment in the animals. The therapeutic effects of IBR733 on U937 (human histiocytic lymphoma) tumor-bearing NPG mice were monitored, and the dose-response relationship was examined to assess the primary pharmacodynamic effects of IBR733 in vivo tumor suppression.

[0192] Ninety-one NPG mice were inoculated with U937 cells via the tail vein. Two days after inoculation, they were randomly divided into seven groups according to body weight. Groups 1 to 7 were the vehicle control group, the anti-CD33-CLL1 bispecific antibody control group (7.5 mg / kg), the IL-15 group (0.5 μg / mouse), and the IL15 + IBR733 low-dose group (2.5 × 10 8 individual cells / kg), IL15+IBR733 medium dose group (5.0×10 8 individual cells / kg), IL15+IBR733 high dose group (1.0×10 9 individual cells / kg), IBR733 medium dose group (5.0×10 8 The total number of cells per group was 13, with 13 animals per group. IBR733 was administered via tail vein injection on days 1, 2, 8, and 9 after tumor inoculation. IL-15 (0.5 μg / animal) was administered intraperitoneally daily from days 1 to 13 after tumor inoculation. Body weight was measured once before group allocation and twice weekly after administration. Bioluminescence signals were detected, and blood samples were collected to determine the percentage of lymphocyte subsets. The last three animals in each group were not administered the drug. Blood was collected from the last three animals in each group on day 2 after tumor inoculation to determine the percentage of CD33 / CLL1 cells. The research results are shown as follows:

[0193] 1) The percentage of CD33 / CLL1 cells in the blood collected from the last three animals in each group was essentially the same, and there was no difference in the percentage of tumor cells between groups.These results suggest that the in vivo efficacy study was successful in establishing a U937 cell (human histiocytic lymphoma cell) intravenous tumor transplant model in NPG mice, and that the percentage of tumor cells was essentially the same at the time of group division.

[0194] 2) The IL-15 + IBR733 high-dose group significantly extended the survival time of the test animals compared with the vehicle control group and other groups, demonstrating a dose-dependent effect. Bioluminescence intensity of tumor cells was measured from day 7, and the mean bioluminescence intensity of tumor cells in the IL-15 + IBR733 high-dose group was reduced by approximately 13.6-fold compared with the vehicle control group, a significant difference (p<0.001), demonstrating an inhibitory effect on tumor growth.

[0195] 3) Based on mortality / moribund observations, the IL-15 + high-dose group demonstrated a significantly prolonged survival time (p<0.001). The longest survival time of the test animals was 21 days. In the vehicle control group, animal death began on day 10 and all had died by day 13. In the antibody control group, animal death began on day 12 and all had died by day 14. In the IL-15 group, animal death began on day 10 and all had died by day 12. In the IL-15 + IBR733 low-dose group, animal death began on day 10 and all had died by day 13. In the IL-15 + IBR733 medium-dose group, animal death began on day 12 and all had died by day 13. In the IBR733 medium-dose group, animal death began on day 11 and all had died by day 13. In the IL-15 + IBR733 high-dose group, animal death began on day 12 and all had died by day 21. The results are shown in Figure 21.

[0196] In vivo pharmacodynamic experiments showed that U937 cells could proliferate in NPG mice after intravenous inoculation. 9When administered intravenously in combination with 1000 cells / kg of IBR733 and IL-15 twice a week for two consecutive weeks, it significantly inhibited the proliferation of U937 cells and significantly extended the survival time of the animals. The animals in this group were in relatively good general condition, and there was no significant difference in body weight compared with other dose groups and the control group, with only a slight weight loss, reflecting better efficacy, safety, and tolerability, and providing a certain reference value for further clinical application.

[0197] Pharmacokinetics (PK) After IBR733 was injected into animals, IBR733[CD3 - CD56 + (CD33-CLL1) + ] cells (i.e., unmetabolized IBR733 cells) and conjugated antibody-depleted NK[CD3 - CD56 + (CD33-CLL1) - ] cells (i.e., IBR733-metabolizing cells). The positivity of IBR733 (CD33-CLL1-raNK) and total NK cells was detected by flow cytometry to assess the distribution and persistence of the cells in the animals.

[0198] The experimental animals were 56 NPG mice (half male and half female) as a model mouse in which human U937-luc tumors were intravenously transplanted. On day 5, 8 mice were assigned to the G1 group (vehicle control), 2 mice to the G2 group (IBR733, 5.0 × 10 7Mice were randomly assigned to one of four groups: 48 mice / mouse (iv, 1.8 μg / mouse), and 48 mice / mouse (ip, 1.8 μg / mouse) + IL-15 (1 dose, 1.8 μg / mouse) once weekly (QW). After treatment, peripheral blood, lung, spleen, and bone marrow samples were collected at the designated time points, and the percentages of IBR733 cells and total NK cells in the blood were determined using flow cytometry (FACS). Mice in the G1 group were bled at 1 hour, 3 days, 7 days, and 11 days, while mice in the G2 group were bled in batches at 1 hour, 4 hours, 8 hours, 1 day, 3 days, 7 days, and 11 days. Fresh tissue samples were collected from the lungs, spleen, and bone marrow at 1 hour, 8 hours, 1 day, 3 days, 7 days, and 11 days after treatment (eight mice per time point, half male and half female) for the G1 group and 11 days for the G2 group (eight mice per time point, half male and half female).

[0199] In a mouse model implanted intravenously with human U937-luc tumors, IBR733 and total NK cells were detected in the peripheral blood, bone marrow, lungs, and spleen 1 to 8 hours after IBR733 injection. The amount of IBR733 distributed in each tissue varied, with more significant accumulation in the lung than in the spleen. The duration of IBR733 in each tissue was approximately 168 hours. In the peripheral blood, IBR733 peaked 4 hours after administration and then gradually decreased. In the bone marrow, IBR733 levels increased continuously after administration, peaked at 72 hours, and then gradually decreased. In the lung and spleen, IBR733 levels peaked 8 hours after administration and then gradually decreased. More significant accumulation was observed in lung cells than in spleen cells. No unexpected deaths occurred during the experimental period, demonstrating good safety.

[0200] Example 6: Efficacy study of conjugate IBR733 on THP-1 tumor-bearing mouse model To establish an in vivo model for drug efficacy testing, human monocytic leukemia cells (THP-1) were inoculated into NCG mice lacking NK cell function after gene knockout. The therapeutic effects of IBR733 on THP-1 tumor-bearing NCG mice were observed, and the dose-response relationship was examined to evaluate the primary pharmacodynamic effects of IBR733 in in vivo antitumor therapy.

[0201] Screening and grouping: Forty-eight female NCG mice were given 5.0 × 10 intravenous injections of the 5.0 × 10 6 Three NCG mice were randomly selected on Day 1 and tumor cells were detected in the peripheral blood and femoral bone marrow using flow cytometry. After tumor cell detection, the mice were divided into two groups according to their weight on Day 2: P1 and P2. Experiment P1 consisted of 27 mice for tumor cell detection, and experiment P2 consisted of 18 mice for survival observation. Experiments P1 and P2 included a vehicle control group and a low-dose group (3.6–10 × 10), respectively. 8 cells / kg), high dose group (1.5×10 9 The number of mice per group was 9 in experiment P1 and 6 in experiment P2. The day of peripheral blood and tissue detection was designated as D1.

[0202] Administration method: The low-dose group and high-dose group in experiment P1 were administered the test article on D2, D5, D9, and D12, respectively, and the low-dose group and high-dose group in experiment P2 were administered the test article on D2, D5, D9, D12, D16, D19, D23, and D26, respectively, and the test article was administered by tail vein injection, and the vehicle control groups in experiments P1 and P2 were administered the formulation buffer solution according to the test article administration day, and the administration day was designated as D2.

[0203] Detection indicators: In experiment P1, blood and femoral bone marrow samples were collected on days 1, 5, 9, and 12, respectively, and THP-1 tumor cells were detected by flow cytometry. In experiment P2, all animals were observed twice daily during the experimental period, and survival time was used as the detection index. Survival curves were plotted at the end of the experiment.

[0204] The research results are shown as follows: 1) Flow cytometry results: Tumor cells were detectable in the peripheral blood and bone marrow of animals in each group on Day 1 before administration, and there was no significant difference in the proportion of tumor cells detected on Day 1 among the vehicle control group, the low-dose test product group, and the high-dose test product group, indicating that the modeling was successful. After administration, on Days 5 and 9, the number of tumor cells in the peripheral blood of each group showed a downward trend, and the number of tumor cells in the femoral bone marrow of each group showed an upward trend. However, the number of tumor cells in the low-dose and high-dose groups was all lower than that in the vehicle control group, and the number of tumor cells in the bone marrow of both the low-dose and high-dose groups was all significantly lower than that in the vehicle control group (P<0.05). The amount of tumor cell inhibition showed a positive correlation with the IBR733 cell dose.

[0205] 2) General clinical observations: In Experiment P2, all animals in the vehicle control, low-dose, and high-dose groups had died by D31, with median survival times of 20 days, 25.5 days, and 31 days, respectively. Compared with the vehicle control group, the survival time of animals in the high-dose group was significantly prolonged (P<0.01), with an extension rate of 55.00%. Compared with the low-dose group, the survival time of animals in the high-dose group was significantly prolonged (P<0.05), with an extension rate of 21.57%. The results are shown in Figure 22.

[0206] Conclusion: In vivo pharmacodynamic experiments demonstrated that human monocytic leukemia cells (THP-1) could proliferate in NCG mice after intravenous inoculation. The dose ranged from 3.6 to 10 × 10 cells / mL twice weekly for four consecutive weeks. 8 cells / kg and 1.5 × 10 9 Intravenous administration of the test product at a dose of 1.5 × 10 cells / kg inhibited the proliferation of human monocytic leukemia cells (THP-1) in animals. The amount of tumor cell inhibition was positively correlated with the dose of IBR733 cells, reaching 1.5 × 10 cells / kg. 9 Intravenous administration of the test product at 100 cells / kg significantly extended the survival time of mice.

[0207] Example 7: Clinical Trial of Conjugate IBR733 in the Treatment of Relapsed / Refractory Acute Myeloid Leukemia The study population in this clinical trial was patients with relapsed / refractory acute myeloid leukemia, a diagnosis specified for IBR733, according to the "China Guidelines for the Diagnosis and Treatment of Relapsed / Refractory Acute Myeloid Leukemia (2017 Edition)." A total of 12 patients were enrolled: 9 men (75.0%) and 3 women (25.0%). The median age was 52.5 years (range, 23-68 years). The acute myeloid leukemia (AML) FAB classification included 7 cases of AML(M2), 2 cases of AML(M5), 2 cases of AML(M1) with MDS transformation, and 1 case of AML(M1). All patients had at least one other comorbidity or medical history in addition to AML. According to literature reports, the incidence of AML increases with age, showing a significant increase from age 50, peaking between ages 60 and 69. The incidence rate in men is significantly higher than in women, and it is rare in children. Therefore, this study's target population is designed to cover major disease populations, and the subjects enrolled to date and those to be enrolled subsequently are somewhat representative, allowing the test drug to exert its maximum effect and enabling a more accurate evaluation of its efficacy. In the future, if possible, related clinical trials may be conducted in minors (under 18 years of age) as necessary.

[0208] From the perspective of study design, the primary objective is to observe safety and tolerability, and the secondary objective is to observe efficacy (treatment efficacy) indicators such as overall survival (OS) and combined complete remission rate (CR+CRi). The principle of dose escalation will be adopted to initially observe the correlation between this cell product and adverse events, and based on this, observe the desired efficacy, obtain initial data on safety and tolerability, and provide a basis for dose setting in subsequent studies.

[0209] Twelve patients enrolled in the IBR733 Injection study had previously received combination chemotherapy or targeted drug therapy for 0.5 to 4 years or more, but had not effectively controlled their disease and had developed clinical resistance to or were intolerant to chemotherapy. Nine patients had previously received chemotherapy and venetoclax targeted drug combination therapy but had failed, including one patient (01-004) who had previously received chemotherapy and two targeted drugs (gilteritinib and venetoclax).

[0210] By the data cutoff date of March 1, 2022, 12 subjects had received various cycles (ranging from 1 to 14 cycles) of IBR733 cell injections. As a result, 2 subjects achieved partial response (PR), 2 subjects had stable disease (SD), 4 subjects had progressive disease (PD), and 4 subjects were not evaluable (NE). Regarding survival, the longest survival time was 14 months, and the subjects continued to benefit. The specific efficacy evaluations of this study were as follows:

[0211] Three subjects (01-001, 01-002, and 01-008) completed 2 to 14 cycles of treatment with IBR733 cell injections, with varying degrees of symptom improvement and clinical benefit observed at different treatment cycles.

[0212] Subject 01-001 received a total of 14 cycles of treatment, with efficacy results of partial response, ongoing clinical benefit, and survival time of 14 months by the data cutoff date of March 1, 2022.

[0213] Subject 01-002 received five cycles of treatment, achieved a partial response, subsequently underwent an allogeneic bone marrow transplant, and survived for 13 months by the data cutoff date of March 1, 2022.

[0214] Subject 01-008 received two cycles of treatment and his disease was controlled and in stable condition.

[0215] Two subjects (01-005, 01-006) had elevated bone marrow blast counts before the second cycle of treatment and received combination chemotherapy. However, after treatment, the patients did not return to the hospital for reexamination and further treatment according to the treatment cycle, became unevaluable, and voluntarily withdrew from the group and chose to drop out of the study.

[0216] Three subjects (01-009, 01-011, and 01-012) experienced disease progression after one to four cycles of IBR733 cell infusion therapy. Subject 01-009 was scheduled to undergo allogeneic hematopoietic stem cell transplantation after one cycle of therapy. Subjects 01-011 and 01-012 received three and four cycles of therapy, respectively, and chose to withdraw from the study due to disease progression.

[0217] Four subjects (01-003, 01-004, 01-007, and 01-010) had clinically terminal disease, received one cycle of treatment, and progressed smoothly through the treatment course. However, due to personal reasons, they did not return to the hospital for reexamination and continued treatment according to the treatment cycle, and therefore chose to drop out of the study, making their disease unevaluable. The detailed treatment status and efficacy evaluation results of the 12 subjects are shown in Table 13 below.

[0218] [Table 13]

[0219] Example 8: IBR733 Injection in Combination with Venetoclax and Azacitidine for the Treatment of Relapsed or Refractory Acute Myeloid Leukemia This study was a single-arm, open-label clinical trial to evaluate the safety, tolerability, and initial efficacy of IBR733 injection in combination with venetoclax and azacitidine for relapsed or refractory acute myeloid leukemia. A total of three subjects with relapsed or refractory acute myeloid leukemia were enrolled in this project by the data cutoff date of April 30, 2022. After the first treatment cycle, ORR reached 100%, with two patients achieving CRi and one patient achieving MLFS. The results of the combination therapy and efficacy assessments for the subjects in this project are shown in Table 14 below.

[0220] [Table 14]

[0221] The patients enrolled in the two IIT trials were all patients with extremely advanced disease who had failed conventional treatment and experienced repeated relapses. Initial clinical responses were observed after one to multiple cycles of IBR733 injection as monotherapy or combination therapy. Existing data on the combination of IBR733 injection with venetoclax and azacitidine provide a foundation for future combination therapy. Based on the past treatment history of several relapsed / refractory subjects (Nos. 003, 004, and 008 in Example 6, and Nos. 001 and 003 in Example 7), no therapeutic benefit was observed with venetoclax and azacitidine combination therapy alone. However, all four relapsed / refractory subjects treated with IBR733 injection in combination with venetoclax and azacitidine demonstrated favorable therapeutic benefit (Example 6, No. 001, achieved PR; Example 7, Nos. 001, 002, and 003, achieved MLFS or CRi).

[0222] Example 9: Evaluation of in vitro cytotoxic activity of anti-CD33 / CLL1 bispecific antibody-natural killer cell conjugates with different L linkers An example of a linker conjugated to the antibody portion of the anti-CD33 / CLL1 bispecific antibody-natural killer cell conjugate of the invention has the following structure: [ka] [In the formula, n is an integer of 0 to 8.] Dibenzoazacyclooctynyl-glutaryl-aminopolyethylene glycol-acetylpiperidine acid pentafluorophenyl ester having the formula:

[0223] In this example, the in vitro cytotoxic activity of conjugates having three antibody moiety linkers, L1 (n=0), L2 (n=3), and L3 (n=1), was tested. The L2 linker is the linker of IBR733 prepared in the above Example 1. The preparation process of the conjugate having L1 and L3 linkers is similar to that of IBR733 in Example 1, except that different L2-2 substrates were selected for preparing the L1 and L3 linkers, and the corresponding substrates are also commercially available.

[0224] The in vitro cytotoxicity test method was similar to that in Example 3, the test cell line was U937, and NK cells (obtained in step (2) of Example 1) and UNK cells (obtained in step (6) of Example 1) were similarly selected as controls.

[0225] As is clear from the results in Figure 22, the cytotoxic activity of the conjugates having the L1, L2, and L3 linkers against U937 cells was higher than that against NK and UNK cells, respectively, and the cytotoxic activity of the conjugate having the L2 linker (i.e., IBR733) was the highest.

[0226] The use of any embodiments or exemplary language (e.g., "such as," "such as," etc.) provided herein is intended to facilitate a better understanding of the invention and does not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element required to practice the invention.

[0227] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, any theory, mechanism, demonstration, or discovery described herein is intended to further enhance the understanding of the present invention, and is not intended to limit the present invention in any way to such theory, mechanism, demonstration, or discovery. While the present invention has been shown and described in detail in the accompanying drawings and the foregoing description, it should be considered illustrative and not limiting of the present invention.

Claims

1. An antibody-natural killer cell (NK cell) conjugate, wherein the antibody is a bispecific antibody comprising a CD33 antigen-binding fragment and a CLL1 antigen-binding fragment, and the bispecific antibody is conjugated to the NK cell via a linker.

2. the CD33 antigen-binding fragment is HCDR1 as set forth in SEQ ID NO:9 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO:9; HCDR2 as set forth in SEQ ID NO: 10 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 10; HCDR3 as set forth in SEQ ID NO: 11 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 11; LCDR1 as set forth in SEQ ID NO: 12 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 12; An LCDR2 set forth in SEQ ID NO: 13 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 13, and comprising an LCDR3 as set forth in SEQ ID NO: 14 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 14; 2. The antibody-natural killer cell (NK cell) conjugate of claim 1, wherein the amino acid sequences of the HCDR and LCDR are as defined by Kabat.

3. 3. The antibody-natural killer cell (NK cell) conjugate of claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the CD33 antigen-binding fragment is set forth in SEQ ID NO: 5 or has at least 80%, 85%, 90%, 95%, or 99% identity to the sequence set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 7 or has at least 80%, 85%, 90%, 95%, or 99% identity to the sequence set forth in SEQ ID NO:

7.

4. the CLL1 antigen-binding fragment is HCDR1 as set forth in SEQ ID NO: 15 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 15; HCDR2 as set forth in SEQ ID NO: 16 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 16; HCDR3 as set forth in SEQ ID NO: 17 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 17; LCDR1 as set forth in SEQ ID NO: 18 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 18; An LCDR2 set forth in SEQ ID NO: 19 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 19, and comprising an LCDR3 as set forth in SEQ ID NO:20 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO:20; The antibody-natural killer cell (NK cell) conjugate of any one of claims 1 to 3, wherein the amino acid sequences of the HCDR and LCDR are as defined by Kabat.

5. 5. The antibody-natural killer cell (NK cell) conjugate of claim 1, wherein the amino acid sequence of the heavy chain variable region of the CLL1 antigen-binding fragment is set forth in SEQ ID NO: 1 or has at least 80%, 85%, 90%, 95%, or 99% identity to the sequence set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 3 or has at least 80%, 85%, 90%, 95%, or 99% identity to the sequence set forth in SEQ ID NO:

3.

6. The antibody-natural killer cell (NK cell) conjugate of any one of claims 1 to 5, wherein the CD33 antigen-binding fragment and the CLL1 antigen-binding fragment are in the form of a single chain antibody (scFv) or a Fab fragment.

7. the bispecific antibody comprises a CD33 antibody arm comprising a CD33 antigen-binding fragment and a CLL1 antibody arm comprising a CLL1 antigen-binding fragment; the CD33 antibody arm comprises a heavy chain variable region set forth in SEQ ID NO:5, a heavy chain constant region set forth in SEQ ID NO:6, a light chain variable region set forth in SEQ ID NO:7, and a light chain constant region set forth in SEQ ID NO:8; and / or the CLL1 antibody arm comprises a heavy chain variable region set forth in SEQ ID NO: 1, a heavy chain constant region set forth in SEQ ID NO: 2, a light chain variable region set forth in SEQ ID NO: 3, and a light chain constant region set forth in SEQ ID NO: 4; The antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 6.

8. the bispecific antibody is a whole antibody, and / or the bispecific antibody is a humanized or fully human antibody, and / or the bispecific antibody is a monoclonal antibody, and / or the bispecific antibody is of the IgG1, IgG2 or IgG4 isotype, and / or the bispecific antibody comprises a light chain constant region of the κ subtype. The antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 7.

9. The NK cells are CD16 ＋ and / or NKG2D ＋ and preferably CD16 ＋ NKG2D ＋ The antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 8, wherein

10. The conjugate CD16 ＋ NKG2D ＋ The proportion of NK cells is at least 90%, preferably CD56 ＋ The antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 9, wherein the proportion of NK cells is at least 95%.

11. The NK cells are obtained from in vitro culture and expansion of peripheral blood mononuclear cell (PBMC)-derived NK cells, or The NK cells are obtained from the in vitro culture and expansion of umbilical cord blood-derived NK cells, or The NK cells are obtained from in vitro derivation, culture, and expansion of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (ESCs); The antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 10.

12. The antibody-natural killer cell (NK cell) conjugate of any one of claims 1 to 11, wherein the bispecific antibody and the NK cell are conjugated via a click chemistry reaction of a linker.

13. 13. The antibody-natural killer cell (NK cell) conjugate of claim 12, wherein the bispecific antibody and the NK cell are conjugated via a first linker and a second linker, wherein the first linker is conjugated to the bispecific antibody, the second linker is conjugated to the NK cell, and the first linker and the second linker are conjugated to each other to form the antibody-NK cell conjugate.

14. 14. The antibody-natural killer cell (NK cell) conjugate of claim 13, wherein the first linker is an active ester capable of forming conjugation to a lysine residue of an antibody via an ester bond to amide bond reaction, the active ester being, for example, a pentafluorophenyl ester, for example, piperidine acid pentafluorophenyl ester.

15. The antibody-natural killer cell (NK cell) conjugate of claim 14, wherein the first linker further comprises a carbon-carbon triple bond structure capable of undergoing a cyclization reaction with the azide group to form a five-membered triazazole ring, for example, the carbon-carbon triple bond structure is an octyne group.

16. The first linker has the following structure: 【Chemistry 1】 [In the formula, n is an integer of 0 to 8.] 16. The antibody-natural killer cell (NK cell) conjugate of claim 15, which is dibenzoazacyclooctynyl-glutaryl-aminopolyethyleneglycol-acetylpiperidine acid pentafluorophenyl ester having the formula:

17. The first linker has the following structure: 【Chemistry 2】 17. The antibody-natural killer cell (NK cell) conjugate of claim 16, which is dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester having the formula:

18. 14. The antibody-natural killer cell (NK cell) conjugate of claim 13, wherein the second linker is an azidoacetylated cyclohexosamine, e.g., azidoacetylated cyclogalactosamine, or azidoacetylated glucosamine.

19. The second linker has the following structure: 【Transformation 3】 19. The antibody-natural killer cell (NK cell) conjugate of claim 18, wherein the conjugate is 1,3,4,6-oxo-tetraacetyl-2-azidoacetamido-2-deoxy-a,b-D-galactose having the formula:

20. 20. The antibody-natural killer cell (NK cell) conjugate of claim 19, wherein the proportion of the second linker in a single α or β configuration is at least 90%.

21. A cell population comprising the antibody-natural killer cell (NK cell) conjugate of any one of claims 1 to 20.

22. CD3 － CD56 ＋ CD16 ＋ and / or the number of cells in said cell population accounts for at least 95%, preferably at least 98%, of the total number of cells in said cell population. － CD56 ＋ NKG2D ＋ 22. The cell population of claim 21, wherein the number of cells represents at least 95%, preferably at least 98%, of the total number of cells in the cell population.

23. CD3 ＋ CD56 ＋ the number of cells accounts for 5% or less of the total number of cells in the cell population; and / or CD3 － CD19 ＋ the number of cells accounts for 2% or less of the total number of cells in the cell population; and / or CD3 ＋ CD4 ＋ and CD3 ＋ CD8 ＋ The number of cells accounts for 2% or less of the total number of cells in the cell population, The cell population of claim 21.

24. 24. The cell population of any one of claims 21 to 23, wherein the antibody-natural killer cell (NK cell) conjugate accounts for at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably at least 99% of all cells in the cell population.

25. A pharmaceutical composition comprising the antibody-natural killer cell (NK cell) conjugate of any one of claims 1 to 20 or the cell population of any one of claims 21 to 24, and a pharmaceutically acceptable carrier.

26. 26. The pharmaceutical composition of claim 25, comprising sodium chloride and / or human serum albumin.

27. 27. The pharmaceutical composition of claim 25 or 26, comprising trehalose, sucrose, dextran, DMSO, or any combination thereof.

28. A pharmaceutical composition according to any one of claims 25 to 27 for use in the treatment of acute myeloid leukemia (AML) in an individual.

29. 29. The pharmaceutical composition of claim 28 for use in the treatment of newly diagnosed acute myeloid leukemia.

30. 29. The pharmaceutical composition of claim 28 for use in the treatment of relapsed or refractory acute myeloid leukemia.

31. 31. The pharmaceutical composition of claim 30, wherein the individual has undergone chemotherapy and / or targeted drug therapy.

32. 32. The pharmaceutical composition of claim 31, wherein the targeted drug is gilteritinib and / or venetoclax.

33. 33. The pharmaceutical composition of any one of claims 25 to 32 for use in combination with venetoclax and / or azacitidine for the combination treatment of relapsed or refractory acute myeloid leukemia.

34. Use of an antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 20 or a cell population according to any one of claims 21 to 24 in the manufacture of a medicament for the treatment of acute myeloid leukemia (AML).

35. 35. The use of claim 34, wherein the medicament is for use in the treatment of initially diagnosed acute myeloid leukemia.

36. 35. The use of claim 34, wherein the medicament is for use in the treatment of relapsed or refractory acute myeloid leukemia.

37. 37. The use of claim 36, wherein the individual has undergone chemotherapy and / or targeted drug therapy.

38. 38. The use of claim 37, wherein the targeted drug is gilteritinib and / or venetoclax.

39. 39. The use of any one of claims 34 to 38, wherein the medicament is for use in combination with venetoclax and / or azacitidine for the combination treatment of relapsed or refractory acute myeloid leukemia.

40. 34. A method of treating acute myeloid leukemia (AML) in an individual, comprising administering to said individual an effective amount of an antibody-natural killer cell (NK cell) conjugate of any one of claims 1 to 20, or a cell population of any one of claims 21 to 24, or a pharmaceutical composition of any one of claims 25 to 33.

41. 41. The method of claim 40, wherein the individual is suffering from initially diagnosed acute myeloid leukemia.

42. 41. The method of claim 40, wherein the individual has relapsed or refractory acute myeloid leukemia.

43. 43. The method of claim 42, wherein the individual has undergone chemotherapy and / or targeted drug therapy.

44. 44. The method of claim 43, wherein the targeted drug is gilteritinib and / or venetoclax.

45. 45. The method of any one of claims 40-44, further comprising administering to the individual venetoclax and / or azacitidine for combination treatment of relapsed or refractory acute myeloid leukemia.

Images