Method for modifying cells and use thereof

Abstract

Provided is a method for preparing a modified cell. The method comprises: 1) contacting a monosaccharide derivative containing a first functional group with an immune cell to obtain an immune cell on which surface the monosaccharide derivative containing the first functional group is expressed; 2) contacting an antigen binding protein with a compound containing a second functional group to obtain the antigen binding protein containing the second functional group; and / or, 3) contacting the immune cell on which surface the monosaccharide derivative containing the first functional group is expressed with the antigen binding protein containing the second functional group, such that the first functional group and the second functional group are connected and reacted to obtain the cell modified with the antigen binding protein on the surface. Also provided are a cell prepared according to the method, and the use thereof in treating diseases.

Description

A method for modifying cells and its application Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a method for modifying cells through sugar metabolism labeling and its application. Background Art

[0002] Cancer incidence and mortality rates are rising annually. With the continued exploration and application of human immunity, immunotherapy research has become increasingly popular. Immune cell therapy, which activates and enhances the immune system to kill tumors, has become the fourth pillar of cancer treatment. Adoptive immune cell therapy, such as chimeric antigen receptor T (CAR-T) cells, has achieved significant results in combating blood cancers, but it is characterized by high production costs, lengthy preparation times, and complex processes.

[0003] Antibody-cell drug conjugates (ACCs) can couple antibodies to the cell surface to improve the targeting of immune cells, thereby specifically killing tumor cells. However, cell therapies, especially allogeneic cell therapies, often lead to graft-versus-host disease (GVHD). Current methods for coupling immune cells to antibodies have long preparation cycles and unstable product quality, failing to meet the needs of rapidly progressive cancer patients. Furthermore, there are concerns about off-target effects in tumor tissue and tumor immune escape.

[0004] Therefore, the development of a new, convenient, and low-cost immune cell-antibody labeling technology that can selectively kill tumor cells without damaging normal cells will greatly promote immune cell therapy.

[0005] Summary of the Invention

[0006] The present application provides a method for preparing modified cells, by metabolic labeling of non-natural sugar molecules, marking molecules targeting tumor antigens on the surface of immune cells through a ligation reaction, thereby enhancing the killing activity of immune cells against tumor cells. The method of the present application can quickly and efficiently modify tumor antigen targeting molecules (e.g., antibodies) on the surface of immune cells (e.g., γδT cells or NK cells). The method of the present application has high labeling efficiency, short cycle, and low economic cost. The modified cells prepared according to the method of the present application have at least one of the following beneficial effects: (1) uniform and stable quality, (2) enhanced enrichment in tumors, (3) low off-target effects and low toxic side effects, (4) strong killing ability on target cells, and (4) significant inhibition of tumor cell growth. The methods, cells, pharmaceutical compositions, etc. provided in the present application have great clinical application value.

[0007] In one aspect, the present application provides a method for preparing a modified cell, comprising step S: contacting an immune cell expressing a monosaccharide derivative comprising a first functional group on its surface with an antigen-binding protein comprising a second functional group, so that the first functional group and the second functional group undergo a ligation reaction to obtain the modified cell; wherein the monosaccharide derivative comprises any one or more structures selected from Formula I to Formula X: Wherein, X is the first functional group, and R1, R2, R3, R4, R5, and R6 are each independently selected from protecting groups that can be hydrolyzed or enzymatically degraded in cells. The purpose is to accelerate the entry of non-natural sugars into cells and expose the hydroxyl groups on the sugars after deprotection. Therefore, any group that meets this function falls within the scope of the present invention.

[0008] In certain embodiments, R1, R2, R3, R4, R5 and R6 are each independently selected from the group consisting of hydrogen, halogen, alkanyl, cycloalkyl, heterocycloalkylalkenyl, alkynyl, aryl, polyethylene glycol, alkylthio, alkylcarbonyl, alkoxycarbonyl and acyl, which are optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, amino, nitro, thiol and C 1-6 In some further embodiments, R1, R2, R3, R4, R5 and R6 are each independently selected from the group consisting of hydrogen, halogen, C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy, C 2～6 Alkoxycarbonyl and acyl, said group being optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxy, amino, nitro, mercapto and C 1-6 alkyl.

[0009] In some embodiments, R1, R2, R3, and R4 in Formula I and Formula II may be the same, for example, selected from acyl and alkoxycarbonyl. In other embodiments, R1, R2, R3, and R4 in Formula I and Formula II may be different, for example, each independently selected from acyl and alkoxycarbonyl.

[0010] In certain embodiments, the monosaccharide derivative comprises any one or more structures selected from the following group: Wherein, X is the first functional group, R1 and R2 are each independently selected from the group consisting of hydrogen, halogen, chain alkyl, cycloalkyl, heterocycloalkyl alkenyl, alkynyl, aryl, polyethylene glycol, alkylthio, alkylcarbonyl, alkoxycarbonyl and acyl, and the group is optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, thiol and C 1-6 In some further embodiments, R1 and R2 are each independently selected from the group consisting of hydrogen, halogen, C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy, C 2～6 Alkoxycarbonyl and acyl, said group being optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxy, amino, nitro, mercapto and C 1-6 In some further embodiments, R1 and R2 are the same, for example, selected from acetyl, propionyl and butyryl. In some further embodiments, R1 and R2 are different, for example, each independently selected from acetyl, propionyl and butyryl.

[0011] In certain embodiments, the monosaccharide derivative comprises any one or more structures selected from the following group: Wherein, X is the first functional group.

[0012] In certain embodiments, the method further comprises step S1: contacting the monosaccharide derivative comprising the first functional group with immune cells to obtain the immune cells expressing the monosaccharide derivative comprising the first functional group on their surfaces.

[0013] In certain embodiments, the method further comprises step S2: contacting the antigen binding protein with a compound comprising a second functional group to obtain the antigen binding protein comprising the second functional group.

[0014] In certain embodiments, the first functional group and / or the second functional group is a group having bioorthogonal reactivity.

[0015] In certain embodiments, the bioorthogonal reaction comprises at least one selected from the group consisting of click chemistry, photocrosslinking, photosensitization, and glycosylation.

[0016] In certain embodiments, the first functional group includes one or more selected from the group consisting of an azide group, an alkynyl group, a trans-cyclooctene group, a triazine group, a tetrazine group, a hydrazide group, a cyclopropene group, an isocyanate group, and a cyclooctyne group.

[0017] In certain embodiments, the second functional group includes one or more selected from the group consisting of an azide group, an alkynyl group, a trans-cyclooctene group, a triazine group, a tetrazine group, a hydrazide group, a cyclopropene group, an isocyanate group, and a cyclooctyne group.

[0018] In certain embodiments, R1, R2, R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy, C 2～6 Alkoxycarbonyl and acyl, said groups being optionally substituted by substituents selected from the group consisting of halogen, hydroxy, amino, nitro, mercapto and C 1-6 Alkyl; R5 is selected from the group consisting of hydrogen, halogen, and substituted or unsubstituted C 1～6 Alkyl, the alkyl group is optionally substituted by a substituent selected from the group consisting of halogen, hydroxy, amino, nitro and mercapto; R6 and R7 are each independently selected from the group consisting of halogen and substituted or unsubstituted C 1～10 Alkyl, the alkyl group is optionally substituted by a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto; R8 is selected from the group consisting of halogen, substituted or unsubstituted C 1～10 Alkyl, phenyl and substituted or unsubstituted C 1～10 Alkylene-phenyl, the alkyl group is optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto; Z1 and Z2 are each independently selected from the group consisting of O and S; and n and m are each independently selected from the group consisting of integers consisting of 1, 2, 3, 4, 5, and 6.

[0019] In certain embodiments, R1 is selected from the group consisting of hydrogen and -C(=O)-C1~6 alkyl. For example, R1 is hydrogen or -C(=O)-CH3. In certain embodiments, R2 is selected from the group consisting of hydrogen and -C(=O)-C1~6 alkyl. For example, R2 is hydrogen or -C(=O)-CH3. In certain embodiments, R3 is selected from the group consisting of hydrogen and -C(=O)-C1~6 alkyl. For example, R3 is hydrogen or -C(=O)-CH3. In certain embodiments, R4 is selected from the group consisting of hydrogen and -C(=O)-C1~6 alkyl. For example, R4 is hydrogen or -C(=O)-CH3. In certain embodiments, R5 is selected from the group consisting of hydrogen and substituted or unsubstituted C 1～6 In certain embodiments, R5 is hydrogen or substituted or unsubstituted C 1～3 For example, R4 is hydrogen or -CH3. In certain embodiments, R6 is selected from the group consisting of hydrogen and substituted or unsubstituted C 1～10 alkyl.

[0020] In certain embodiments, R7 is selected from the group consisting of hydrogen and substituted or unsubstituted C 1～10 In certain embodiments, R8 is selected from the group consisting of methyl, ethyl, and -CH2-phenyl.

[0021] In certain embodiments, m is 1, 2, or 3.

[0022] In certain embodiments, n is 1, 2, or 3.

[0023] In certain embodiments, in Formula I, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl, R2 is selected from hydrogen and -C(=O)-C1~6 alkyl, R3 is selected from hydrogen and -C(=O)-C1~6 alkyl, R4 is selected from hydrogen and -C(=O)-C1~6 alkyl, and R1, R2, R3 and R4 are not hydrogen at the same time.

[0024] In certain embodiments, in Formula II, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl, R2 is selected from hydrogen and -C(=O)-C1~6 alkyl, R3 is selected from hydrogen and -C(=O)-C1~6 alkyl, R4 is selected from hydrogen and -C(=O)-C1~6 alkyl, and R1, R2, R3 and R4 are not hydrogen at the same time.

[0025] In certain embodiments, in Formula III, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl, R2 is selected from hydrogen and -C(=O)-C1~6 alkyl, R3 is selected from hydrogen and -C(=O)-C1~6 alkyl, R4 is selected from hydrogen and -C(=O)-C1~6 alkyl, and R1, R2, R3 and R4 are not hydrogen at the same time.

[0026] In certain embodiments, in Formula IV, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R6 is selected from C1~ 10 Alkyl, R7 is selected from C1~ 10 Alkyl, Z1 is selected from O and S, Z2 is selected from O and S, n is 1, 2 or 3, m is 1, 2 or 3, and R1, R2 and R3 are not hydrogen at the same time.

[0027] In certain embodiments, in Formula V, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R8 is selected from: C1-C3 alkyl, phenyl, C1-C3 alkylene-phenyl, and R1, R2 and R3 are not hydrogen at the same time.

[0028] In certain embodiments, in Formula VI, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and C1~6 alkyl (preferably -CH3), and R1, R2, R3 and R4 are not hydrogen at the same time, or R1, R2, R3, R4, R5 are not hydrogen at the same time. In a specific embodiment, at most four of R1, R2, R3, R4, and R5 are hydrogen.

[0029] In certain embodiments, in Formula VII, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and C1~6 alkyl (preferably -CH3), and R1, R2, R3 and R4 are not hydrogen at the same time, or R1, R2, R3, R4, R5 are not hydrogen at the same time. In a specific embodiment, at most four of R1, R2, R3, R4, and R5 are hydrogen.

[0030] In certain embodiments, in Formula VIII, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and C1~6 alkyl (preferably -CH3), and R1, R2, R3 and R4 are not hydrogen at the same time, or R1, R2, R3, R4, R5 are not hydrogen at the same time. In a specific embodiment, at most four of R1, R2, R3, R4, and R5 are hydrogen.

[0031] In certain embodiments, in Formula VI, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and C1~6 alkyl (preferably -CH3), and R1, R2, R3 and R4 are not hydrogen at the same time, or R1, R2, R3, R4, R5 are not hydrogen at the same time. In a specific embodiment, at most four of R1, R2, R3, R4, and R5 are hydrogen.

[0032] In certain embodiments, in Formula V, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), and R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3). In a specific embodiment, R1, R2, R3 and R4 are all hydrogen. In a specific embodiment, R1, R2, R3 and R4 are all -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3).

[0033] In certain embodiments, the monosaccharide derivative comprising a first functional group is selected from any one of the following Table A:

[0034] Table A

[0035] In certain embodiments, the first functional group is an azido group, and the second functional group is a cyclooctyne group. In certain embodiments, the first functional group is an azido group, and the second functional group is selected from the group consisting of OCT, ALO, MOFO, DBCO, DIFO, DIMAC, and BCN. In certain embodiments, the first functional group is an azido group, and the second functional group is

[0036] In certain embodiments, the monosaccharide derivative is selected from any one of the following groups:

[0037] In certain embodiments, the compound comprising a second functional group further comprises a group capable of covalently linking to a thiol group. Preferably, the group capable of covalently linking to a thiol group is a succinimide active ester group, a maleimide group, or a maleic anhydride group.

[0038] In certain embodiments, step S2 comprises contacting the antigen binding protein with a compound comprising the structure represented by Formula L1, Formula L2 or Formula L3 to obtain the antigen binding protein comprising the second functional group:

[0039] Wherein, Y represents the second functional group, R L Selected from the group consisting of polyethylene glycol, amide group and alkyl ether group, preferably, R L Selected from C 1～20 Alkylene or C 1～20 Alkylene ether group.

[0040] In certain embodiments, the compound of formula L1 is DBCO-PEG4-NHS: In certain embodiments, the compound of formula L2 is DBCO-PEG4-MAL:

[0041] In certain embodiments, the immune cells include NK cells, DC cells, NKT cells, monocytes, macrophages, T cells and / or B cells. In certain embodiments, the immune cells are NK cells. In certain embodiments, the immune cells are T cells, particularly γδT cells. In certain embodiments, the γδT cells include Vγ9Vδ2 and / or Vδ1T cells.

[0042] In certain embodiments, the antigen binding protein comprises an antibody or an antigen binding fragment thereof.

[0043] In certain embodiments, the antibody includes a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0044] In certain embodiments, the antigen binding protein comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0045] In certain embodiments, the antigen binding protein is capable of specifically binding to a tumor antigen. In certain embodiments, the tumor antigen comprises a tumor-associated antigen and / or a tumor-specific antigen. In certain embodiments, the tumor antigen is selected from the group consisting of CD20 and / or claudin 18.2.

[0046] In certain embodiments, the antigen-binding protein is an anti-CD20 antibody. For example, the anti-CD20 antibody is rituximab. For example, the anti-CD20 antibody comprises HCDRs 1 to 3 of rituximab. For example, the anti-CD20 antibody comprises LCDRs 1 to 3 of rituximab. For example, the anti-CD20 antibody comprises the light chain variable region of rituximab. For example, the anti-CD20 antibody comprises the heavy chain variable region of rituximab.

[0047] In certain embodiments, the antigen-binding protein is an anti-claudin 18.2 antibody. For example, the anti-claudin 18.2 antibody is the hu7v3 antibody. For example, the anti-claudin 18.2 antibody comprises HCDRs 1 to 3 of the hu7v3 antibody. For example, the anti-claudin 18.2 antibody comprises the heavy chain variable region of the hu7v3 antibody.

[0048] In certain embodiments, the immune cells are derived from a human sample. In certain embodiments, the immune cells are derived from human peripheral blood.

[0049] In certain embodiments, in step S of the method, the contact time between the immune cells expressing the monosaccharide derivative comprising the first functional group on their surface and the antigen-binding protein comprising the second functional group is about 10 minutes to 60 minutes, for example, about 20 minutes to 40 minutes, or about 30 minutes. In certain embodiments, in step S of the method, the concentration of the second functional group contained in the antigen-binding protein is about 10-30 μM, for example, about 20 μM, and the density of the immune cells expressing the monosaccharide derivative comprising the first functional group on their surface does not exceed 1×10 7 pieces / mL.

[0050] In certain embodiments, in step S1 of the method, the contact time between the monosaccharide derivative comprising the first functional group and the immune cells is about 2 hours to 48 hours, for example, about 16 hours to 32 hours, for example, about 24 hours.

[0051] In certain embodiments, in step S2 of the method, the contact time between the antigen binding protein and the compound comprising the structure represented by Formula L1 and / or Formula L2 is about 30 minutes to 6 hours, for example, about 1 hour to 2 hours, for example, about 1 hour.

[0052] In another aspect, the present application provides modified cells prepared according to the method. In certain embodiments, the modified cells are antibody-modified γδT cells.

[0053] In another aspect, the present application provides a modified cell, wherein the modified cell is linked to an antigen-binding protein on its surface via a monosaccharide derivative containing an azide group, wherein the structure of the monosaccharide derivative containing an azide group is selected from one or more of the following groups:

[0054] Wherein, X is an azide group (-N3),

[0055] R1, R2, R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy, C 2～6 Alkoxycarbonyl and acyl, said groups being optionally substituted by substituents selected from the group consisting of halogen, hydroxy, amino, nitro, mercapto and C 1-6 alkyl;

[0056] R5 is selected from the group consisting of hydrogen, halogen, and substituted or unsubstituted C 1～6 an alkyl group optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto;

[0057] R6 and R7 are each independently selected from the group consisting of halogen and substituted or unsubstituted C 1～10 an alkyl group optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto;

[0058] R8 is selected from the group consisting of halogen, substituted or unsubstituted C 1～10 Alkyl, phenyl and substituted or unsubstituted C 1～10 Alkylene-phenyl, said alkyl being optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto;

[0059] Z1 and Z2 are each independently selected from: O and S;

[0060] n and m are each independently selected from the following integers: 1, 2, 3, 4, 5 and 6.

[0061] In certain embodiments, R1 is selected from the group consisting of hydrogen and -C(=O)-C1-6 alkyl. For example, R1 is hydrogen or -C(=O)-CH3.

[0062] In certain embodiments, R2 is selected from the group consisting of hydrogen and -C(=O)-C1-6 alkyl. For example, R2 is hydrogen or -C(=O)-CH3.

[0063] In certain embodiments, R3 is selected from the group consisting of hydrogen and -C(=O)-C1-6 alkyl. For example, R3 is hydrogen or -C(=O)-CH3.

[0064] In certain embodiments, R4 is selected from the group consisting of hydrogen and -C(=O)-C1-6 alkyl. For example, R4 is hydrogen or -C(=O)-CH3.

[0065] In certain embodiments, R5 is selected from the group consisting of hydrogen and substituted or unsubstituted C 1～6 In certain embodiments, R5 is hydrogen or substituted or unsubstituted C 1～3 Alkyl. For example, R4 is hydrogen or -CH3.

[0066] In certain embodiments, R6 is selected from the group consisting of hydrogen and substituted or unsubstituted C 1～10 alkyl.

[0067] In certain embodiments, R7 is selected from the group consisting of hydrogen and substituted or unsubstituted C 1～10 alkyl.

[0068] In certain embodiments, R8 is selected from the group consisting of methyl, ethyl, and -CH2-phenyl.

[0069] In certain embodiments, m is 1, 2, or 3.

[0070] In certain embodiments, n is 1, 2, or 3.

[0071] In certain embodiments, in Formula I, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl, R2 is selected from hydrogen and -C(=O)-C1~6 alkyl, R3 is selected from hydrogen and -C(=O)-C1~6 alkyl, R4 is selected from hydrogen and -C(=O)-C1~6 alkyl, and R1, R2, R3 and R4 are not hydrogen at the same time.

[0072] In certain embodiments, in Formula II, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl, R2 is selected from hydrogen and -C(=O)-C1~6 alkyl, R3 is selected from hydrogen and -C(=O)-C1~6 alkyl, R4 is selected from hydrogen and -C(=O)-C1~6 alkyl, and R1, R2, R3 and R4 are not hydrogen at the same time.

[0073] In certain embodiments, in Formula III, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl, R2 is selected from hydrogen and -C(=O)-C1~6 alkyl, R3 is selected from hydrogen and -C(=O)-C1~6 alkyl, R4 is selected from hydrogen and -C(=O)-C1~6 alkyl, and R1, R2, R3 and R4 are not hydrogen at the same time.

[0074] In certain embodiments, in Formula IV, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R6 is selected from C1~ 10 Alkyl, R7 is selected from C1~ 10 Alkyl, Z1 is selected from O and S, Z2 is selected from O and S, n is 1, 2 or 3, m is 1, 2 or 3, and R1, R2 and R3 are not hydrogen at the same time.

[0075] In certain embodiments, in Formula V, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R8 is selected from: C1-C3 alkyl, phenyl, C1-C3 alkylene-phenyl, and R1, R2 and R3 are not hydrogen at the same time.

[0076] In certain embodiments, in Formula VI, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and C1~6 alkyl (preferably -CH3), and R1, R2, R3 and R4 are not hydrogen at the same time, or R1, R2, R3, R4, R5 are not hydrogen at the same time. In a specific embodiment, at most four of R1, R2, R3, R4, and R5 are hydrogen.

[0077] In certain embodiments, in Formula VII, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and C1~6 alkyl (preferably -CH3), and R1, R2, R3 and R4 are not hydrogen at the same time, or R1, R2, R3, R4, R5 are not hydrogen at the same time. In a specific embodiment, at most four of R1, R2, R3, R4, and R5 are hydrogen.

[0078] In certain embodiments, in Formula VIII, R1 is selected from hydrogen and -C(=O)-C1-6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1-6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1-6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and -C(=O)-C1-6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and C1-6 alkyl (preferably -CH3), and R1, R2, R3 and R4 are not hydrogen at the same time, or R1, R2, R3, R4, R5 are not hydrogen at the same time. In a specific embodiment, up to four of R1, R2, R3, R4, and R5 are hydrogen.

[0079] In certain embodiments, in Formula VI, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and C1~6 alkyl (preferably -CH3), and R1, R2, R3 and R4 are not hydrogen at the same time, or R1, R2, R3, R4, R5 are not hydrogen at the same time. In a specific embodiment, at most four of R1, R2, R3, R4, and R5 are hydrogen.

[0080] In certain embodiments, in Formula V, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), and R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3). In a specific embodiment, R1, R2, R3 and R4 are all hydrogen. In a specific embodiment, R1, R2, R3 and R4 are all -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3).

[0081] In certain embodiments, the monosaccharide derivative comprising a first functional group is selected from any one of Table A.

[0082] In certain embodiments, the immune cells include NK cells, DC cells, NKT cells, monocytes, macrophages, T cells and / or B cells. In certain embodiments, the immune cells are NK cells. In certain embodiments, the immune cells are T cells, particularly γδT cells. In certain embodiments, the γδT cells include Vγ9Vδ2 and / or Vδ1T cells.

[0083] In certain embodiments, the antigen binding protein comprises an antibody or an antigen binding fragment thereof.

[0084] In certain embodiments, the antibody includes a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0085] In certain embodiments, the antigen binding protein comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0086] In certain embodiments, the antigen binding protein can specifically bind to a tumor antigen. In certain embodiments, the tumor antigen includes a tumor-associated antigen and / or a tumor-specific antigen.

[0087] In some embodiments, the antigen binding protein can specifically bind to a tumor antigen selected from the group consisting of CEA (carcinoembryonic antigen), CA-125 (cancer antigen 125), CA-19-9 (cancer antigen 19-9), CA-15-3 (cancer antigen 15-3), PSA (prostate specific antigen), HER2 (human epidermal growth factor receptor 2), EGFR (epidermal growth factor receptor), PD-L1 (programmed death ligand 1), CTLA-4 (cytotoxic T lymphocyte-associated protein 4), CD56 (neural cell adhesion molecule), MUC1 (mucin 1), Glypican-3 (glycoprotein 3), WT1 (Wilms tumor protein 1), NY-ESO-1 (New York esophageal squamous cell carcinoma 1), 5T4 (tumor-associated antigen 5T4), α-Fetoprotein (alpha-fetoprotein), Gastrin-releasing peptide (gastrin-releasing peptide), Carcinoembryonic Antigen (carcinoembryonic antigen), HER2 (human epidermal growth factor receptor 2), EGFR (epidermal growth factor receptor), PD-L1 (programmed death ligand 1), CTLA-4 (cytotoxic T lymphocyte-associated protein 4), CD20 (B lymphocyte surface antigen), CD30 (activated lymphocyte surface antigen), CD56 (neural cell adhesion molecule), CD38 (monoclonal antibody target on the T cell surface), CD19 (monoclonal antibody target on the B lymphocyte surface), CD33 (monoclonal antibody target on the myeloid cell surface), CD70 (T cell co-stimulatory molecule), CD47 (immunosuppressive molecule), CD117 (c-Kit, stem cell factor receptor), CD44 (cell surface adhesion molecule), CD123 (IL-3 receptor α chain), CD52 (monoclonal antibody target on the T cell surface), CD34 (monoclonal antibody target on the hematopoietic stem cell surface), CD138 (B cell maturation antigen), CD79b (B cell surface antigen) and BCMA (B cell maturation antigen).

[0088] In certain embodiments, the tumor antigen is selected from the group consisting of CD20 and / or claudin18.2.

[0089] In certain embodiments, the antigen-binding protein comprises an antibody heavy chain variable region HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, or the HCDR3 comprises an amino acid sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. For example, the CDRs utilize the Kobat numbering scheme.

[0090] In certain embodiments, the antigen-binding protein comprises an antibody heavy chain variable region HCDR2, wherein the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, or the HCDR2 comprises an amino acid sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. For example, the CDRs utilize the Kobat numbering scheme.

[0091] In certain embodiments, the antigen-binding protein comprises an antibody heavy chain variable region HCDR1, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, or the HCDR1 comprises an amino acid sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. For example, the CDRs utilize the Kobat numbering scheme.

[0092] In certain embodiments, the antigen-binding protein comprises an antibody heavy chain variable region, which comprises the amino acid sequence shown in SEQ ID NO: 4, or the antibody heavy chain variable region comprises an amino acid sequence having more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 4.

[0093] In certain embodiments, the antigen-binding protein comprises an antibody light chain variable region LCDR3, wherein the LCDR3 comprises the amino acid sequence of SEQ ID NO: 7, or the LCDR3 comprises an amino acid sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to the amino acid sequence of SEQ ID NO: 7. For example, the CDRs utilize the Kobat numbering scheme.

[0094] In certain embodiments, the antigen-binding protein comprises an antibody light chain variable region LCDR2, wherein the LCDR2 comprises the amino acid sequence of SEQ ID NO: 6, or the LCDR2 comprises an amino acid sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to the amino acid sequence of SEQ ID NO: 6. For example, the CDRs utilize the Kobat numbering scheme.

[0095] In certain embodiments, the antigen-binding protein comprises an antibody light chain variable region LCDR1, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 5, or the LCDR1 comprises an amino acid sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to the amino acid sequence of SEQ ID NO: 5. For example, the CDRs utilize the Kobat numbering scheme.

[0096] In certain embodiments, the antigen-binding protein comprises an antibody light chain variable region, which comprises the amino acid sequence shown in SEQ ID NO: 8, or the antibody light chain variable region comprises an amino acid sequence having more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0097] In certain embodiments, the antigen binding protein comprises rituximab.

[0098] In certain embodiments, the antigen-binding protein comprises an antibody heavy chain variable region HCDR3, wherein the HCDR3 comprises the amino acid sequence of SEQ ID NO: 11, or the HCDR3 comprises an amino acid sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to the amino acid sequence of SEQ ID NO: 11. For example, the CDRs utilize the IMGT numbering scheme.

[0099] In certain embodiments, the antigen-binding protein comprises an antibody heavy chain variable region HCDR2, wherein the HCDR2 comprises the amino acid sequence of SEQ ID NO: 10, or the HCDR2 comprises an amino acid sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to the amino acid sequence of SEQ ID NO: 10. For example, the CDRs utilize the IMGT numbering scheme.

[0100] In certain embodiments, the antigen-binding protein comprises an antibody heavy chain variable region HCDR1, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, or the HCDR1 comprises an amino acid sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. For example, the CDRs utilize the IMGT numbering scheme.

[0101] In certain embodiments, the antigen-binding protein comprises an antibody heavy chain variable region, which comprises the amino acid sequence shown in SEQ ID NO: 12, or the antibody heavy chain variable region comprises an amino acid sequence having more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 12.

[0102] In certain embodiments, the antigen binding protein is a VHH.

[0103] In certain embodiments, the antigen-binding protein comprises an antibody heavy chain, which comprises the amino acid sequence shown in SEQ ID NO: 13, or the antibody heavy chain comprises an amino acid sequence having more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 13.

[0104] In certain embodiments, the antigen binding protein comprises an Fc, for example, an Fc from IgG1.

[0105] In certain embodiments, the antigen binding protein is a fusion protein of VHH and Fc.

[0106] In certain embodiments, the antigen binding protein comprises a Hu7v3 antibody.

[0107] In certain embodiments, the cell has an azide group expressed on its surface via a sugar metabolism pathway by taking up the monosaccharide derivative.

[0108] In certain embodiments, the cell is linked to the antigen binding protein with a second functional group via the azide group of the monosaccharide derivative, preferably via a bond formed by a linking reaction between the azide group and the second functional group.

[0109] In certain embodiments, the second functional group comprises

[0110] In certain embodiments, the structure of is shown in Formula L1, Formula L2 or Formula L3:

[0111] Wherein, Y is the second functional group, R L Selected from the group consisting of polyethylene glycol, amide group and alkyl ether group, preferably, R L Selected from C 1～20 Alkylene or C 1～20 Alkylene ether group.

[0112] In certain embodiments, the structure of the second functional group is selected from the group consisting of:

[0113] In another aspect, the present application provides a pharmaceutical composition comprising the modified cells, and optionally a pharmaceutically acceptable adjuvant.

[0114] On the other hand, the present application provides use of the modified cells in preparing a drug for treating and / or alleviating tumors.

[0115] In another aspect, the present application provides a method for treating and / or alleviating tumors, comprising administering the modified cells to a subject in need thereof.

[0116] In certain embodiments, the tumor comprises a solid tumor. In certain embodiments, the tumor comprises a hematological tumor.

[0117] In certain embodiments, the tumor comprises lung cancer, breast cancer, colorectal cancer, prostate cancer, stomach cancer, liver cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma, lymphoma, leukemia, brain tumor, bone cancer, kidney cancer, bladder cancer, esophageal cancer, nasopharyngeal cancer, melanoma, skin cancer, laryngeal cancer, oral cancer, tongue cancer, gallbladder cancer, bile duct cancer, skin cancer, testicular cancer, uterine cancer, endometrial cancer, renal pelvis cancer, renal cell carcinoma, bladder cancer, glioma, neuroblastoma, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, myeloid leukemia, myelodysplastic syndrome, soft tissue sarcoma, osteosarcoma, liposarcoma, neurofibrosarcoma, angiosarcoma, gastrointestinal stromal tumor, thymoma, thymic carcinoma, pituitary tumor, retinoblastoma and / or glioblastoma.

[0118] In certain embodiments, the tumor comprises a tumor that overexpresses CD20. In certain embodiments, the tumor comprises a lymphoma. In certain embodiments, the tumor comprises a B-lymphoma.

[0119] In certain embodiments, the tumor comprises a tumor that highly expresses claudin 18.2. In certain embodiments, the tumor comprises gastric cancer, colon cancer, esophageal cancer, and / or pancreatic cancer.

[0120] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0122] FIG1 is a schematic diagram showing the process of cell preparation in the present application.

[0123] Figures 2 to 7 show the structures of exemplary non-natural sugars.

[0124] FIG8 shows the flow cytometry statistics of γδT cells after incubation with 42 sugars for 48 hours.

[0125] FIG9 shows a flow cytometric statistical graph of rituximab labeled after γδT cells were incubated with 42 sugars.

[0126] FIG10 shows the flow cytometry statistics of Hu7v3 labeled after γδT cells were incubated with 42 sugars.

[0127] FIG11 shows a statistical graph of the killing detection of γδT cells after incubation with 42 sugars and labeling with rituximab.

[0128] FIG12 shows the statistical graph of Hu7v3 killing detection after γδT cells were incubated with 42 sugars.

[0129] FIG13 shows the flow cytometry statistics of T cells after incubation with 42 sugars for 48 hours.

[0130] FIG14 shows a flow cytometric statistical graph of rituximab labeled after T cells were incubated with 42 sugars.

[0131] FIG15 shows a flow cytometry statistical graph of Hu7v3 labeled after T cells were incubated with 42 sugars.

[0132] FIG16 shows a statistical graph of T cells incubated with 42 sugars and labeled with rituximab for killing assay.

[0133] FIG17 shows a statistical graph of Hu7v3 killing detection after T cells were incubated with 42 sugars.

[0134] FIG18 shows the flow cytometry statistics of NK cells after incubation with 42 sugars for 48 hours.

[0135] FIG19 shows the flow cytometry statistics of NK cells incubated with 42 sugars and labeled with rituximab.

[0136] FIG20 shows the flow cytometry statistics of Hu7v3 labeled after NK cells were incubated with 42 sugars.

[0137] FIG21 shows a statistical graph of NK cell killing detection after incubation with 42 sugars and labeling with rituximab.

[0138] FIG22 shows the statistical graph of Hu7v3 killing detection after NK cells were incubated with 42 kinds of sugars.

[0139] Figure 23 shows the bioluminescent imaging of γδT cells after administration to mice with CD20 antibodies linked to carbohydrates No. 20, 22, and 25. PBS is the solvent control group, and V2 is the unlabeled antibody control group.

[0140] FIG24 shows the survival rate of mice after administration of γδT cells to which CD20 antibodies were linked via carbohydrates Nos. 20, 22, and 25, and PBS was used as the control group.

[0141] Figure 25 shows the bioluminescent imaging of γδT cells after administration to mice with CD20 antibodies linked to carbohydrates 3, 29, 23, 5 and 15, PBS is the solvent control group, and V2 is the unlabeled antibody control group.

[0142] FIG26 shows the survival rate of mice after administration of γδT cells with CD20 antibodies linked to carbohydrates 3, 29, 23, 5, and 15 to mice, with PBS serving as the control group.

[0143] Figure 27 shows the bioluminescent imaging of γδT cells after administration to mice with CD20 antibodies linked to carbohydrates 12, 8, 14, 41 and 42, PBS is the solvent control group, and V2 is the unlabeled antibody control group.

[0144] FIG28 shows the survival rate of mice after administration of γδT cells with CD20 antibodies linked to carbohydrates Nos. 12, 8, 14, 41, and 42 to mice, with PBS serving as the control group.

[0145] Figures 29 to 31 show the survival rate of mice after γδT cells with Claudin18.2 antibodies linked to sugars 29, 3, 22, 12, 5, 15, 20, 23, 25, 8, 41, 42, and 14 were administered to mice. PBS is the solvent control group, and γδT represents the cell control group without antibody linkage. DETAILED DESCRIPTION

[0146] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0147] Definition of terms

[0148] In this application, the term "bioorthogonal reaction" generally refers to a group of reactions that can occur in a biological environment with minimal effects on biomolecules or minimal interference with biochemical processes. Bioorthogonal reactions generally have one or more of the following characteristics: (1) they can be carried out at physiological temperature and pH, (2) they selectively provide high yields of products, (3) they are not affected by water or endogenous nucleophiles, electrophiles, reducing agents or oxidizing agents in complex biological environments, (4) they can form stable reaction products, (5) they react rapidly, and (6) they can involve functional groups that do not exist naturally in biological systems. Bioorthogonal reactions that can be applied to the methods of this application may include: azide-alkynyl cycloaddition reaction (SPAAC), the reaction of nitrones with cyclic alkynes, the reaction of aldehydes or ketones to form oximes or hydrazones, the Diels-Alder reaction of tetrazines with cyclic alkenes or cyclic alkynes, norbornene cycloaddition reaction, oxa-norbornadiene cycloaddition reaction, tetracycloalkane coupling reaction, and Staudinger coupling reaction. The first or second functional group of the present invention can be a group capable of participating in the above-mentioned bioorthogonal reaction or a derivative thereof. The group obtained after the orthogonal reaction of the first or second functional group can be referred to as a "linker obtained by the bioorthogonal reaction." For example, an exemplary linker can be the structure obtained by the reaction of an azide group and DBCO:

[0149] In this application, the term "antigen binding protein" generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or backbone portion that allows the portion that binds to the antigen to adopt a conformation that promotes binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include, but are not limited to, antibodies, antigen binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, VHH, scFv, di-scFv and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they exhibit the desired antigen binding activity.

[0150] In this application, the term "antibody" generally refers to an immunoglobulin that can undergo a specific binding reaction with the corresponding antigen. The antibody can be secreted by an immune cell (e.g., an effector B cell). The antibody can be a monoclonal antibody (including a full-length monoclonal antibody comprising two light chains and two heavy chains), a polyclonal antibody, a multispecific antibody (e.g., a bispecific antibody), a humanized antibody, a fully human antibody, a chimeric antibody, and / or a camelized single domain antibody. An "antibody" can generally comprise a protein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four framework regions (FRs), arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable domains of native heavy and light chains each comprise four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), most of which adopt a β-sheet configuration and are connected by three CDRs, forming loops connecting and, in some cases, forming part of the β-sheet structure. The CDRs in each chain are brought together in close proximity by the FR regions and, together with the CDRs from the other chain, form the antigen binding site of the antibody.

[0151] In this application, the term "γδT cells" generally refers to T cells whose T cell receptors (TCRs) are composed of γ chains and δ chains. TCRγ chains include but are not limited to Vδ1, Vδ2, Vδ3 and Vδ5. TCRδ chains include but are not limited to Vγ2, Vγ3, Vγ4, Vγ5, Vγ8 and Vγ9. The term "γδT cells" includes different γδT cell types formed by the combination of different TCRδ chains and TCRγ chains. For example, γδT cells expressing TCRs containing δ chain variable region 1 (Vδ1) can be referred to as Vδ1T cells. Vδ1T cells can be divided into Vγ2Vδ1T, Vγ3Vδ1T, Vγ4Vδ1T, Vγ5Vδ1T, Vγ8Vδ1T and Vγ9Vδ1T according to the type of γ chain variable region. For example, γδT cells expressing TCRs containing γ chain variable region 9 (Vγ9) and δ chain variable region 2 (Vδ2) can be referred to as Vγ9Vδ2T cells. γδT cells can be obtained from peripheral blood. The γδT cells can be mammalian γδT cells, such as rodent γδT cells and primate γδT cells. In a specific embodiment, the γδT cells are human γδT cells. In a specific embodiment, the γδT cells are mouse γδT cells. In a specific embodiment, the γδT cells are monkey γδT cells. The term also includes γδT cells obtained from the body, as well as γδT cells after passage in vitro or ex vivo culture. The γδT cells can be engineered γδT, for example, γδT into which exogenous genes have been transferred, γδT cells with endogenous genes knocked out, and for example, CARγδT cells and TCRγδT cells.

[0152] In this application, the term "monosaccharide derivative" generally refers to a compound derived from a monosaccharide. In this application, a monosaccharide derivative may refer to a compound in which some groups are substituted, for example, acylated, compared to a naturally occurring hydrolyzed monosaccharide. Monosaccharide derivatives can be obtained by hydrolysis to obtain natural sugars, for example, glucose, galactose, mannose, fucose or sialic acid. In particular, the monosaccharide derivatives of the present application can be products obtained by replacing the hydrogen atoms attached to the carbon atoms of a natural monosaccharide and / or replacing the hydrogen atoms on the hydroxyl groups. For example, the monosaccharide derivatives of the present application can be acetylated products of natural monosaccharides. The monosaccharide derivatives of the present application can be hydrolyzed to obtain N-acetylgalactosamine, N-acetylmannosamine or sialic acid.

[0153] In this application, the term "alkyl" generally includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (e.g., isopropyl, tert-butyl, isobutyl, etc.). The term alkyl may further include alkyl groups whose main chain carbon atoms are substituted with one or more carbon atoms of oxygen, nitrogen, sulfur, or phosphorus. The alkyl groups described herein may contain 1-20, 1-12, 1-10, 1-8, or 1-6 carbon atoms.

[0154] In this application, the term "alkenyl" generally refers to any cyclic or acyclic branched or unbranched unsaturated carbon chain portion, which has one or more double bonds. In this application, the term "alkenyl" generally refers to any cyclic or acyclic branched or unbranched unsaturated carbon chain portion, which has one or more triple bonds. The alkenyl groups described herein may contain 2-20, 2-12, 2-10, 2-8 or 2-6 carbon atoms.

[0155] In this application, the term "alkynyl" generally refers to an unsaturated straight or branched chain alkynyl group, such as ethynyl, 1-propynyl, propargyl, butynyl, etc. The alkynyl group can be substituted or unsubstituted. The alkynyl group described herein can contain 2-20, 2-12, 2-10, 2-8, or 2-6 carbon atoms.

[0156] In this application, as known to those skilled in the art, terms such as "alkyl", "alkenyl", "cycloalkyl", etc. may be preceded by an identifier to indicate the number of atoms present in the group in a particular case, for example, C1-C6 alkyl, etc. The subscript number after "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group having three carbon atoms (e.g., n-propyl, isopropyl). For example, the term "C 1～6 The term "alkyl" includes alkyl groups containing 1 to 6 carbon atoms.

[0157] In this application, the term "aryl" generally refers to a 3- to 12-membered substituted or unsubstituted monocyclic aromatic group, in which each atom of the ring can be carbon (i.e., a carbocyclic aryl) or in which one or more atoms can be a heteroatom (i.e., a heteroaryl).

[0158] The term "heteroatom" is art-recognized and refers to an atom of any element other than carbon or hydrogen. Exemplary heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur, and selenium.

[0159] In the present application, the compounds of the present application include their tautomers, mesomorphs, racemates, enantiomers, and / or diastereomers of the compounds. In the present application, the term "diastereomer" generally refers to stereoisomers with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, for example, melting points, boiling points, spectral properties and reactivity. In the present application, the term "mesomorph" generally refers to an asymmetric atom in a molecule, but has symmetry factors that make the total optical rotation in the molecule zero. The term "racemate" or "racemic mixture" refers to a composition consisting of two enantiomeric substances in equimolar amounts.

[0160] In the present application, some atoms of the compounds of the present application may appear in more than one isotopic form. For example, hydrogen may appear in the form of protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H), carbon can exist in three different isotopes ( 12 C. 13 C and 14 C) naturally occurring. Examples of isotopes that can be incorporated into the compounds of the present application also include, but are not limited to 15 N. 18 O. 17 O. 18 F. 32 P. 33 P. 129 I. 131 I. 123 I. 124 I. 125 I, or similar isotopes. Unless otherwise indicated, the structures described herein may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds that are identical to the structures described herein except for the replacement of hydrogen atoms by deuterium or tritium, or the replacement of carbon atoms by carbon 13 or carbon 14 are within the scope of this application.

[0161] In this application, the term "immune cell" generally refers to cells that can participate in or are associated with an immune response. Immune cells can include all white blood cells and non-leukocyte antigen-presenting cells. The term includes individual cells, cell lines, or cell cultures. The cells include not only specific cells but also the progeny of these cells.

[0162] In this application, the term "pharmaceutically acceptable adjuvant" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Typically, a physiologically acceptable carrier is a pH buffered aqueous solution.

[0163] As used herein, the term "treatment" refers to administering to a patient an internal or external therapeutic agent, such as a cell comprising any modification of the present application or a pharmaceutical composition comprising modified cells, wherein the patient has one or more symptoms of the disease, and it is known that the therapeutic agent has a therapeutic effect on these symptoms. Typically, the patient is administered an amount of the therapeutic agent that effectively alleviates one or more symptoms of the disease (therapeutically effective amount). The desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and regressing or improving the prognosis. For example, if one or more symptoms associated with cancer are alleviated or eliminated, including but not limited to, reducing (or destroying) cancer cell proliferation, reducing symptoms from the disease, improving the quality of life of those individuals with the disease, reducing the dosage of other drugs needed to treat the disease, delaying the progression of the disease, and / or prolonging individual survival, the individual is successfully "treated".

[0164] In this application, the term "administer" generally refers to delivering a substance to a subject in need thereof by any route known in the art. Pharmaceutical carriers and formulations or compositions are also well known in the art. Routes of administration may include intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral, and / or mucosal.

[0165] In the present application, the term "disease associated with abnormal expression" of a target generally means that the occurrence and / or progression of the disease is associated with the expression level of the target. For example, "high expression" of a target means that the expression level of a target in cells from a disease area, such as a specific tissue or organ of a patient, is increased relative to the expression level of normal cells from the tissue or organ, i.e., high expression. Or, for example, "low expression" of a target means that the expression level of a target in cells from a disease area, such as a specific tissue or organ of a patient, is decreased relative to the expression level of normal cells from the tissue or organ, i.e., low expression. Or, for example, cells from a disease area, such as a specific tissue or organ of a patient, express a target, i.e., positive. Or, for example, cells from a disease area, such as a specific tissue or organ of a patient, do not express a target, i.e., negative. For example, the characteristics of target expression can be determined by standard assays known in the art.

[0166] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0167] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0168] Detailed Description of the Invention

[0169] Methods for modifying cells

[0170] In one aspect, the present application provides a method for modifying a cell, the method comprising one or more of the following steps:

[0171] 1) contacting a monosaccharide derivative comprising a first functional group with immune cells to obtain immune cells expressing the monosaccharide derivative comprising the first functional group on their surfaces;

[0172] 2) contacting the antigen binding protein with a compound comprising a second functional group to obtain an antigen binding protein comprising a second functional group; and / or,

[0173] 3) contacting the immune cell expressing the monosaccharide derivative containing the first functional group on its surface with the antigen binding protein containing the second functional group, so that the first functional group and the second functional group undergo a connection reaction to obtain a cell with the antigen binding protein modified on its surface.

[0174] Step S1

[0175] In the present application, the method may include step S1: contacting a monosaccharide derivative comprising a first functional group with an immune cell to obtain an immune cell that expresses a monosaccharide derivative comprising a first functional group on its surface. The monosaccharide derivative may be derived from mannose, galactose, fucose, xylose, glucose, mannosamine, galactosamine, glucosamine and / or sialic acid. The first functional group may be connected to the monosaccharide molecule through a suitable spacer, for example, an amide bond or an alkylamino bond. The first functional group is connected to the monosaccharide molecule at the Cl position, C2 position, C3 position, C4 position or C5 position of the monosaccharide molecule, and optionally may be further acylated on the remaining one or more Cs. In one embodiment, the monosaccharide molecule may contain a hydrolyzable substituent at one or more of the Cl position, C2 position, C3 position, C4 position or C5 position that does not contain the first functional group, and the hydrolyzable substituent contributes to the hydrophobicity of the polymer and can be hydrolyzed and converted into hydroxyl groups once entering the cell. An example of a hydrolyzable substituent is Wherein, R is an alkyl group, for example, R is a methyl group.

[0176] For example, the monosaccharide derivative may be selected from the group consisting of: Wherein, X is the first functional group, and R1, R2, R3, R4, R5 and R6 are each independently selected from a protecting group that can be hydrolyzed or enzymatically degraded in cells, the purpose of which is to accelerate the entry of non-natural sugars into cells and expose the hydroxyl groups on the sugars after deprotection.

[0177] Any group that fulfills this function falls within the scope of the present invention.

[0178] In certain embodiments, R1, R2, R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy, C 2～6 Alkoxycarbonyl and acyl, said groups being optionally substituted by substituents selected from the group consisting of halogen, hydroxy, amino, nitro, mercapto and C 1-6 alkyl;

[0179] R5 is selected from the group consisting of hydrogen, halogen, and substituted or unsubstituted C 1～6 an alkyl group optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto;

[0180] R6 and R7 are each independently selected from the group consisting of halogen and substituted or unsubstituted C 1～10 an alkyl group optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto;

[0181] R8 is selected from the group consisting of halogen, substituted or unsubstituted C 1～10 Alkyl, phenyl and substituted or unsubstituted C 1～10 Alkylene-phenyl, said alkyl being optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto;

[0182] Z1 and Z2 are each independently selected from: O and S;

[0183] n and m are each independently selected from the following integers: 1, 2, 3, 4, 5 and 6.

[0184] In certain embodiments, R1 is selected from the group consisting of hydrogen and -C(=O)-C1-6 alkyl. For example, R1 is hydrogen or -C(=O)-CH3.

[0185] In certain embodiments, R2 is selected from the group consisting of hydrogen and -C(=O)-C1-6 alkyl. For example, R2 is hydrogen or -C(=O)-CH3.

[0186] In certain embodiments, R3 is selected from the group consisting of hydrogen and -C(=O)-C1-6 alkyl. For example, R3 is hydrogen or -C(=O)-CH3.

[0187] In certain embodiments, R4 is selected from the group consisting of hydrogen and -C(=O)-C1-6 alkyl. For example, R4 is hydrogen or -C(=O)-CH3.

[0188] In certain embodiments, R5 is selected from the group consisting of hydrogen and substituted or unsubstituted C 1～6 In certain embodiments, R5 is hydrogen or substituted or unsubstituted C 1～3 Alkyl. For example, R4 is hydrogen or -CH3.

[0189] In certain embodiments, R6 is selected from the group consisting of hydrogen and substituted or unsubstituted C 1～10 alkyl.

[0190] In certain embodiments, R7 is selected from the group consisting of hydrogen and substituted or unsubstituted C 1～10 alkyl.

[0191] In certain embodiments, R8 is selected from the group consisting of methyl, ethyl, and -CH2-phenyl.

[0192] In certain embodiments, m is 1, 2, or 3.

[0193] In certain embodiments, n is 1, 2, or 3.

[0194] In certain embodiments, in Formula I, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl, R2 is selected from hydrogen and -C(=O)-C1~6 alkyl, R3 is selected from hydrogen and -C(=O)-C1~6 alkyl, R4 is selected from hydrogen and -C(=O)-C1~6 alkyl, and R1, R2, R3 and R4 are not hydrogen at the same time.

[0195] In certain embodiments, in Formula II, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl, R2 is selected from hydrogen and -C(=O)-C1~6 alkyl, R3 is selected from hydrogen and -C(=O)-C1~6 alkyl, R4 is selected from hydrogen and -C(=O)-C1~6 alkyl, and R1, R2, R3 and R4 are not hydrogen at the same time.

[0196] In certain embodiments, in Formula III, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl, R2 is selected from hydrogen and -C(=O)-C1~6 alkyl, R3 is selected from hydrogen and -C(=O)-C1~6 alkyl, R4 is selected from hydrogen and -C(=O)-C1~6 alkyl, and R1, R2, R3 and R4 are not hydrogen at the same time.

[0197] In certain embodiments, in Formula IV, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R6 is selected from C1~ 10 Alkyl, R7 is selected from C1~ 10 Alkyl, Z1 is selected from O and S, Z2 is selected from O and S, n is 1, 2 or 3, m is 1, 2 or 3, and R1, R2 and R3 are not hydrogen at the same time.

[0198] In certain embodiments, in Formula V, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R8 is selected from: C1-C3 alkyl, phenyl, C1-C3 alkylene-phenyl, and R1, R2 and R3 are not hydrogen at the same time.

[0199] In certain embodiments, in Formula VI, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and C1~6 alkyl (preferably -CH3), and R1, R2, R3 and R4 are not hydrogen at the same time, or R1, R2, R3, R4, R5 are not hydrogen at the same time. In a specific embodiment, at most four of R1, R2, R3, R4, and R5 are hydrogen.

[0200] In certain embodiments, in Formula VII, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and C1~6 alkyl (preferably -CH3), and R1, R2, R3 and R4 are not hydrogen at the same time, or R1, R2, R3, R4, R5 are not hydrogen at the same time. In a specific embodiment, at most four of R1, R2, R3, R4, and R5 are hydrogen.

[0201] In certain embodiments, in Formula VIII, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and C1~6 alkyl (preferably -CH3), and R1, R2, R3 and R4 are not hydrogen at the same time, or R1, R2, R3, R4, R5 are not hydrogen at the same time. In a specific embodiment, at most four of R1, R2, R3, R4, and R5 are hydrogen.

[0202] In certain embodiments, in Formula VI, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R4 is selected from hydrogen and C1~6 alkyl (preferably -CH3), and R1, R2, R3 and R4 are not hydrogen at the same time, or R1, R2, R3, R4, R5 are not hydrogen at the same time. In a specific embodiment, at most four of R1, R2, R3, R4, and R5 are hydrogen.

[0203] In certain embodiments, in Formula V, R1 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R2 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), R3 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3), and R4 is selected from hydrogen and -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3). In a specific embodiment, R1, R2, R3 and R4 are all hydrogen. In a specific embodiment, R1, R2, R3 and R4 are all -C(=O)-C1~6 alkyl (preferably -C(=O)-CH3).

[0204] In one embodiment, the monosaccharide derivative may comprise any one or more structures selected from the following group: Wherein, X is the first functional group.

[0205] In one embodiment, the monosaccharide derivative can be selected from any one of the structures shown in Table A above.

[0206] The monosaccharide derivative may also include its tautomer, meso-racemate, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof.

[0207] In the present application, the first functional group may be a group having bioorthogonal reaction activity, and the bioorthogonal reaction may include at least one selected from the group consisting of click chemistry, photocrosslinking, photosensitivity, and glycosylation.

[0208] In the present application, the first functional group may be selected from the group consisting of: azide, alkynyl, trans-cyclooctene, triazine, tetrazine, hydrazide, cyclopropene, isocyanate, and cyclooctyne. In the present application, the first functional group may be selected from the group consisting of: azide, dibenzocyclooctyne (DBCO), tetrazine, trans-cyclooctene, tetrazine, and norbornene. Examples of the above structures are as follows:

[0209] In the present application, the monosaccharide derivative containing the first functional group can be expressed on the cell membrane surface through the sugar metabolism pathway, so that the first functional group is embedded in the cell membrane surface, thereby obtaining a cell modified with the first functional group. In addition to carbohydrates, the first functional group can also be coupled to lipids (choline, cholesterol, or fatty acids) or amino acids (such as methionine, alanine, or phenylalanine) on the cell membrane surface.

[0210] In the present application, the monosaccharide derivative comprising a first functional group is contacted with immune cells for about 2 hours to 48 hours, for example, about 16 hours to 32 hours, for example, about 24 hours. In the present application, the contacting of the monosaccharide derivative comprising a first functional group with immune cells is carried out at room temperature or at 37°C.

[0211] In the present application, the immune cells may be selected from one or more of the following groups: NK cells, DC cells, NKT cells, monocytes, macrophages, T cells and / or B cells. In one embodiment, the immune cells are NK cells. In another embodiment, the immune cells are T cells. In a more specific embodiment, the immune cells are γδ T cells.

[0212] The method may include contacting the cells with an effective amount of a monosaccharide derivative comprising a first functional group. An effective amount may refer to a monosaccharide derivative comprising the first functional group comprising at least 10% of the cell surface glycoproteins. For example, among the cell surface glycoproteins, more than 10% of the glycoproteins are monosaccharide derivatives comprising the first functional group, preferably more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or more. For each monosaccharide derivative comprising the first functional group and each cell type, a person skilled in the art can easily determine the amount of monosaccharide derivative comprising the first functional group required for metabolic labeling of the cells.

[0213] In the present application, the purity of the immune cells can be identified and then co-cultured with the monosaccharide derivative containing the first functional group for 24 hours; thereby obtaining immune cells expressing the first functional group on the surface.

[0214] In the present application, immune cells expressing the first functional group on their surface can be verified. For example, the immune cells can be incubated with biotin or a fluorescent protein coupled to a functional group capable of undergoing a bioorthogonal reaction with the first functional group, and then the fluorescent signal can be detected to characterize the degree of labeling of the first functional group on the immune cell surface.

[0215] Step S2

[0216] In the present application, the method may include step S2: contacting the antigen binding protein with a compound comprising a second functional group to obtain the antigen binding protein comprising the second functional group.

[0217] In the present application, the antigen-binding protein may target a tumor antigen. The tumor antigen may be a tumor-specific antigen or a tumor-associated antigen. Tumor antigens may be overexpressed or abnormally expressed cellular proteins, products of mutated oncogenes and tumor suppressor genes, products of other mutated genes, tumor antigens produced by oncogenic viruses, oncofetal antigens, cell surface glycolipids and glycoproteins, or cell type-specific differentiation antigens. In the present application, the antigen-binding protein may target a protein on the surface of B lymphocytes, for example, CD20. In the present application, the antigen-binding protein may target a protein on the surface of B lymphocytes, for example, CD20. In the present application, the antigen-binding protein may target a claudin protein, for example, claudin 18.2. In the present application, the antigen-binding protein may be an anti-CD20 antibody. In the present application, the antigen-binding protein may be an anti-claudin 18.2 antibody. For example, the antigen-binding protein of the present application may comprise a heavy chain variable region and / or a light chain variable region. For example, the antigen-binding protein of the present application may comprise a heavy chain and / or a light chain.

[0218] The antibodies of the present application can be prepared using techniques well known in the art, such as the hybridoma method, recombinant DNA technology, phage display technology, synthetic technology, or a combination of such technologies, or other techniques known in the art. Variants can refer to amino acid sequence mutants of the antibody, as well as covalent derivatives of the native polypeptide, provided that the biological activity equivalent to that of the native polypeptide is retained. Amino acid sequence mutants generally differ from the native amino acid sequence in that one or more amino acids in the native amino acid sequence are substituted, or one or more amino acids are deleted and / or inserted into the polypeptide sequence. Deletion mutants include fragments of the native polypeptide and N-terminal and / or C-terminal truncation mutants. Typically, amino acid sequence mutants have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% homology to the native sequence.

[0219] In the present application, the second functional group may be a group that can undergo a bioorthogonal reaction with the first functional group. In the present application, the second functional group may be selected from the following group: an azido group, an alkynyl group, a trans-cyclooctene, a triazine, a tetrazine, a hydrazide, a cyclopropene, an isocyanate, and a cyclooctyne. In the present application, the second functional group may be selected from the following group: an azido group, a dibenzocyclooctyne (DBCO), a bicyclononyne (BCN), a tetrazine, a trans-cyclooctene (TCO), and a norbornene, and the above-mentioned structural examples are as follows:

[0220] In one embodiment, one of the first functional group and the second functional group may be an azido group, and the other may be dibenzocyclooctyne (DBCO). In one embodiment, one of the first functional group and the second functional group may be a tetrazine, and the other may be trans-cyclooctene (TCO). In one embodiment, one of the first functional group and the second functional group may be a tetrazine, and the other may be norbornene. In one embodiment, one of the first functional group and the second functional group may be BCN, and the other may be an azido group.

[0221] The second functional group can be connected to the antigen binding protein through a linker, such as a disulfide bond, a hydrazone, a bond, or an enzyme-cleavable bond. In the present application, the compound comprising the second functional group can include a group capable of reacting with an amino acid, and the second functional group is connected to the amino acid of the antigen binding protein through the group. For example, the second functional group can be connected to the antigen binding protein through maleimide or succinimide. In one embodiment, the structure of the compound comprising the second functional group can be Formula L1, Formula L2, or Formula L3: Wherein, Y represents the second functional group, R L is selected from the group consisting of polyethylene glycol, amide, -C(O)- and / or chain alkyl, and combinations thereof. L Can be selected from the following group: polyethylene glycol, amide group and alkyl ether group. L Can be selected from C 1～20 Alkylene or C 1～20 Alkylene ether group

[0222] In one embodiment, when the first functional group is an azide group, the second functional group may be DBCO, and the compound optionally comprising the second functional group is

[0223] The concentration ratio of the antigen-binding protein and the second functional group in the method described herein can be 1:1 to 1:10, for example, 1:5. In the present application, the contact time between the antigen-binding protein and the compound comprising the second functional group can be about 30 minutes to 6 hours, for example, about 1 hour to 2 hours, for example, about 1 hour. In the present application, the contact between the antigen-binding protein and the compound comprising the second functional group can be carried out at room temperature.

[0224] Step S

[0225] The method described herein includes step S: contacting the immune cell expressing a monosaccharide derivative containing a first functional group on its surface with an antigen-binding protein containing a second functional group, thereby causing a ligation reaction between the first and second functional groups to produce a cell surface-modified with the antigen-binding protein. The pairing of the first and second functional groups is freely selectable by those skilled in the art, including but not limited to the combinations listed above. In one specific example, the first functional group can be an azido group, and the second functional group can be DBCO.

[0226] The contacting can be performed under conditions that allow bioorthogonal reactions to occur, for example, under mild conditions, including neutral pH, aqueous solution and ambient temperature, and low reactant concentrations. In some embodiments, the contacting allows the antigen binding protein to be modified on the cell surface while retaining the structural integrity, function or activity of the antigen binding protein. In other embodiments, the antigen binding protein retains some structure or undergoes some structural changes while retaining some of its function or activity.

[0227] The contact time sufficient to allow the first functional group and the second functional group to undergo a ligation reaction, thereby modifying the antigen-binding protein on the cell surface, can be about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, or more than about 60 minutes. In one embodiment, a suitable contact time is about 30 minutes.

[0228] In the present application, the contacting in step S can be performed at room temperature. In the present application, the contacting in step S can be performed at 37°C.

[0229] Depending on the type of antigen-binding protein and cells, a person skilled in the art can easily determine the contact ratio of the immune cells containing the monosaccharide derivative containing the first functional group and the antigen-binding protein containing the second functional group. In certain embodiments, the concentration of the antigen-binding protein containing the second functional group can be about 10 μM to about 30 μM, and the density of the immune cells containing the monosaccharide derivative containing the first functional group does not exceed 2×10 7 In certain embodiments, the concentration of the antigen-binding protein comprising the second functional group may be about 15 μM to about 25 μM, and the density of the immune cells of the monosaccharide derivative comprising the first functional group does not exceed 1.5×10 7 In a specific embodiment, the concentration of the antigen binding protein comprising the second functional group can be about 20 μM, and the density of the immune cells of the monosaccharide derivative comprising the first functional group does not exceed 1×10 7 pieces / mL.

[0230] In the present application, the cells of the surface modified antigen-binding protein can be verified. For example, the corresponding antigen with a detectable marker is co-incubated with the modified immune cells, and then the signal intensity is detected to characterize the degree of antigen-binding protein modified on the surface of the immune cells. The detectable marker can be biotin, which is detected by conjugating AF488-containing streptavidin to characterize the labeling of cell surface antigen-binding proteins, for example, by laser confocal imaging. In the present application, the labeling of the antigen-binding protein can be detected on the surface of at least 10% (for example, at least 20% or more, at least 30% or more, at least 40% or more, at least 50% or more, at least 60% or more, at least 70% or more, at least 80% or more, at least 90% or more or more) of the cells.

[0231] Modified cells

[0232] In another aspect, the present application provides a modified cell prepared by the methods described herein. In a specific example, the modified cell is an antibody-cell conjugate. Using the methods described herein, the cell is surface-conjugated to an antibody via a monosaccharide derivative.

[0233] On the other hand, the application provides a kind of modified cell, which is modified with antigen binding proteins on the cell surface, for example, modified on the non-natural sugar molecules on the cell surface by bioorthogonal reactions, and the non-natural sugar molecules can be expressed on the cell surface by sugar metabolism pathways. The cell can be an epithelial cell, fibroblast, neuronal cell, endothelial cell or immune cell. In certain embodiments, the cell is an immune cell. The example of immune cell can include but is not limited to peripheral blood leukocytes, spleen leukocytes, lymph node leukocytes, hybridoma cells, T cells (cytotoxic / suppressor cells, helper cells, memory cells, immature cells and sensitized cells), B cells (memory cells and immature cells), monocytes, macrophages, granulocytes (basophils, eosinophils and neutrophils), natural killer cells, natural suppressor cells, thymocytes and dendritic cells.

[0234] The cell surface has an azide group expressed on the surface through a sugar metabolism pathway by absorbing a non-natural sugar derivative containing an azide group. The azide group is connected to an antibody via a linker compound. One end of the linker compound has a first active group that can react with a sulfhydryl or amino group, and the other end has a second active group that can undergo a bioorthogonal reaction with the azide group. The first active group reacts and connects with the sulfhydryl or amino group on the antibody, and the second group reacts and connects with the azide group.

[0235] In one embodiment, the cell is a T cell. Examples of T cells may include but are not limited to CD4 + T cells, CD8 +T cells, Treg cells, memory T cells, natural killer T cells, mucosal-associated invariant T cells (MAIT), αβ-T cells, γδ T cells, and engineered versions of these cells, such as CAR T cells and TCR T cells.

[0236] In one embodiment, the cell is a γδ T cell, for example, a Vγ9Vδ2 T cell, or for another example, a Vδ1 T cell.

[0237] In one embodiment, the cell is a CAR T cell.

[0238] In one embodiment, the cell is a NK cell.

[0239] The immune cells modified by the methods described herein have enhanced targeting function. For example, when modified with antibodies targeting specific tumor antigens, the modified cells can efficiently target cells (e.g., tumor cells) expressing the tumor antigen. Compared with cells not modified with antibodies, the ability of cells with the surface modified antibodies to target tumor antigens can be enhanced by at least 10%, or at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or higher. The targeting ability can be detected by flow cytometry, laser confocal imaging or other methods known in the art. The targeting ability can be detected by in vivo enrichment experiments, and the cells with the surface modified antibodies are more likely to be enriched in tumors expressing the antigen. The immune cells modified by the methods described herein can reduce off-target effects and have safety.

[0240] The immune cells modified by the methods described herein have enhanced cell killing function. For example, when modified with antibodies targeting specific tumor antigens, the modified cells can efficiently kill cells (e.g., tumor cells) expressing the tumor antigen. Compared with cells not modified with antibodies, the ability of cells with the antibody on the surface to kill tumor cells expressing the corresponding antigen can be enhanced by at least 10%, or at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or higher. The killing ability can be detected by in vitro or in vivo methods. For example, it can be detected by an LDH detection kit. For example, an animal model can be used to detect by in vivo methods.

[0241] Pharmaceutical composition and treatment method

[0242] In another aspect, the present application provides a method for treating and / or alleviating tumors, comprising administering to a subject in need thereof modified cells prepared according to the methods described herein. Appropriate antigen-binding proteins can be selected based on the type of tumor to be treated and / or alleviated, and then modified cells targeting specific tumor antigens can be prepared according to the methods of the present application.

[0243] For example, the tumor can be a B lymphocyte tumor. For example, the tumor can be a tumor with high expression of CD20. For example, the tumor can be a CD20 positive tumor. For example, the CD20 positive tumor can include B cell lymphoma or leukemia, including but not limited to: diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone B cell lymphoma (MZL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), lymphoplasmacytic lymphoma (LPL), plasma cell lymphoma (PCL), primary central nervous system lymphoma (PCNSL).

[0244] For another example, the tumor may be a tumor that highly expresses claudin 18.2. For example, the tumor may be a claudin 18.2-positive tumor. For example, claudin 18.2-positive tumors may include, but are not limited to, gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, bile duct cancer, liver cancer, breast cancer, and / or lung cancer.

[0245] The tumor may be a primary tumor or a metastatic tumor.

[0246] The modified cells described herein can be administered alone or as a pharmaceutical composition in combination with a pharmaceutically acceptable adjuvant. For example, they can be administered in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0247] The cells or pharmaceutical compositions of the present application can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. Preferably, the pharmaceutical compositions of the present application can be formulated for intravenous administration.

[0248] The modified cells described herein can be co-administered with one or more other anti-tumor therapies (eg, surgery, chemotherapy, radiotherapy, targeted therapy, or immune checkpoint inhibitors).

[0249] The dosage and frequency of administration of the cells of the present application will be determined by practical factors such as the patient's condition, the type and severity of the patient's disease, and taking into account individual differences in the patient's age, weight, tumor size, degree of infection or metastasis, and condition.

[0250] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method, and use of the present application, and are not intended to limit the scope of the present invention.

[0251] Example

[0252] Example 1 Preparation of compounds with orthogonal reactive groups

[0253] According to the literature 1 (Cheng, B., Wang, CT, Hao, Y., Wang, JK, Xia, XQ, Zhang, H., He, RD, Zhang, SR, Dai, P., Chen, 29(11),e202203054), Document 2(Han, SF, Collins, BE, Bengtson, P., Paulson, JC (2005) Homomultimeric complexes of CD22 in B cells revealed by protein-glycan cross-linking.Nat.Chem.Biol.1(2),93-7), Document 3(Luchansky, SJ, Goon, S., Bertozzi, CR (2004) Expanding the diversity of unnatural The monosaccharide derivatives of the present application were obtained by the synthesis method of reference 4 (Cheng, B., Dong, L., Zhu, YT, Huang, RB, Sun, YT, You, QC, Song, QT, Paton, JC, Paton, AW, Chen, X. (2019) 9-Azido Analogues of Three Sialic Acid Forms for Metabolic Remodeling of Cell-Surface Sialoglycans. ACS Chem. Biol. 14 (10), 2141-2147.). For example. The monosaccharide derivatives of the present application can also be obtained in a commercial manner, taking Ac4ManNAz (Sigma-Aldrich, Product No. 900917) and Ac4GalNAz (Sigma-Aldrich, Product No. 900915) as examples. A total of 42 monosaccharide derivatives with orthogonal reactive groups were obtained, and the structures are shown in Figures 2 to 7, wherein Figure 2 shows 5 GalNAz derivatives, Figure 3 shows 5 GlcNAz derivatives, Figure 4 shows 5 ManNAz derivatives, Figure 5 shows 13 ManNAz-6-P derivatives, Figure 6 shows 12 azidosialic acid derivatives, and Figure 7 shows 2 azidofucoses.

[0254] To investigate the labeling efficiency of 42 non-natural sugars, γδ T cells, T cells, and NK cells were labeled with the corresponding concentrations of each non-natural sugar. Flow cytometry was used to analyze the labeling efficiency of each cell group 48 hours after labeling, with results expressed as fluorescence intensity (MFI). The cells were then labeled with rituximab and Hu7v3, both of which were already labeled with DBCO. One hour after labeling, the cells were removed and the antibody labeling efficiency was analyzed by flow cytometry. Cytotoxicity assays were then performed on Raji and SNU-620 cell lines to investigate the differences between cells labeled with different non-natural sugars. Specific examples are as follows.

[0255] Example 2 Antibody Labeling DBCO

[0256] (1) Antibody desalting: Take out an appropriate amount of two antibodies that need to be labeled with DBCO (rituximab targeting CD20 and Hu7v3 antibody targeting Claudin18.2) for desalting, that is, take out 4 desalting columns from each group, wash them 5 times with 1×PBS, add 2.5mL of antibody to each desalting column, wait for the antibody to flow out, add 0.5mL of 1×PBS, wait for the 1×PBS to flow out, take out the desalting column from the test tube rack, wipe it with a paper towel, wipe the liquid under the desalting column dry, place the desalting column on top of a 15mL centrifuge tube, add 3mL of 1×PBS to each column, collect the antibody, and after the antibody desalting is completed, wash the desalting column 5 times with 1×PBS and 5 times with pure water, and store it at 4°C;

[0257] (2) Antibody labeling with DBCO: Select the A280 plate on a UV spectrophotometer to detect the collected antibody concentration. Calculate the molar concentration of the two antibodies by dividing by the extinction coefficient and add the corresponding 5 equivalents of DBCO. Label at 30°C for 1 hour.

[0258] (3) Desalting after antibody labeling with DBCO: Desalt the antibody labeled with DBCO again according to the same steps as the antibody desalting, replace the storage medium with PBS, and detect the concentration of the antibody labeled with DBCO after desalting.

[0259] Example 3 ACC preparation

[0260] The non-natural sugar with an azide group prepared in Example 1 was added to the culture medium of γδT cells, T cells or NK cells, and the culture was continued for 48 hours.

[0261] Specifically, cells cultured to day 11 were labeled with corresponding concentrations of 42 non-natural sugars, a total of 42 groups, which served as experimental groups, while the control group did not add any non-natural sugars;

[0262] Table 1 Labeled concentrations of non-natural sugars

[0263] (2) DBCO-PEG-biotin and SA-488 were used as primary and secondary antibodies, and flow cytometry (Wellgrow) was used to detect whether the cell surface was labeled with orthogonal groups.

[0264] Specifically, 1×10 cells were taken from each group. 5 Cells were centrifuged, washed, and resuspended in EP tubes at 300g for 3 minutes, after which the supernatant was discarded and the cells were resuspended in 1 mL of flow cytometry loading buffer (PBS containing 1% serum). After centrifugation at 300g for 3 minutes, the cells were resuspended in 200 μL of flow cytometry loading buffer. 5 μL of 10 mM DBCO-PEG4-Biotin antibody was added to all groups and incubated at room temperature in the dark for 30 minutes. After incubation, the cells were centrifuged, washed, and resuspended. 2 μL of SA-488 antibody was added to all groups and incubated at room temperature in the dark for 30 minutes. After incubation, the cells were centrifuged, washed, and resuspended, and transferred to corresponding labeled flow cytometry tubes for flow cytometry analysis. The results are shown in Figures 8 (γδ T cells), 13 (T cells), and 18 (NK cells). Compared with the control group, although the labeling efficiency of various non-natural sugars on each cell type varied, all cells were labeled.

[0265] 3.2 Cell Marker Antibodies

[0266] (1) After 48 h of non-natural sugar labeling, the experimental and control cells were removed and centrifuged at 300 g for 3 min. After centrifugation, they were washed with 10 mL of culture medium to remove residual sugars.

[0267] (2) Cells were plated at 1×10 7 density, 20 μM concentration of labeled rituximab, and 1×10 7 The cells were labeled with Hu7v3 at a density of 20 μM and 20 μM, and incubated at 37°C for 1 h. After incubation, the cells were centrifuged and the supernatant was discarded. The cells were washed once with culture medium, and the cells were counted and resuspended in an appropriate volume of culture medium.

[0268] 3.3 Flow Cytometry

[0269] FITC Anti-Human IgG Fc antibody was used for flow cytometry detection of both antibodies.

[0270] ① All groups took out 1×10 5 Place cells in an EP tube, centrifuge at 300g for 3 min, remove the supernatant, add 1 mL of flow cytometry loading buffer to each tube, resuspend the cells, and wash once at 300g for 3 min.

[0271] ②After washing, add 200 μL of flow cytometry loading buffer to each tube to resuspend the cells;

[0272] ③ Add 1 μL of FITC Anti-Human IgG Fc antibody to all groups, flick the bottom of the tube to evenly distribute the antibody, and incubate at room temperature in the dark for 30 minutes;

[0273] ④ After incubation, centrifuge at 300g for 3 minutes to remove the supernatant, resuspend the cells in 1 mL of flow cytometry loading buffer, and centrifuge at 300g for 3 minutes to remove the supernatant and wash away the residual antibodies.

[0274] ⑤ Resuspend the cells with 500 μL flow cytometry loading buffer, transfer to the corresponding labeled flow cytometry tube, and perform flow cytometry detection.

[0275] Flow cytometry was used to detect the two antibodies, rituximab and Hu7v3, labeled on the cells.

[0276] The results are shown in Figures 9, 10, 14, 15, 19 and 20. Compared with the control group, it was found that γδT cells, T cells and NK cells could all be labeled with the two antibodies.

[0277] Example 4 Detection of target cell killing efficiency

[0278] (1) Calcein-AM labeling of target cells:

[0279] ① Cell counting: Gently blow the cultured Raji cells and SNU-620 cells evenly in a clean bench, transfer all the cells to a 15 mL centrifuge tube, and centrifuge at 300 g for 3 min.

[0280] ② After centrifugation, spray the tube walls and cap with alcohol and place in a clean bench. Open the cap of a 15 mL centrifuge tube in the clean bench, remove the supernatant, and resuspend in 1 mL of the corresponding complete culture medium. Mix thoroughly by pipetting. Remove 10 μL and mix with 10 μL of AOPI staining solution in a 1:1 ratio. Count 12 μL using a cell counter. Record the number of viable cells and viability.

[0281] ③According to the counting results, take an appropriate amount and transfer it to a labeled 15mL centrifuge tube.

[0282] ④ Add 10 mL of tumor killing buffer to each centrifuge tube, centrifuge at room temperature, 300g, for 5 min, discard the supernatant, and then gently tap the bottom of the centrifuge tube with your fingertips to disperse the cell clumps.

[0283] ⑤Add tumor killing buffer, resuspend the cells, and adjust the cell density to 1×10 6 cells / mL, then add Calcein-AM solution to the cell suspension in the centrifuge tube (make sure to mix Calcein-AM before use) at a volume ratio of 1:100. Gently pipette and mix thoroughly. Incubate the cell suspension in a 37°C incubator for 30 minutes.

[0284] ⑥ After incubation, add 10 mL of tumor killing buffer to the centrifuge tube to wash the cells. Centrifuge at room temperature, 300g, for 5 minutes, and discard the supernatant. Repeat the washing operation twice. After each aspiration of the supernatant, gently tap the bottom of the centrifuge tube with your fingers to disperse the cells. Finally, add an appropriate volume of tumor killing buffer to the centrifuge tube to resuspend the cells. At this time, the cell suspension density is 1×10 5 cells / mL.

[0285] ⑦ Among them, cells labeled with rituximab were used to kill Raji cells, and cells labeled with Hu7v3 were used to kill SNU-620 cells, and the killing efficiency of the two antibodies was tested respectively.

[0286] (2) Preparation of γδT cells, T cells, and NK cells

[0287] ① Cell counting: In a biosafety cabinet, gently blow the cells labeled with two antibodies, take out 10 μL and mix it with 10 μL of AOPI staining solution in a 1:1 ratio. Take 12 μL and count it using a cell counter, and record the number of live cells and the viability.

[0288] ② According to the above counting results, the labeling effector target ratio is 10:1, which is 1×10 5 cells / well, make 4 replicate wells, i.e. take out 4×10 5 Transfer the γδT cells, T cells, and NK cells to a labeled 15 mL centrifuge tube.

[0289] ③ Add 10 mL of tumor killing buffer to each centrifuge tube and centrifuge at room temperature at 300g for 5 minutes. Discard the supernatant and gently tap the bottom of the centrifuge tube with your fingertips to disperse the cell clumps. Repeat this operation once.

[0290] ④ Add tumor killing buffer to the centrifuge tube and adjust the effective cell density to 1×10 6 cells / mL.

[0291] (3) Co-culture of γδT cells, T cells, NK cells and target cells

[0292] ② Add 100 μL of γδT cells, T cells, and NK cells to the corresponding labeled wells of a U-shaped 96-well plate. Make four replicate wells for each effector target ratio; add 100 μL of lysis buffer and 100 μL of tumor killing buffer to the corresponding labeled wells.

[0293] ③ Take 100 μL of target cells labeled with Calcein-AM and add them to the corresponding labeled wells.

[0294] ④ Place the U-bottom 96-well plate in a 37°C electric constant temperature incubator and incubate for 2.5 hours.

[0295] (4) Reading the results

[0296] ① Take the 96-well plate out of the incubator, incubate at 400g for 10 min, and take 150 μL of supernatant from each well (be careful to take the upper layer of the liquid to avoid the sediment at the bottom) and transfer it to a 96-well plate with a black bottom (the sample positions should be the same as marked in the table).

[0297] ② Measure the absorbance of each well using the fluorescence colorimetric method in the microplate reader (488 excitation, 520 emission).

[0298] (5) Data processing and statistical analysis of results

[0299] Result calculation formula:

[0300] Specific lysis percentage = (fluorescence value of the experimental group - fluorescence value of the spontaneous release group) / (fluorescence value of the maximum release group - fluorescence value of the spontaneous release group) × 100%.

[0301] Fluorescence value of the experimental group = the average fluorescence value of target cells and effector cells at a specific concentration

[0302] Fluorescence value of spontaneous release group = average fluorescence value of target cells and tumor killing buffer

[0303] Fluorescence value of the maximum release group = average fluorescence of target cells and lysate

[0304] The results are shown in Tables 2 to 7, as well as Figures 11, 12, 16, 17, 21 and 22. The results show that after γδT cells, NK cells and T cells were labeled with two antibodies, their killing efficiency was higher than that of unlabeled cells (control group), and all had a targeted killing effect.

[0305] Table 2 The results of rituximab killing of Raji cells by γδT cells labeled with 42 sugars

[0306] Table 3 The results of killing SNU-620 cells by Hu7v3 antibody after γδT cells were labeled with 42 kinds of sugars

[0307] Table 4 The results of NK cells labeled with 42 sugars and then labeled with rituximab to kill Raji cells

[0308] Table 5 The results of killing SNU-620 cells by NK cells labeled with 42 sugars and then labeled with Hu7v3 antibody

[0309] Table 6 The results of T cells labeled with 42 sugars and then labeled with rituximab to kill Raji cells

[0310] Table 7 The results of T cell killing of SNU-620 cells after labeling with 42 sugars and Hu7v3 antibody

[0311] Example 5 In vivo tumor inhibition experiment in mouse model

[0312] The γδT cells labeled with rituximab targeting CD20 (CD20-γδT) prepared in Example 3 were effector cells that inhibited tumors in a mouse model, and the tumor cells in the mice were Raji-Luc cells.

[0313] To expand Raji-Luc cells, 6 × 10 cells were injected into the tail vein of NPG mice 2 days in advance. 5 cells; the drugs were administered on days 0, 2, 4, and 6, respectively. The PBS group was administered with 100 μL PBS, and the other groups were administered with the corresponding 5×10 6 The cells were dissolved in 100uL PBS and administered; bioluminescence imaging of the mice was performed on days 8, 16, and 24; and the death of the mice was recorded.

[0314] Bioluminescence imaging (Figures 23, 25, and 27) demonstrates that the CD20-γδT product prepared with 13 saccharides significantly inhibited tumor growth in mice compared to the control group. Survival curves (Figures 24, 26, and 28) demonstrate that the CD20-γδT product prepared with 13 saccharides significantly prolonged mouse survival compared to the PBS group.

[0315] The γδT cells labeled with hu7v3 targeting Claudin18.2 prepared in Example 3 (Claudin18.2-γδT) were used as effector cells for tumor suppression in a mouse model, and the tumor cells in the mice were SNU-620-Luc cells.

[0316] SNU-620-Luc cells were expanded and injected into the tail vein of NSG mice 2 days in advance, with 5×10 cells per mouse. 6 cells; the drugs were administered on days 0, 2, 4, 6, and 8, respectively. The PBS group was administered with 100 μL PBS, and the other groups were administered with the corresponding 5×10 6 The cells were dissolved in 100uL PBS and administered; the tumor volume was measured and calculated on days 0, 8, 16, 24, 32, 40, and 48; and the death of the mice was recorded.

[0317] From the tumor volume data (Figures 29 to 31), compared with the control group, it can be concluded that the Claudin18.2-γδT products prepared from 13 sugars can significantly inhibit tumor growth in mice.

[0318] Figure legend: "20-CD20-γδT" represents γδT linked to the CD20 antibody via the non-natural sugar numbered 20. Similarly, PBS is the solvent control group, and γδT represents the cell control group without antibody connection.

Claims

1. A method for preparing a modified cell, comprising: Step S: contacting an immune cell expressing a monosaccharide derivative containing a first functional group on its surface with an antigen binding protein containing a second functional group, so that the first functional group and the second functional group undergo a connection reaction to obtain the modified cell; wherein, The monosaccharide derivative comprises any one or more structures selected from Formula I to Formula X: in, X is the first functional group, R1, R2, R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy, C 2～6 Alkoxycarbonyl and acyl, said group being optionally substituted by a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, mercapto and C 1-6 alkyl; R5 is selected from the group consisting of hydrogen, halogen, and substituted or unsubstituted C 1～6 an alkyl group optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto; R6 and R7 are each independently selected from the group consisting of halogen and substituted or unsubstituted C 1～10 an alkyl group optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto; R8 is selected from the group consisting of halogen, substituted or unsubstituted C 1～10 Alkyl, phenyl and substituted or unsubstituted C 1～10 Alkylene-phenyl, the alkyl group optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto; Z1 and Z2 are each independently selected from: O and S; n and m are each independently selected from the following integers: 1, 2, 3, 4, 5 and 6. 2 . The method according to claim 1 , further comprising step S1 : contacting the monosaccharide derivative comprising the first functional group with immune cells to obtain the immune cells expressing the monosaccharide derivative comprising the first functional group on the surface.

3. The method according to any one of claims 1 to 2, further comprising step S2: contacting the antigen binding protein with a compound comprising a second functional group to obtain the antigen binding protein comprising the second functional group.

4. The method according to any one of claims 1 to 3, wherein R1, R2, R3 and R4 are each independently selected from the group consisting of: hydrogen and -C(=O)-C 1～6 Alkyl, preferably hydrogen and -C(=O)-CH3.

5. The method according to any one of claims 1 to 4, wherein R6 and R7 are each independently selected from substituted or unsubstituted C 1～10 alkyl.

6. The method according to any one of claims 1 to 5, wherein R8 is selected from the group consisting of: methyl, ethyl and -CH2-phenyl.

7. The method according to any one of claims 1 to 6, wherein n and m are each independently selected from the following integers: 1, 2 and 3.

8. The method according to any one of claims 1 to 7, wherein the first functional group and / or the second functional group is a group having bioorthogonal reaction activity, preferably, the bioorthogonal reaction comprises at least one selected from the group consisting of click chemistry, photocrosslinking, photosensitization and glycosylation.

9. The method according to claim 8, wherein the first functional group comprises one or more selected from the group consisting of an azide group, an alkynyl group, a trans-cyclooctene group, a triazine group, a tetrazine group, a hydrazide group, a cyclopropene group, an isocyanide group, and a cyclooctyne group.

10. The method according to any one of claims 8-9, wherein the second functional group comprises one or more selected from the group consisting of an azide group, an alkynyl group, a trans-cyclooctene group, a triazine group, a tetrazine group, a hydrazide group, a cyclopropene group, an isocyanide group, and a cyclooctyne group.

11. The method according to any one of claims 1 to 10, wherein X is an azido group.

12. The method according to any one of claims 1 to 11, wherein the first functional group is an azido group, and the second functional group is 13. The method according to any one of claims 1 to 12, wherein the monosaccharide derivative comprising a first functional group is selected from any one of the following groups:

14. The method according to any one of claims 3 to 13, wherein the compound comprising a second functional group further comprises a group capable of being covalently linked to a thiol group, preferably, the group capable of being covalently linked to a thiol group is a succinimide active ester group, a maleimide group or a maleic anhydride group.

15. The method according to any one of claims 3 to 14, wherein the structure of the compound comprising the second functional group is as shown in Formula L1, Formula L2 or Formula L3: in, Y is the second functional group, R L Selected from the group consisting of polyethylene glycol, amide group and alkyl ether group, preferably, R L Selected from C 1～20 Alkylene or C 1～20 Alkylene ether group.

16. The method according to claim 15, wherein the compound of formula L1 in step S2 is DBCO-PEG4-NHS:

17. The method according to claim 16, wherein the compound of formula L2 in step S2 is DBCO-PEG4-MAL:

18. The method according to any one of claims 1-17, wherein the immune cells comprise NK cells, DC cells, NKT cells, monocytes, macrophages, T cells and / or B cells.

19. The method according to any one of claims 1-18, wherein the immune cell is a T cell.

20. The method according to any one of claims 1-19, wherein the immune cells are γδ T cells. The method according to claim 20 , wherein the γδ T cells comprise Vγ9Vδ2 T cells and / or Vδ1 T cells.

22. The method of any one of claims 1-21, wherein the antigen binding protein comprises an antibody or an antigen binding fragment thereof.

23. The method of claim 22, wherein the antigen binding protein comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

24. The method according to any one of claims 1-23, wherein the antigen binding protein is capable of specifically binding to a tumor antigen.

25. The method of claim 24, wherein the tumor antigen comprises a tumor-associated antigen and / or a tumor-specific antigen.

26. The method of claim 24 or 25, wherein the tumor antigen is selected from the group consisting of CD20 and claudin18.

2.

27. The method according to any one of claims 1-26, wherein the immune cells are derived from human peripheral blood.

28. The method according to any one of claims 1 to 27, wherein the contact time in step S is about 10 minutes to 60 minutes.

29. The method according to any one of claims 2 to 28, wherein the contact time in step S1 is about 2 hours to 48 hours.

30. The method according to any one of claims 3 to 29, wherein the contact time in step S2 is about 30 minutes to 6 hours.

31. A modified cell prepared according to the method of any one of claims 1-30.

32. A modified cell, which is linked to an antigen binding protein on its surface via a monosaccharide derivative containing an azide group, wherein: The structure of the monosaccharide derivative containing an azide group is selected from one or more of the following groups: in, X is an azide group, R1, R2, R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy, C 2～6 Alkoxycarbonyl and acyl, said group being optionally substituted by a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, mercapto and C 1-6 alkyl; R5 is selected from the group consisting of hydrogen, halogen, and substituted or unsubstituted C 1～6 an alkyl group optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto; R6 and R7 are each independently selected from the group consisting of halogen and substituted or unsubstituted C 1～10 an alkyl group optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto; R8 is selected from the group consisting of halogen, substituted or unsubstituted C 1～10 Alkyl, phenyl and substituted or unsubstituted C 1～10 Alkylene-phenyl, the alkyl group optionally substituted with a substituent selected from the group consisting of halogen, hydroxy, amino, nitro, and mercapto; Z1 and Z2 are each independently selected from: O and S; n and m are each independently selected from the following integers: 1, 2, 3, 4, 5 and 6.

33. The method of claim 32, wherein R1, R2, R3 and R4 are each independently selected from the group consisting of hydrogen and -C(=O)-C 1～6 Alkyl, preferably hydrogen and -C(=O)-CH3.

34. The modified cell according to any one of claims 32-33, wherein R6 and R7 are each independently selected from substituted or unsubstituted C 1～10 alkyl.

35. The modified cell of any one of claims 32-34, wherein R8 is selected from the group consisting of methyl, ethyl and -CH2-phenyl.

36. The modified cell of any one of claims 32-35, wherein n and m are each independently selected from the following integers: 1, 2, and 3.

37. The modified cell according to any one of claims 32-36, wherein R1, R2, R3 and R4 are each independently selected from the group consisting of: hydrogen and -C(=O)-C 1～6 Alkyl, preferably hydrogen and -C(=O)-CH3.

38. The modified cell according to any one of claims 32-37, wherein R6 and R7 are each independently selected from substituted or unsubstituted C 1～10 alkyl.

39. The modified cell of any one of claims 32-38, wherein R8 is selected from the group consisting of: methyl, ethyl, and -CH2-phenyl.

40. The modified cell of any one of claims 32-39, wherein n and m are each independently selected from the following integers: 1, 2, and 3.

41. The modified cell according to any one of claims 32-40, wherein the monosaccharide derivative comprising a first functional group is selected from any one of the following groups:

42. The modified cell according to any one of claims 32 to 41, wherein the cell is an immune cell, preferably an immune cell capable of eliciting a cell-mediated immune response.

43. The modified cell according to any one of claims 32-42, wherein the cell is a NK cell (Natural Killer cell), a T cell and / or a γδ T cell.

44. The modified cell according to any one of claims 32-43, wherein the cell has an azide group expressed on the surface via a sugar metabolism pathway by absorbing the monosaccharide derivative.

45. The modified cell according to any one of claims 32-44, wherein the cell is linked to the antigen binding protein with a second functional group via the azide group of the monosaccharide derivative, preferably via a bond formed by a linking reaction between the azide group and the second functional group.

46. ​​A modified cell according to any one of claims 32-45, wherein the second functional group comprises 47. The modified cell according to any one of claims 32-46, wherein the structure of the second functional group is as shown in Formula L1, Formula L2 or Formula L3: in, Y is the second functional group, R L Selected from the group consisting of polyethylene glycol, amide group and alkyl ether group, preferably, R L Selected from C 1～20 Alkylene or C 1～20 Alkylene ether group.

48. The modified cell according to any one of claims 32-47, wherein the structure of the second functional group is selected from the group consisting of:

49. The modified cell of any one of claims 32-38, wherein the antigen binding protein comprises an antibody or an antigen binding fragment thereof.

50. The modified cell of any one of claims 32-49, wherein the antigen binding protein comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

51. The modified cell of any one of claims 32-50, wherein the antigen binding protein is capable of specifically binding to a tumor antigen.

52. The modified cell of any one of claims 32-51, wherein the tumor antigen comprises a tumor-associated antigen and / or a tumor-specific antigen.

53. The modified cell of any one of claims 32-52, wherein the antigen binding protein targets CD20 and claudin18.

2.

54. The modified cell according to any one of claims 32-53, wherein the antigen binding protein comprises an antibody heavy chain variable region HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

3.

55. The modified cell according to any one of claims 32-54, wherein the antigen binding protein comprises an antibody heavy chain variable region HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:

2.

56. The modified cell according to any one of claims 32-55, wherein the antigen binding protein comprises an antibody heavy chain variable region HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:

1.

57. The modified cell according to any one of claims 32-56, wherein the antigen binding protein comprises an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:

4.

58. The modified cell according to any one of claims 32-57, wherein the antigen binding protein comprises an antibody light chain variable region LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

7.

59. The modified cell according to any one of claims 32-58, wherein the antigen binding protein comprises an antibody light chain variable region LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:

6.

60. The modified cell according to any one of claims 32-59, wherein the antigen binding protein comprises an antibody light chain variable region LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:

5.

61. The modified cell according to any one of claims 32-60, wherein the antigen binding protein comprises an antibody light chain variable region, and the antibody light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

8.

62. The modified cell according to any one of claims 32-61, wherein the antigen binding protein comprises an antibody heavy chain variable region HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

11.

63. The modified cell according to any one of claims 31-62, wherein the antigen binding protein comprises an antibody heavy chain variable region HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:

10.

64. The modified cell according to any one of claims 32-63, wherein the antigen binding protein comprises an antibody heavy chain variable region HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:

9.

65. The modified cell according to any one of claims 32-64, wherein the antigen binding protein comprises an antibody heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:

12.

66. A pharmaceutical composition comprising the modified cell of any one of claims 31-65.

67. Use of the cell according to claim 66 in the preparation of a medicament for treating and / or alleviating a tumor.

68. The use according to claim 67, wherein the tumor comprises a tumor with high expression of CD20.

69. The use according to claim 67, wherein the tumor comprises a tumor with high expression of claudin 18.2.

Images