Method for evaluating generation of ADA by antibody-immune agonist conjugate and use thereof

Abstract

The present invention relates to the field of biomedicine, and in particular to a method for evaluating generation of an ADA by an immune agonist conjugate and a use thereof.

Description

Methods for evaluating ADA production by antibody-immunostimulant conjugates and their applications Technical Field

[0001] This article relates to the biomedical field, particularly to the methods and applications for evaluating antibody-drug antibody (ADA) conjugates. Background Technology

[0002] Immunotherapy, a novel approach to cancer treatment, has demonstrated significant efficacy. Immune checkpoint inhibitors, represented by monoclonal antibodies such as CLTA-4 and PD-1 / L1, are primarily T-cell-based therapies and have been approved for treating various cancers. Simultaneously, efforts are being made to explore other mechanisms by which the immune system fights cancer. Targeting myeloid cells (primarily macrophages and dendritic cells (DCs)) has emerged as a promising therapeutic direction. Activating macrophages and DCs through agonists or macrophage checkpoint inhibitors not only enhances their ability to phagocytose and clear tumor cells but also strengthens their antigen-presenting function, thereby more effectively stimulating adaptive anti-tumor immunity. However, the side effects caused by systemic administration of immune agonists limit their application in cancer. Therefore, antibody-immune agonist conjugates (AIACs) not only effectively enhance the immune system's ability to attack and clear tumor cells but also reduce systemic nonspecific immune side effects.

[0003] However, compared to traditional antibody drugs, antibody-immunostimulant conjugates (AICs), while enhancing the anti-tumor effects of the immune system, also amplify drug-related immune risks. The development of drug antibodies is currently one of the biggest challenges facing AICs in clinical trials. Therefore, understanding the immunogenicity of new drug candidates in the pre-clinical stage would make clinical research safer and increase the success rate of development.

[0004] Therapeutic protein drugs often exhibit species-specific differences, and predicting human immunogenicity based on animal immunogenicity studies has limitations. Compared to traditional therapeutic protein drugs, antibody-immunoagonal conjugates (AICs) may further amplify interspecies immune differences, leading to more complex and difficult-to-interpret immunogenicity-related responses. Furthermore, preclinical studies often utilize computer simulations to predict drug immunogenic epitopes and the immunogenicity potential of preclinical therapeutic candidates; however, this method is limited to conventional peptide drugs, and computer tools struggle to predict the immunogenicity potential of AICs. T-cell proliferation assays are another technique that can be used to predict the immunogenicity potential of preclinical therapeutic candidates, but they are currently not used for the immunogenicity assay of antibody-immunoagonal conjugates.

[0005] In conventional methods, the immunogenicity of a drug is determined by co-incubating the test drug with frozen peripheral blood mononuclear cells (PBMCs) for at least 5-7 days, or even longer, and by measuring the BrdU (5-bromo-2′-deoxyuridine)-positive CD4+. + The detection method uses T cells. However, this method is time-consuming and produces weak positive signals. Furthermore, using IL2-secreting T cells results in a low percentage of positive results, large errors, and difficulty in accurately comparing the immunogenicity of different drugs. Therefore, there is a need in the art for a method to assess antibody-immunoagonal conjugate-induced ADA production that is more time-efficient and reliable. Summary of the Invention

[0006] On one hand, the present invention provides a method for evaluating the production of drug antibodies (ADA) induced by immune agonist conjugates, comprising the following steps:

[0007] 1) Collect PBMCs (peripheral blood mononuclear cells) collected within 24 hours, remove CD8 cells and CD25 cells to obtain PBMCs. CD8-CD25- cell;

[0008] 2) Incubate the PBMC CD8-CD25- Cells were incubated and then co-cultured with an immune agonist conjugate.

[0009] 3) EdU (5-ethynyl-2'-deoxyuridine) was used for labeling, and proliferating CD4 cells were screened after labeling. + CD3 + T cells (Th cells), also known as EdU + CD4 + CD3 + T cells;

[0010] 4) Through the EdU + CD4 + CD3 + T (proliferating CD4) + CD3 + T cells account for a significant portion of total CD4+. + CD3 + The percentage of T cells was used to assess the probability of ADA production induced by immune agonist conjugates.

[0011] In some embodiments, the immune agonist conjugate comprises a targeting molecule, a linker, and an immune agonist. In some embodiments, the targeting molecule is an antibody or an antigen-binding fragment thereof.

[0012] In some implementations, EdU in step 4) + CD4 + CD3 + T cells account for a significant portion of total CD4+.+ CD3 + Data processing of T cell percentage: EdU obtained from co-culture of immune agonist conjugates. + CD4 + CD3 + T cells account for a significant portion of total CD4+. + CD3 + The percentage of T cells divided by EdU in the blank control group + CD4 + CD3 + T cells account for a significant portion of total CD4+. + CD3 + The percentage of T cells is used to obtain the immune stimulation coefficient.

[0013] In some implementations, the immune agonist conjugate is an antibody-immunoagonist conjugate.

[0014] In some implementation schemes, multiple positive and negative controls with known clinical immunogenicity are set to establish a critical value for the immunostimulation coefficient. When the immunostimulation coefficient obtained from co-culturing the immunostimulation conjugate is greater than the critical value, the immunostimulation conjugate will induce drug antibody production with a high probability; when the immunostimulation coefficient obtained from co-culturing the immunostimulation conjugate is less than or equal to the critical value, the immunostimulation conjugate will induce or not induce drug antibody production with a low probability.

[0015] In some embodiments, the positive control is selected from one or more of the following groups: keyhole limpet hemocyanin (KLH), the structure of formula (I-1), and the structure of formula (I-2);

[0016] In formula (I-1), A is trastuzumab, z is 4, and the drug-antibody ratio (DAR) is approximately 4.

[0017] In formula (I-2), Ab is pertuzumab, z is 4, and the drug-antibody ratio (DAR) is approximately 4.

[0018] In some embodiments, the negative control is selected from one or more of the following groups: etanercept, structures of formula (I-3), and structures of formula (I-4);

[0019] In formula (I-3), Ab is trastuzumab, z is 4, and its drug-antibody ratio (DAR) is approximately 4.

[0020] In formula (I-4), A is the modified trastuzumab, with GALPETGG (SEQ ID NO:1) introduced into the C-terminus of the trastuzumab light chain, where GALPETGG (SEQ ID NO:2) is the recognition sequence of the ligase donor substrate, and GA is the spacer sequence; z is 2, and its drug-antibody ratio (DAR) is approximately 1.74.

[0021] In some implementations, the high probability is 90%-100%. In some implementations, the high probability is 90%, 91%, 92%, 93%, 93.5%, 94%, 94.3%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0022] In some embodiments, the low probability is 0-10% (e.g., 0.001%-10%). In some embodiments, the low probability is 0.001%, 0.008%, 0.1%, 0.8%, 1.4%, 2.6%, 3.3%, 4.2%, 5%, 5.5%, 5.8%, 6%, 7.1%, 7.3%, 8.4%, 9.8%, or 10%.

[0023] In some embodiments, the number of positive controls is at least two, such as two, three, four, five, six, seven, eight, or nine. In some embodiments, the number of negative controls is at least two, such as two, three, four, five, six, seven, eight, or nine.

[0024] In some implementations, the number of PBMC cell-derived donors is at least two, such as two, three, four, five, six, seven, eight, nine, ten, eleven, or twenty.

[0025] In some implementations, the probability of clinically generating ADA after administration of the immune agonist conjugate is M / T; where M is the number of patients who received the medication with an immune stimulation coefficient greater than the critical value of the immune stimulation coefficient, and T is the total number of patients who received the medication.

[0026] In some embodiments, the critical value of the immunostimulation coefficient is 1.2-2.5. In some embodiments, the critical value of the immunostimulation coefficient is 1.2, 1.29, 1.3, 1.42, 1.48, 1.5, 1.54, 1.58, 1.59, 1.6, 1.62, 1.63, 1.64, 1.66, 1.7, 1.75, 1.8, 1.9, 2.0, 2.05, 2.14, 2.25, 2.3, 2.42, or 2.5.

[0027] In some implementations, in step 2), PBMC CD8-CD25-The co-culture time of cells with the immune agonist conjugate is 66-78 hours. In some implementation schemes, PBMCs... CD8-CD25- The co-culture times of cells with the immune agonist conjugate are 66 h, 66.8 h, 67 h, 68 h, 68.4 h, 68.5 h, 69 h, 69.4 h, 69.6 h, 70 h, 70.5 h, 70.8 h, 71 h, 71.4 h, 71.8 h, 71.9 h, 72 h, 72.2 h, 72.3 h, 72.4 h, 72.8 h, 73 h, 73.4 h, 74 h, 74.5 h, 75 h, 75.8 h, 76 h, 77 h, or 78 h. In some implementations, PBMCs... CD8-CD25- The co-culture time of cells and immune agonist conjugates is 70-74 hours.

[0028] In some embodiments, the immune agonist conjugate includes a structure of formula (II):

[0029] Where n is an integer from 2 to 10, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0030] Lm2 is or mixtures thereof;

[0031] x is selected from hydrogen, OH, NH2, an amino acid fragment containing 1-10 amino acids, or a nucleotide fragment containing 1-10 nucleotides;

[0032] Lk is L 1 -L 2 -L 3 The combination;

[0033] L 1 and L 3 Each is selected independently from:

[0034] -CH2-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-; and C 1-4 Alkylene groups in combination with one or more of the following groups: -CH2-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-;

[0035] L 2 Does not exist or C 7-34 Alkylene, wherein one or more of the -CH2- structures in the alkylene are optionally replaced by -O-, and the alkylene is optionally replaced by -NH-, -OH or -NH(CO)CH3;

[0036] Y and W are either absent independently, or p-aminobenzyloxycarbonyl groups ( PABC) or spacers or combinations thereof containing 1-10 amino acids (preferably 1-6 amino acids, more preferably 1-4 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids);

[0037] B2 is selected from the following groups or combinations thereof: -(CH2) k C(O)-、-(CH2) k C(O)-Val-Cit-PABC-、-(CH2) k C(O)-Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d -、-(CH2) k1 C(O)-NH-(C2H4-O) j -(CH2) k2 C(O)-Lys-、-(CH2) k C(O)-NH-(C2H4-O) j -、-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -NH-(C2H4-O) j -、-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -;

[0038] a and b are each independently 0 or 1;

[0039] Each k, k1, and k2 is an integer from 0 to 10, preferably 0, 1, or 2; especially 1 or 2.

[0040] d is an integer from 1 to 10, especially 1 or 2;

[0041] j is an integer from 1 to 10, especially 1, 3 or 4;

[0042] R 1 and R 2 Each is independently selected from: hydrogen, -OH, -NH2, -C 1-6 Alkyl, -OC 1-6 Alkyl, -NH-C 1- 6-alkyl, -C 1-6 Alkyl-NH2, -N(C) 1-6 alkyl)-C 1-6 Alkyl, -C 1-6 Alkyl-OC1-6 Alkyl, -C 1-6 Alkyl-NH-C 1-6 Alkyl, -C(O)-C 1-6 Alkyl group, -NHC(O)-C 1-6 Alkyl, -C(O)-NH2, -C(O)NH-C 1-6 Alkyl, -C(O)N(C) 1-6 alkyl)-C 1-6 Alkyl group, -S(=O)2-C 1-6 Alkyl group, -NHS(=O)2-C 1-6 Alkyl group, -S(=O)2O-C 1- 6-alkyl, -S(=O)2NH-C 1-6 Alkyl groups and -S(=O)2N(C) 1-6 alkyl)-C 1-6 alkyl;

[0043] PL is an immune agonist;

[0044] z is an integer or non-integer from 1 to 20. For example, z is 1, 2, 3, 5, 6, 8, 9, 10 or 20.

[0045] A is a target molecule; preferably, the target molecule is an antibody or antigen-binding fragment; the target molecule is modified to contain a Sortase enzyme recognition sequence, and the target molecule is coupled to (Gly)n under the action of Sortase enzyme, where n is an integer from 2 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0046] In some embodiments, the targeting molecule is linked to the sortase enzyme recognition sequence via a spacer sequence. In some embodiments, the spacer sequence is GA or (GGGGS). o (SEQ ID NO:3); where o is an integer from 1 to 4.

[0047] In some embodiments, the immune agonist conjugate comprises a structure of formula (II-1), a structure of formula (II-2), or a mixture thereof:

[0048] in,

[0049] A, B2, PL, and z are as defined above.

[0050] In some implementations, Lk is L 1 -L 2 -L 3 L 1 It is -NH-, L 3 It is -C(O)-, L 2It is -(C2H4-O) i -C2H4-.

[0051] In some implementations, i is an integer from 2 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10, especially 4.

[0052] In some implementations, B2 is -(CH2). k C(O)-. In some implementations, B2 is -(CH2). k C(O)-Val-Cit-PABC. In some implementations, B2 is -(CH2). k C(O)-Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d - In some implementations, B2 is -(CH2). k C(O)-NH-(C2H4-O) j - In some implementations, B2 is -(CH2). k1 C(O)-NH-(C2H4-O) j -(CH2) k2 C(O)-Lys-. In some implementations, B2 is -(CH2). k C(O)-(NH-CR 1 R 2 -C(O)) d -NH-(C2H4-O) j - In some implementations, B2 is -(CH2). k C(O)-(NH-CR 1 R 2 -C(O)) d -

[0053] In some implementations, each k, k1, and k2 is independently 1 or 2.

[0054] In some implementations, Y and W are independently selected from leucine (Leu), glutamine (Gln), PABC, Phe-Lys-PABC, Val-Cit-PABC, and Val-Lys-PABC.

[0055] In some implementations, a and b are 0.

[0056] In some implementation schemes, R 1 and R 2 Each is independently hydrogen or -C 1-6 Alkyl; preferably, R 1 and R 2Each is either hydrogen or -C independently. 1-6 Alkyl; more preferably, R 1 and R 2 They are all methyl groups.

[0057] In some implementations, x is NH2.

[0058] In some embodiments, the immune agonist is selected from imidazoquinoline. In some embodiments, the immune agonist has the structure of formula i.

[0059] in,

[0060] Each R 9 Independently selected from hydrogen, halogen, -C 1-7 Alkyl-OC(O)-C 1-7 Alkyl, -C 1-7 Alkyl-OC(O)-C 2-7 Alkenyl and 5-7 membered heterocyclic groups;

[0061] R 10 and R 11 Each is independently selected from hydrogen and -C. 1-7 alkyl;

[0062] R 12 Selected from -C 1-7 Alkyl, -C 1-7 Alkoxy-C 1-7 Alkyl or -C 1-7 Alkyl-C 1-7 Alkoxy;

[0063] R 13 Selected from hydrogen or -C 1-7 Alkyl, the -C 1-7 The alkyl group may optionally be substituted with a substituent selected from -OH and -NH2;

[0064] u can be 1, 2, 3, or 4.

[0065] In some implementation schemes, R 9 It is hydrogen. In other embodiments, R 9 It is a 5-7 membered heterocyclic group. In some specific implementations, R 9 It is piperazine-based. In other embodiments, R... 10 For hydrogen. In some other implementations, R 11 It is hydrogen.

[0066] In some implementation schemes, R 12 Selected from -C 1-7 Alkyl or -C 1-7 Alkyl-C 1-7Alkyl group. In some specific implementations, R 12 Selected from Methyl, ethyl, or butyl. In some embodiments, R 12 for In other implementations, R 12 It is methyl. In some other embodiments, R... 12 It is ethyl. In some other embodiments, R 12 It is butyl.

[0067] In some implementation schemes, R 13 Selected from hydrogen, In some implementation schemes, R 13 It is hydrogen. In other embodiments, R 13 for In some other implementation schemes, R 13 for

[0068] In some embodiments, the immune agonist is selected from any one of compounds i-1 to i-5:

[0069] In some embodiments, the immune agonist has the structure of formula ii:

[0070] in,

[0071] L 4 Selected from -CH2-, -NH-, -O-, or -C(O)-;

[0072] R 14 Selected from -C 1-7 Alkyl, -C 1-7 Alkoxy, -C 1-7 Alkyl-OC 1-7 Alkyl or -C 1-7 Alkyl-OC(O)-C 1-7 alkyl;

[0073] R 15 and R 16 Each is independently selected from hydrogen or -C. 1-7 alkyl;

[0074] R 17 Selected from -NH2, -OH, -C 1-7 Alkyl, -C 1-7 Alkoxy or NH-C 1-7 alkyl;

[0075] R 18Selected from -CH2-aryl or -CH2-heteroaryl, wherein the aryl and the heteroaryl are each optionally substituted independently by a substituent selected from -C(O)OH and / or

[0076] In some implementations, L 4 It is -O-.

[0077] In some implementation schemes, R 14 Selected from -C 1-7 Alkyl or -C 1-7 Alkyl-OC 1-7 Alkyl group. In some specific embodiments, R 14 For butyl. In some other specific embodiments, R 14 for

[0078] In some implementation schemes, R 15 It is hydrogen. In other embodiments, R 16 It is hydrogen.

[0079] In some implementation schemes, R 17 It is -OH.

[0080] In some specific implementation schemes, R 18 Selected from

[0081] In some embodiments, the immune agonist is selected from compounds ii-1 or ii-2:

[0082] In some embodiments, the immune agonist has the structure of formula iii:

[0083] in,

[0084] R 19 Selected from -OH, -NH2, -C 1-7 Alkyl, -C 1-7 Alkoxy or -NH-C 1-7 alkyl;

[0085] R 20 Selected from -CH2-aryl, wherein the aryl group is optionally substituted by two substituents selected from: -OH, -C 1-7 Alkoxy, -C 1-7 alkyl-piperidinyl and -C 1-7 Alkyl-piperazinyl.

[0086] In some implementation schemes, R 19 -NH-C 1-7Alkyl group. In some specific embodiments, R 19 for

[0087] In other specific implementation schemes, R 20 for

[0088] In some embodiments, the immune agonist is compound iii-1:

[0089] In some embodiments, the immune agonist has the structure of formula iv:

[0090] in,

[0091] L 5 Selected from -CH2-, -NH-, -C(O)-, -NHC(O)-, or -C(O)NH-;

[0092] R 21 Selected from Where B is a heteroaryl ring;

[0093] R 22 Selected from hydrogen or -C 1-7 alkyl;

[0094] R 23 and R 24 Each is independently selected from hydrogen or -C. 1-7 alkyl;

[0095] L 6 Selected from -CH2- or -C(O)-;

[0096] R 25 Selected from -N(C 1-7 Alkyl)(C 1-7 alkyl);

[0097] R 26 R 27 and R 28 Each is independently selected from hydrogen or -C. 1-7 alkyl;

[0098] v is 1, 2, or 3.

[0099] In some specific implementation schemes, L 5 Selected from -NHC(O)- or -C(O)NH-. In some other specific embodiments, L 5 It is -NHC(O)-.

[0100] In some embodiments, B is a pyridine ring. In some specific embodiments, R... 21for

[0101] In some specific implementation schemes, R 23 For hydrogen. In some other specific implementations, R 24 It is hydrogen.

[0102] In some implementations, L 6 It is -C(O)-.

[0103] In some implementation schemes, R 25 for

[0104] In some implementation schemes, R 26 It is hydrogen. In other embodiments, R 27 For hydrogen. In some other implementations, R 28 It is hydrogen.

[0105] In some embodiments, the immune agonist is compound IV-1:

[0106] In some embodiments, the immune agonist conjugate is selected from one or a mixture of the following structures:

[0107] in,

[0108] Lm2, B2, PL and z are as defined above;

[0109] The modified target molecule A contains a Sortase enzyme recognition sequence. Under the action of the Sortase enzyme, the target molecule A is coupled with (Gly)n, where n is an integer from 2 to 10.

[0110] In some embodiments, the immune agonist conjugate is selected from the following structures:

[0111] in,

[0112] z is as defined above.

[0113] In some embodiments, the immune agonist conjugate comprises a compound with the structure (AC102-6-1), a compound with the structure (AC102-6-1'), or a mixture thereof:

[0114] The value of z is 0.6-4, for example, 0.6, 0.7, 0.8, 0.9, 1, 1.3, 1.4, 1.73, 1.8, 1.9, 2, 2.1, 2.2, 2.4, 3 or 4.

[0115] In some embodiments, the immune agonist conjugate comprises a compound with the structure (AC102-8-1), a compound with the structure (AC102-8-1'), or a mixture thereof:

[0116] The value of z is 0.6-4, for example, 0.6, 0.7, 0.8, 0.9, 1, 1.3, 1.4, 1.73, 1.8, 1.9, 2, 2.1, 2.2, 2.4, 3 or 4.

[0117] In some embodiments, the immune agonist conjugate is selected from compounds with the structures shown in formula (I-1), formula (I-2), or formula (I-3); wherein A or Ab is a target molecule; preferably, the target molecule is an antibody or antigen-binding fragment.

[0118] In some embodiments, the sortase types include sorting enzyme A, sorting enzyme B, sorting enzyme C, sorting enzyme D, Lactobacillus plantarum sorting enzyme, etc. (see patent US20110321183A). In some embodiments, the sortase enzyme recognition sequence is LPETGG.

[0119] In some implementations, the target for the targeted molecule recognition is selected from one or more of the following groups: CD19, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KIT, CD123, CD138, CD142, CD174, CD227 / MUC1, CD352, CLDN18.2, DLL3, ErbB2 / HER2, CN33, GPNMB, ENPP3, Nectin-4 EGFRvIII, SLC44A4 / AGS-5, mesothelin, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, cadherin 6, FGFR2, FGFR3, CA6, CanAg, integrin αV, TDGF1, Ephrin A4, Trop2, PTK7, NOTCH3, C4.4A, FLT3, ROR1, ROR2, and ROR1 / 2.

[0120] In some embodiments, the targeting molecule is an antibody or its antigen-binding fragment. In some embodiments, the antibody is trastuzumab or pertuzumab. In some embodiments, the antibody is linked to a sortase enzyme recognition sequence via a spacer sequence. In some embodiments, the spacer sequence is GA or (GGGGS). o Where o is an integer from 1 to 4, such as 1, 2, 3, or 4. In some embodiments, the antibody heavy or light chain is terminated with a Sortase enzyme recognition sequence. In some embodiments, the C-terminus of the antibody heavy or light chain is sequentially connected with a spacer sequence and a Sortase enzyme recognition sequence. In some embodiments, the Sortase enzyme recognition sequence is LPETGG.

[0121] On the other hand, the present invention also provides a kit for evaluating the production of ADA induced by an immune agonist conjugate, the kit comprising: PBMC cells collected within 24 hours and an EdU detection reagent.

[0122] In some implementations, the immune agonist conjugate is an antibody-immunoagonist conjugate.

[0123] In some embodiments, the kit further includes: for screening PBMCs CD8-CD25- Cellular material; preferably, the material used for screening PBMCs CD8-CD25- The material used for the cells is magnetic beads.

[0124] In some embodiments, the kit further includes: for screening CD4 + CD3 + Materials for T cells; preferably, those used for screening CD4 + CD3 + The materials used for T cells are CD3 fluorescent antibodies and CD4 fluorescent antibodies.

[0125] In some embodiments, the kit further includes a positive control and a negative control. In some embodiments, the positive control is selected from one or more of the following: KLH, a compound with the structure of formula (I-1) and a compound with the structure of formula (I-2); in formula (I-1), A is trastuzumab, z is 4, and its DAR value is approximately 4; in formula (I-2), Ab is pertuzumab, z is 4, and its DAR value is approximately 4.

[0126] In some embodiments, the negative control is selected from one or more of the following groups: etanercept, compounds of formula (I-3) and formula (I-4); in formula (I-3), Ab is trastuzumab, z is 4, and its DAR value is approximately 4; in formula (I-4), A is modified trastuzumab with GALPETGG introduced into the C-terminus of the trastuzumab light chain, wherein LPETGG is the recognition sequence of the ligase donor substrate, and GA is the spacer sequence; z is 2, and its DAR value is approximately 1.74. Beneficial effects

[0127] Currently, there is no market-developed method for evaluating the clinical induction of ADA by immune agonist conjugates (ADAs), and assessment methods for ADA induction by ADA in clinical applications of ADA by immune agonist conjugates (especially antibody-immunoagonist conjugates) remain lacking. This invention provides a method for efficiently evaluating the clinical induction of ADA by immune agonist conjugates. This method shortens the culture time in T cell proliferation assays and increases the detection rate of positive signals, enabling rapid evaluation of the immunogenicity of immune agonist conjugates. This provides crucial reference for the early screening and clinical research of immune agonist conjugates (especially antibody-immunoagonist conjugates), and can significantly improve the success rate of ADA development. Attached Figure Description

[0128] Figure 1 shows the test drug and PBMCs. CD8-CD25- CD4 proliferated after co-culturing for 24 hours, 48 ​​hours, and 72 hours + CD3 + T cells account for a significant portion of total CD4+. + CD3 + The proportion of T cells.

[0129] Figure 2 shows the test drug and PBMCs. CD8-CD25- CD4 proliferation was achieved after co-culturing for 72, 96, and 120 hours. + CD3 + T cells account for a significant portion of total CD4+. + CD3 + The proportion of T cells.

[0130] Figure 3 shows the test drug and PBMCs from multiple donors. CD8-CD25- CD4 proliferation after 72 hours of co-culturing + CD3 + T cells account for a significant portion of total CD4+. + CD3 + The proportion of T cells; in the figure, "donor" represents the donor.

[0131] Figure 4 shows the test drug and PBMCs from multiple donors. CD8-CD25-Immunostimulation coefficients of each test drug after 72 hours of co-culture. Detailed Implementation

[0132] definition

[0133] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The term "technique" as used herein refers to techniques generally understood in the art, including variations and equivalent substitutions that are obvious to one of ordinary skill in the art. While it is believed that those skilled in the art will readily understand the following terms, the following definitions will better illustrate this disclosure. When a trade name is used herein, it refers to the corresponding product or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference.

[0134] When a specific quantity, concentration, or other value or parameter is described in the form of a range, preferred range, or preferred upper or lower limit, it should be understood that it is equivalent to specifically disclosing any range formed by combining any upper or preferred value with any lower or preferred value, whether or not the range is explicitly stated. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range as well as all integers and fractions (decimals) within that range. For example, the statement "i is an integer from 2 to 20" means that i is any integer from 2 to 20, such that i can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Other similar statements should be understood in a similar manner.

[0135] Unless otherwise stated herein, the singular forms such as “a” and “described” include the plural forms. The expressions “a or more” or “at least one” can refer to 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0136] When the terms “about” and “approximately” are used in conjunction with numerical variables, they generally mean that the value of the variable and all values ​​of the variable are within the experimental error range (e.g., within the 95% confidence interval of the mean) or within ±10% of the specified value, or within a wider range.

[0137] The terms “optional” or “optionally” mean that the events described below may or may not occur, and the description includes the possibility that the events or circumstances may or may not occur.

[0138] The expressions “comprising” or similar expressions “including,” “containing,” and “having” are open-ended and do not exclude additional unstated elements, steps, or components. The expression “consisting of” excludes any unspecified elements, steps, or components. The expression “substantially constitutes” means that the scope is limited to the specified elements, steps, or components, as well as elements, steps, or components that are optionally present and do not substantially affect the essential and novel features of the claimed subject matter. It should be understood that the expression “comprising” encompasses the expressions “substantially constitutes” and “consisting of”.

[0139] The term "targeting molecule" refers to a molecule that has an affinity for a specific target (such as a receptor, cell surface protein, cytokine, etc.). Targeting molecules can deliver payloads to specific sites in the body through targeted delivery. Targeting molecules can recognize one or more targets. The specific target site is defined by the target recognized by the targeting molecule. For example, a targeting molecule that targets a receptor can deliver a payload to a site containing a large portion of the receptor. Examples of targeting molecules include, but are not limited to, antibodies, antibody fragments, binding proteins against a given antigen, antibody mimics, scaffold proteins with affinity for a given target, ligands, etc.

[0140] As used herein, the term "antibody" is used broadly and specifically includes intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they possess the desired biological activity. Antibodies can belong to any subtype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass and can be derived from any suitable species. In some embodiments, antibodies are of human or mouse origin. Antibodies can also be fully humanized antibodies, humanized antibodies, or chimeric antibodies prepared by recombinant methods.

[0141] In this article, monoclonal antibodies are used to refer to antibodies obtained from a substantially homogeneous group of antibodies; that is, the individual antibodies constituting this group are identical, except for a small number of possible natural mutations. Monoclonal antibodies exhibit high specificity for antigenic sites. The term "monoclonal" signifies that the antibody is derived from a substantially homogeneous group of antibodies and should not be interpreted as requiring specific methods to prepare the antibody.

[0142] A complete or full-length antibody essentially contains one or more antigen-binding variable regions and one or more light chain constant regions (C10, C20, C30, C40, C50, C60, C70, C80, C9 ... L ) and one or more heavy chain constant regions (C H The one or more heavy chain constant regions may include C, depending on the antibody subtype. H 1. C H 2. C H 3 and / or C H4. The antigen-binding variable region (also known as the fragment variable region or Fv fragment) typically contains the light chain variable region (V... L ) and heavy chain variable region (V H Constant regions can be those with a natural sequence (such as a constant region with a human natural sequence) or a variant of its amino acid sequence. Variable regions recognize and interact with target antigens. Constant regions can be recognized and interacted with by the immune system.

[0143] Antibody fragments may contain a portion of a complete antibody, preferably an antigen-binding region or a variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and others derived from V... H and C H The Fab fragment consists of a domain-1 Fd fragment, an Fv fragment, a single-domain antibody (dAb) fragment, and a separated complementarity-determining region (CDR). The Fab fragment is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment with the same structure generated, for example, through recombinant expression. The Fab fragment contains a light chain (containing V... L and C L ) and another chain, wherein the other chain contains a variable structural domain (V) of the heavy chain. H ) and the constant region structural domain of the heavy chain (C H 1) The F(ab')2 fragment is an antibody fragment obtained by digesting immunoglobulins with pepsin at pH 4.0–4.5, or a fragment with the same structure generated, for example, through recombinant expression. The F(ab')2 fragment essentially comprises two Fab fragments, each heavy chain containing several additional amino acids, including cysteine ​​residues that form disulfide bonds linking the two fragments. The Fab' fragment is a fragment containing half of the F(ab')2 fragment (one heavy chain and one light chain). Antibody fragments may comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linker units. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, bifunctional antibodies, Fd and Fd' fragments, and other fragments, including modified fragments. Antibody fragments typically contain at least or about 50 amino acids and typically contain at least or about 200 amino acids. Antigen-binding fragments may include any antibody fragment inserted into an antibody framework (e.g., by substituting a corresponding region) to produce an immune-specific binding to an antigen.

[0144] The antibodies according to the present invention can be prepared using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or other techniques known in the art. For example, genetically engineered recombinant antibodies (or antibody mimics) can be expressed using a suitable culture system (e.g., E. coli or mammalian cells). Engineering can refer to, for example, introducing a ligase-specific recognition sequence at the end of the antibody.

[0145] Immune agonists are agonists that can induce or enhance an immune response against a tumor, such as by activating immune cells, including but not limited to dendritic cells (DCs), B cells, macrophages, NK cells, and T cells. Non-limiting examples of immune agonists known in the art include TLR agonists, including but not limited to agonists of TLR7 and / or TLR8 and / or TLR9 (e.g., imiquimod, remiquimod, 852A, and VTX-2337); and STING agonists (e.g., ADU-S100 and MK-1454).

[0146] The term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group composed of carbon and hydrogen atoms, which is linked to the rest of the molecule by a single bond. Alkyl groups can contain 1 to 20 carbon atoms (C1-C2). 20 Alkyl groups, such as C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C3 alkyl, C4 alkyl, and C3-C6 alkyl. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers. A divalent group is a group obtained by removing a hydrogen atom from a carbon atom having a free valence electron, from the corresponding monovalent group. A divalent group has two linkage sites that connect to the rest of the molecule. For example, "alkylene" or "trialkylene" refers to a straight-chain or branched saturated divalent hydrocarbon group. Examples of alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), and pentylene (-C5H6-). 10 -), Hexyl (-C6H) 12 -), 1-methylethylidene (-CH(CH3)CH2-), 2-methylethylidene (-CH2CH(CH3)-), methylpropylene, ethylpropylene, etc.

[0147] As used herein, when one group is combined with another, the connection of the groups can be linear or branched, as long as a chemically stable structure is formed. The structure formed by such combinations can be connected to other parts of the molecule via any suitable atom in the structure, preferably by designated chemical bonds. For example, when describing C… 1-4When an alkylene group is combined with any one of the following groups: -CH2-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-, C 1-4 Alkyl groups can form linear linkages with the above groups, such as C 1-4 Alkylene-CH2-, C 1-4 Alkylene-NH-, C 1-4 Alkylene-C(O)-, C 1-4 Alkylene-NHC(O)-, C 1-4 Alkylene -C(O)NH-, -CH2-C 1-4 Alkylene, -NH-C 1-4 Alkylene, -C(O)-C 1-4 Alkylene, -NHC(O)-C 1-4 Alkylene, -C(O)NH-C 1-4 Alkylene. The resulting divalent structure can be further attached to other parts of the molecule.

[0148] The term "heterocycle" (and its variations, such as "heterocyclic" or "heterocyclic group"), used alone or in combination with other terms, broadly refers to a single aliphatic ring, typically having 3 to 12 ring atoms, containing at least 2 carbon atoms and one or more, preferably 1 to 3, heteroatoms independently selected from oxygen, sulfur, nitrogen, and phosphorus, and combinations containing at least one of the aforementioned heteroatoms. Alternatively, the heterocycle defined above can be a polycyclic system (e.g., a bicyclic system) in which two or more rings may be fused, bridged, or spirocyclically linked together, wherein at least one such ring contains one or more heteroatoms independently selected from oxygen, sulfur, nitrogen, and phosphorus.

[0149] The heterocyclic group can be, for example, a four-membered ring, such as an azirrobutyl or oxobutyl ring; or a five-membered ring, such as tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoalkyl, pyrazolyl, pyrrolinyl, oxopyrrolyl, 2-oxoimidazolidin-1-yl; or a six-membered ring, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazinyl, 1,1-dioxo-1,2-thiazohexane-2-yl or trithiaalkyl; or a seven-membered ring, such as a diazacycloheptatrienyl ring. Optionally, the heterocyclic group can be benzofused.

[0150] Heterocyclic groups can be, but are not limited to, bicyclic heterocyclic groups, such as five-membered fused five-membered rings, such as hexahydrocyclopentane[c]pyrrole-2(1H)-yl; or five-membered fused six-membered rings, such as hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl.

[0151] As mentioned above, heterocycles can be unsaturated, meaning they can contain one or more double bonds, but are not limited to this. For example, unsaturated heterocycles containing nitrogen atoms can be 1,6-dihydropyrimidine, 1,2-dihydropyrimidine, 1,4-dihydropyrimidine, 1,6-dihydropyridine, 1,2-dihydropyridine, 1,4-dihydropyridine, 2,3-dihydro-1H-pyrrole, 3,4-dihydro-1H-pyrrole, 2,5-dihydro-1H-pyrrole, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl, or 4H-[1,4]thiazinyl rings; unsaturated heterocycles containing oxygen atoms can be 2H-pyran, 4H-pyran, or 2,3-dihydrofuran; and unsaturated heterocycles containing sulfur atoms can be 2H-thiopyran or 4H-thiopyran. Heterocycles can be, but are not limited to, benzofused heterocycles, such as dihydroisoquinoline rings.

[0152] The term "heteroaryl" should be understood to preferably refer to a monovalent monocyclic, bicyclic, or tricyclic aromatic ring system ("5- to 10-membered heteroaryl") having 5, 6, 7, 8, 9, or 10 ring atoms, particularly 5, 6, 9, or 10 ring atoms, and at least one of the ring atoms (preferably 1-4, more preferably 1, 2, or 3) can be the same or different heteroatoms, such as oxygen, nitrogen, or sulfur ring systems. Furthermore, in each case, the heteroaryl can be a benzofused heteroaryl. Specifically, the heteroaryl group is selected from the group consisting of: thiophene, furanyl, pyrrole, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, etc., and their benzo[derivatives], such as benzofuranyl, benzothiophene, benzooxazolyl, benzoisooxazolyl, benzoimidazolyl, benzotriazolyl, indazole, indolyl, isindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc., and their benzo[fused] derivatives, such as quinolinyl, quinazolinyl, isoquinolinyl, etc.; or acrylonitrile, indolazinyl, purine, etc., and their benzo[derivatives]; or cinolinyl, phthalazinyl, quinazolinyl, quinolinyl, naphthidyl, carbazole, acrylonitrile, etc.

[0153] The method of the present invention

[0154] On one hand, the present invention provides a method for evaluating the production of drug antibodies induced by an immune agonist conjugate, comprising the following steps:

[0155] 1) Collect PBMCs (peripheral blood mononuclear cells) collected within 24 hours, remove CD8 cells and CD25 cells to obtain PBMCs. CD8-CD25- cell;

[0156] 2) Incubate the PBMC CD8-CD25- Cells were incubated and then co-cultured with an immune agonist conjugate.

[0157] 3) EdU (5-ethynyl-2'-deoxyuridine) was used for labeling, and proliferating CD4 cells were screened after labeling. + CD3 + T cells (Th cells), also known as EdU + CD4 + CD3 + T cells;

[0158] 4) Through the EdU + CD4 + CD3 + T cells account for a significant portion of total CD4+. + CD3 + The percentage of T cells was used to assess the probability of ADA production induced by immune agonist conjugates.

[0159] In some embodiments, the immune agonist conjugate comprises a targeting molecule, a linker, and an immune agonist. In some embodiments, the targeting molecule is an antibody or an antigen-binding fragment thereof.

[0160] In some implementation schemes, total CD4 + CD3 + T cells were screened using CD3 and CD4 fluorescent antibody labeling.

[0161] In some implementations, EdU in step 4) + CD4 + CD3 + T cells account for a significant portion of total CD4+. + CD3 + Data processing of T cell percentage: EdU obtained from co-culture of immune agonist conjugates. + CD4 + CD3 + T cells account for a significant portion of total CD4+. + CD3 + The percentage of T cells divided by EdU in the blank control group + CD4 + CD3 + T cells account for a significant portion of total CD4+. + CD3 + The percentage of T cells is used to obtain the immune stimulation coefficient.

[0162] In some implementations, the immune agonist conjugate is an antibody-immunoagonist conjugate.

[0163] In some implementation schemes, multiple positive and negative controls with known clinical immunogenicity are set to establish a critical value for the immunostimulation coefficient. When the immunostimulation coefficient obtained from co-culturing the immunostimulation conjugate is greater than the critical value, the immunostimulation conjugate will induce drug antibody production with a high probability; when the immunostimulation coefficient obtained from co-culturing the immunostimulation conjugate is less than or equal to the critical value, the immunostimulation conjugate will induce or not induce drug antibody production with a low probability.

[0164] In some embodiments, the immunostimulation coefficient threshold is set such that at least 90% of the positive control samples have an immunostimulation coefficient greater than the threshold. In some embodiments, the immunostimulation coefficient threshold is set such that at least 90% of the negative control samples have an immunostimulation coefficient less than or equal to the threshold. In some embodiments, at least 90% of the KLH test samples have an immunostimulation coefficient greater than the threshold (e.g., 10 out of 11 samples meet this requirement, i.e., 10 / 11). In some embodiments, at least 90% of the test samples of the compound of formula (I-1) have an immunostimulation coefficient greater than the threshold (e.g., all 11 samples meet this requirement, i.e., 11 / 11). In some embodiments, at least 90% of the test samples of the compound of formula (I-2) have an immunostimulation coefficient greater than the threshold (e.g., all 11 samples meet this requirement, i.e., 11 / 11). In some embodiments, at least 90% of the test samples of compound (I-3) have an immunostimulation coefficient less than or equal to the immunostimulation coefficient threshold (e.g., 10 out of 11 samples meet the threshold, i.e., 10 / 11). In some embodiments, at least 90% of the test samples of compound (I-4) or AC102-6-1-1 have an immunostimulation coefficient less than or equal to the immunostimulation coefficient threshold (e.g., 10 out of 11 samples meet the threshold, i.e., 10 / 11).

[0165] In some implementations, the positive control is a drug with a clinically probable probability of producing ADA at least 90%. In other implementations, the negative control is a drug with a clinically probable probability of producing ADA less than 10%.

[0166] In some implementation schemes, the clinical probability of ADA being generated after administration of the immune agonist conjugate is M / T; where M is the number of patients who received the medication with an immune stimulation coefficient greater than the critical value of the immune stimulation coefficient, and T is the total number of patients who received the medication.

[0167] In some implementations, the positive control is selected from one or more of the following groups: keyhole limpet hemocyanin (KLH), the structure of formula (I-1), and the structure of formula (I-2);

[0168] In formula (I-1), A is trastuzumab, z is 4, and the drug-antibody ratio (DAR) is approximately 4.

[0169] In formula (I-2), Ab is pertuzumab, z is 4, and the drug-antibody ratio (DAR) is approximately 4.

[0170] In some implementations, the negative control is selected from one or more of the following groups: etanercept, the structure of formula (I-3), and the structure of formula (I-4);

[0171] In formula (I-3), Ab is trastuzumab, z is 4, and its DAR is approximately 4;

[0172] In formula (I-4), A is the modified trastuzumab, with GALPETGG (SEQ ID NO:1) introduced into the C-terminus of the trastuzumab light chain, where GALPETGG (SEQ ID NO:2) is the recognition sequence of the ligase donor substrate, and GA is the spacer sequence; z is 2, and its DAR is approximately 1.74.

[0173] In some implementations, the high probability is 90%-100%. In some implementations, the high probability is 90%, 91%, 92%, 93%, 93.5%, 94%, 94.3%, 95%, 96%, 97%, 98%, or 100%.

[0174] In some embodiments, the low probability is 0-10% (e.g., 0.001%-10%). In some embodiments, the low probability is 0.001%, 0.008%, 0.1%, 0.8%, 1.4%, 2.6%, 3.3%, 4.2%, 5%, 5.5%, 5.8%, 6%, 7.1%, 7.3%, 8.4%, 9.8%, or 10%.

[0175] In some embodiments, the positive controls are at least two, such as two, three, four, five, six, seven, eight, or nine. In some embodiments, the negative controls are at least two, such as two, three, four, five, six, seven, eight, or nine.

[0176] In some implementations, the number of PBMC cell donors is at least two, such as two, three, four, five, six, seven, eight, nine, ten, eleven, or twenty.

[0177] In some embodiments, the clinical probability of ADA occurring after administration of the immune agonist conjugate to patients is M / T; where M is the number of patients whose immunostimulation coefficient is greater than the critical value of the immunostimulation coefficient, and T is the total number of patients administered the conjugate. In some embodiments, the critical value of the immunostimulation coefficient is 1.2-2.5. In some embodiments, the critical value of the immunostimulation coefficient is 1.2, 1.29, 1.3, 1.42, 1.48, 1.5, 1.54, 1.58, 1.59, 1.6, 1.62, 1.63, 1.64, 1.66, 1.7, 1.75, 1.8, 1.9, 2.0, 2.05, 2.14, 2.25, 2.3, 2.42, or 2.5.

[0178] In some implementations, in step 2), PBMC CD8-CD25- The co-culture time of cells with the immune agonist conjugate is 66-78 hours. In some implementation schemes, PBMCs... CD8-CD25- The co-culture time of cells with the immune agonist conjugate is 66 h, 66.8 h, 67 h, 68 h, 68.4 h, 68.5 h, 69 h, 69.4 h, 69.6 h, 70 h, 70.5 h, 70.8 h, 71 h, 71.4 h, 71.8 h, 71.9 h, 72 h, 72.2 h, 72.3 h, 72.4 h, 72.8 h, 73 h, 73.4 h, 74 h, 74.5 h, 75 h, 75.8 h, 76 h, 77 h, or 78 h. In some preferred embodiments, PBMCs CD8- CD25- The co-culture time of cells and immune agonist conjugates is 70-74 hours.

[0179] The immune agonist and immune agonist conjugate of the present invention

[0180] The present invention also provides immune agonists for use in the methods of the present invention, and corresponding immune agonist conjugates.

[0181] In some embodiments, the immune agonist is selected from imidazoquinoline. In some embodiments, the immune agonist has the structure of formula i:

[0182] in,

[0183] Each R 9 Independently selected from hydrogen, halogen, -C 1-7 Alkyl-OC(O)-C 1-7 Alkyl, -C 1-7 Alkyl-OC(O)-C 2-7 Alkenyl and 5-7 membered heterocyclic groups;

[0184] R 10 and R 11Each is independently selected from hydrogen and -C. 1-7 alkyl;

[0185] R 12 Selected from -C 1-7 Alkyl, -C 1-7 Alkoxy-C 1-7 Alkyl or -C 1-7 Alkyl-C 1-7 Alkoxy;

[0186] R 13 Selected from hydrogen or -C 1-7 Alkyl, the -C 1-7 The alkyl group may optionally be substituted with a substituent selected from -OH and -NH2;

[0187] u can be 1, 2, 3, or 4.

[0188] In some implementation schemes, R 9 It is hydrogen. In other embodiments, R 9 It is a 5-7 membered heterocyclic group. In some specific implementations, R 9 It is piperazine-based. In other embodiments, R... 10 For hydrogen. In some other implementations, R 11 It is hydrogen.

[0189] In some implementation schemes, R 12 Selected from -C 1-7 Alkyl or -C 1-7 Alkyl-C 1-7 Alkyl group. In some specific implementations, R 12 Selected from Methyl, ethyl, or butyl. In some embodiments, R 12 for In other implementations, R 12 It is methyl. In some other embodiments, R... 12 It is ethyl. In some other embodiments, R 12 It is butyl.

[0190] In some implementation schemes, R 13 Selected from hydrogen, In some implementation schemes, R 13 It is hydrogen. In other embodiments, R 13 for In some other implementation schemes, R 13 for

[0191] In some embodiments, the immune agonist is selected from any one of compounds i-1 to i-5:

[0192] In some implementations, the immune agonist has the structure of formula ii:

[0193] in,

[0194] L 4 Selected from -CH2-, -NH-, -O-, or -C(O)-;

[0195] R 14 Selected from -C 1-7 Alkyl, -C 1-7 Alkoxy, -C 1-7 Alkyl-OC 1-7 Alkyl or -C 1-7 Alkyl-OC(O)-C 1-7 alkyl;

[0196] R 15 and R 16 Each is independently selected from hydrogen or -C. 1-7 alkyl;

[0197] R 17 Selected from -NH2, -OH, -C 1-7 Alkyl, -C 1-7 Alkoxy or -NH-C 1-7 alkyl;

[0198] R 18 Selected from -CH2-aryl or -CH2-heteroaryl, wherein the aryl and the heteroaryl are each optionally substituted independently by a substituent selected from -C(O)OH and / or

[0199] In some implementations, L 4 It is -O-.

[0200] In some implementation schemes, R 14 Selected from -C 1-7 Alkyl or -C 1-7 Alkyl-OC 1-7 Alkyl group. In some specific embodiments, R 14 For butyl. In some other specific embodiments, R 14 for

[0201] In some implementation schemes, R 15 It is hydrogen. In other embodiments, R 16 It is hydrogen.

[0202] In some implementation schemes, R 17 It is -OH.

[0203] In some specific implementation schemes, R 18 Selected from

[0204] In some implementations, the immune agonist is selected from compounds ii-1 or ii-2:

[0205] In some implementations, the immune agonist has the structure of formula iii:

[0206] in,

[0207] R 19 Selected from -OH, -NH2, -C 1-7 Alkyl, -C 1-7 Alkoxy or -NH-C 1-7 alkyl;

[0208] R 20 Selected from -CH2-aryl, wherein the aryl group is optionally substituted by two substituents selected from: -OH, -C 1-7 Alkoxy, -C 1-7 alkyl-piperidinyl and -C 1-7 Alkyl-piperazinyl.

[0209] In some implementation schemes, R 19 -NH-C 1-7 Alkyl group. In some specific embodiments, R 19 for

[0210] In other specific implementation schemes, R 20 for

[0211] In some embodiments, the immune agonist is compound iii-1:

[0212] In some implementations, the immune agonist has the structure of formula iv:

[0213] in,

[0214] L 5 Selected from -CH2-, -NH-, -C(O)-, -NHC(O)-, or -C(O)NH-;

[0215] R 21 Selected from Where B is a heteroaryl ring;

[0216] R 22 Selected from hydrogen or -C 1-7 alkyl;

[0217] R 23 and R 24 Each is independently selected from hydrogen or -C. 1-7 alkyl;

[0218] L 6 Selected from -CH2- or -C(O)-;

[0219] R 25 Selected from -N(C 1-7 Alkyl)(C 1-7 alkyl);

[0220] R 26 R 27 and R 28 Each is independently selected from hydrogen or -C. 1-7 alkyl;

[0221] v is 1, 2, or 3.

[0222] In some specific implementation schemes, L 5 Selected from -NHC(O)- or -C(O)NH-. In some other specific embodiments, L 5 It is -NHC(O)-.

[0223] In some embodiments, B is a pyridine ring. In some specific embodiments, R... 21 for

[0224] In some specific implementation schemes, R 23 For hydrogen. In some other specific implementations, R 24 It is hydrogen.

[0225] In some implementations, L 6 It is -C(O)-.

[0226] In some implementation schemes, R 25 for

[0227] In some implementation schemes, R 26 It is hydrogen. In other embodiments, R 27 For hydrogen. In some other implementations, R 28 It is hydrogen.

[0228] In some implementations, the immune agonist is compound IV-1:

[0229] In some implementations, the immune agonist conjugate includes a structure of formula (II):

[0230] Where n is an integer from 2 to 10, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0231] Lm2 is or mixtures thereof;

[0232] x is selected from hydrogen, OH, NH2, an amino acid fragment containing 1-10 amino acids, or a nucleotide fragment containing 1-10 nucleotides;

[0233] Lk is L 1 -L 2 -L 3 The combination;

[0234] L 1 and L 3 Each is selected independently from:

[0235] -CH2-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-; and C 1-4 Alkylene groups in combination with one or more of the following groups: -CH2-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-;

[0236] L 2 Does not exist or C 7-34 Alkylene, wherein one or more of the -CH2- structures in the alkylene are optionally replaced by -O-, and the alkylene is optionally replaced by -NH-, -OH or -NH(CO)CH3;

[0237] Y and W are either absent independently, or p-aminobenzyloxycarbonyl groups ( PABC) or spacers or combinations thereof containing 1-10 amino acids (preferably 1-6 amino acids, more preferably 1-4 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids);

[0238] B2 is selected from the following groups or combinations thereof: -(CH2) k C(O)-、-(CH2) k C(O)-Val-Cit-PABC-、-(CH2) k C(O)-Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d -、-(CH2) k1 C(O)-NH-(C2H4-O) j -(CH2) k2 C(O)-Lys-、-(CH2) kC(O)-NH-(C2H4-O) j -、-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -NH-(C2H4-O) j -、-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -;

[0239] a and b are each independently 0 or 1;

[0240] Each k, k1, and k2 is an integer from 0 to 10, preferably 0, 1, or 2; especially 1 or 2.

[0241] d is an integer from 1 to 10, especially 1 or 2;

[0242] j is an integer from 1 to 10, especially 1, 3 or 4;

[0243] R 1 and R 2 Each is independently selected from: hydrogen, -OH, -NH2, -C 1-6 Alkyl, -OC 1-6 Alkyl, -NH-C 1- 6-alkyl, -C 1-6 Alkyl-NH2, -N(C) 1-6 alkyl)-C 1-6 Alkyl, -C 1-6 Alkyl-OC 1-6 Alkyl, -C 1-6 Alkyl-NH-C 1-6 Alkyl, -C(O)-C 1-6 Alkyl group, -NHC(O)-C 1-6 Alkyl, -C(O)-NH2, -C(O)NH-C 1-6 Alkyl, -C(O)N(C) 1-6 alkyl)-C 1-6 Alkyl group, -S(=O)2-C 1-6 Alkyl group, -NHS(=O)2-C 1-6 Alkyl group, -S(=O)2O-C 1- 6-alkyl, -S(=O)2NH-C 1-6 Alkyl groups and -S(=O)2N(C) 1-6 alkyl)-C 1-6 alkyl;

[0244] PL is an immune agonist;

[0245] z is an integer or non-integer from 1 to 20. For example, z is 1, 2, 3, 5, 6, 8, 9, 10 or 20.

[0246] A is a target molecule; preferably, the target molecule is an antibody or antigen-binding fragment; the target molecule is modified to contain a Sortase enzyme recognition sequence, and the target molecule is coupled to (Gly)n under the action of Sortase enzyme, where n is an integer from 2 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0247] In some embodiments, the targeting molecule is linked to the sortase enzyme recognition sequence via a spacer sequence. In some embodiments, the spacer sequence is GA or (GGGGS). o (SEQ ID NO:3); where o is an integer from 1 to 4.

[0248] In some embodiments, the immune agonist conjugate comprises a structure of formula (II-1), a structure of formula (II-2), or a mixture thereof:

[0249] in,

[0250] A, B2, PL, and z are as defined above.

[0251] In some implementations, Lk is L 1 -L 2 -L 3 L 1 It is -NH-, L 3 It is -C(O)-, L 2 It is -(C2H4-O) i -C2H4-.

[0252] In some implementations, i is an integer from 2 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10, especially 4.

[0253] In some implementations, B2 is -(CH2). k C(O)-. In some implementations, B2 is -(CH2). k C(O)-Val-Cit-PABC. In some implementations, B2 is -(CH2). k C(O)-Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d - In some implementations, B2 is -(CH2). k C(O)-NH-(C2H4-O) j- In some implementations, B2 is -(CH2). k1 C(O)-NH-(C2H4-O) j -(CH2) k2 C(O)-Lys-. In some implementations, B2 is -(CH2). k C(O)-(NH-CR 1 R 2 -C(O)) d -NH-(C2H4-O) j - In some implementations, B2 is -(CH2). k C(O)-(NH-CR 1 R 2 -C(O)) d -

[0254] In some implementations, each k, k1, and k2 is independently 1 or 2.

[0255] In some implementations, Y and W are independently selected from leucine (Leu), glutamine (Gln), PABC, Phe-Lys-PABC, Val-Cit-PABC, and Val-Lys-PABC.

[0256] In some implementations, a and b are 0.

[0257] In some implementation schemes, R 1 and R 2 Each is independently hydrogen or -C 1-6 Alkyl; preferably, R 1 and R 2 Each is either hydrogen or -C independently. 1-6 Alkyl; more preferably, R 1 and R 2 They are all methyl groups.

[0258] In some implementations, x is NH2.

[0259] In some embodiments, the immune agonist conjugate is selected from one or a mixture of the following structures:

[0260] in,

[0261] Lm2, B2, PL and z are as defined above;

[0262] The modified target molecule A contains a Sortase enzyme recognition sequence. Under the action of the Sortase enzyme, the target molecule A is coupled with (Gly)n, where n is an integer from 2 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0263] In some implementations, the immune agonist conjugate is selected from the following structures:

[0264] in,

[0265] z is as defined above.

[0266] In some embodiments, the immune agonist conjugate comprises a compound with the structure (AC102-6-1), a compound with the structure (AC102-6-1'), or a mixture thereof:

[0267] The value of z is 0.6-4, for example, 0.6, 0.7, 0.8, 0.9, 1, 1.3, 1.4, 1.73, 1.8, 1.9, 2, 2.1, 2.2, 2.4, 3 or 4.

[0268] In some embodiments, the immune agonist conjugate comprises a compound with the structure (AC102-8-1), a compound with the structure (AC102-8-1'), or a mixture thereof:

[0269] The value of z is 0.6-4, for example, 0.6, 0.7, 0.8, 0.9, 1, 1.3, 1.4, 1.73, 1.8, 1.9, 2, 2.1, 2.2, 2.4, 3 or 4.

[0270] In some embodiments, the immune agonist conjugate is selected from compounds with the structures shown in formula (I-1), formula (I-2), or formula (I-3); wherein A or Ab is a target molecule; preferably, the target molecule is an antibody or antigen-binding fragment.

[0271] In some embodiments, the sortase types include sortase A, sortase B, sortase C, sortase D, and Lactobacillus plantarum sortase. In some embodiments, the sortase enzyme recognition sequence is LPETGG.

[0272] In some implementations, the target for the targeted molecule recognition is selected from one or more of the following groups: CD19, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KIT, CD123, CD138, CD142, CD174, CD227 / MUC1, CD352, CLDN18.2, DLL3, ErbB2 / HER2, CN33, GPNMB, ENPP3, Nectin-4 EGFRvIII, SLC44A4 / AGS-5, mesothelin, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, cadherin 6, FGFR2, FGFR3, CA6, CanAg, integrin αV, TDGF1, Ephrin A4, Trop2, PTK7, NOTCH3, C4.4A, FLT3, ROR1, ROR2, and ROR1 / 2.

[0273] In some embodiments, the targeting molecule is an antibody or its antigen-binding fragment. In some embodiments, the antibody is trastuzumab or pertuzumab. In some embodiments, the antibody is linked to a sortase enzyme recognition sequence via a spacer sequence. In some embodiments, the spacer sequence is GA or (GGGGS). o Where o is an integer from 1 to 4, such as 1, 2, 3, or 4. In some embodiments, a sortase recognition sequence is attached to the end of the antibody heavy or light chain. In some embodiments, a spacer sequence and a sortase recognition sequence are sequentially attached to the C-terminus of the antibody heavy or light chain. In some embodiments, the sortase recognition sequence is LPETGG.

[0274] The reagent kit of the present invention

[0275] On the other hand, the present invention also provides a kit for evaluating the production of ADA induced by an immune agonist conjugate, the kit comprising: PBMC cells collected within 24 hours and an EdU detection reagent.

[0276] In some implementations, the immune agonist conjugate is an antibody-immunoagonist conjugate.

[0277] In some implementations, the kit also includes: for screening PBMCs CD8-CD25-Cellular material; preferably, the material used for screening PBMCs CD8-CD25- The material used for the cells is magnetic beads.

[0278] In some implementations, the kit also includes: a method for screening CD4. + CD3 + Materials for T cells; preferably, those used for screening CD4 + CD3 + The materials used for T cells are CD3 fluorescent antibodies and CD4 fluorescent antibodies.

[0279] In some embodiments, the kit further includes a positive control and a negative control. In some embodiments, the positive control is selected from one or more of the following: KLH, compounds of formula (I-1), and compounds of formula (I-2); in formula (I-1), A is trastuzumab, z is 4, and its DAR value is approximately 4; in formula (I-2), Ab is pertuzumab, z is 4, and its DAR value is approximately 4.

[0280] In some implementations, the negative control is selected from one or more of the following groups: etanercept, compounds of formula (I-3) and formula (I-4); in formula (I-3), Ab is trastuzumab, z is 4, and its DAR value is approximately 4; in formula (I-4), A is modified trastuzumab with GALPETGG introduced into the C-terminus of the trastuzumab light chain, wherein LPETGG is the recognition sequence of the ligase donor substrate, and GA is the spacer sequence; z is 2, and its DAR value is approximately 1.74.

[0281] Example

[0282] To more clearly illustrate the objectives and technical solutions of this invention, the invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Specific experimental methods not mentioned in the following embodiments were performed according to conventional experimental methods.

[0283] Drug preparation methods

[0284] 1) Preparation of AC102-6-1-1: DAR is approximately 1.74. It can be prepared with reference to patents WO2024051747A and WO2022188740A.

[0285] 2) Preparation of NJH395 analogue: Its structure is formula (I-1), the antibody is trastuzumab, and the DAR is about 4; for specific preparation methods, please refer to patents WO2017072662, US2017121421 and WO2018198091.

[0286] 3) Preparation of SBT6050 analogue: Its structure is formula (I-2), the antibody is pertuzumab, and the DAR is about 4; for specific preparation methods, please refer to patent WO2018170179.

[0287] 4) BDC1001 analogues: Their structure is formula (I-3). The main difference between them and BDC1001 is that PEG6 is used instead of the PEG10 linker in BDC1001, resulting in a DAR of approximately 4. The preparation method can be found in patent WO2019222676A. Theoretically, BDC1001 analogues have similar ADA clinical outcomes to BDC1001.

[0288] Detection methods

[0289] 1) PBMC sorting: Select PBMCs freshly collected within 24 hours, centrifuge at 800g for 5 minutes, discard the supernatant, and sort every 10 7 Each cell was resuspended in 80 μl of PBS. 20 μl of (CD8) microbeads (Miltenyi Biotec) / 10 cells were added. 7 Cells, mix well, and incubate at 4°C in the dark for 15 min. After incubation, pass the cells through a column and collect all cell suspensions that have passed through the sorting column to remove CD8 from PBMCs. + Cells, to obtain PBMCs CD8- Cells. To the obtained PBMCs CD8- Add 20 μl (CD25) microbeads (Miltenyi Biotec) / 10 to the cells 7 Cells, mixed well, incubated at 4°C in the dark for 15 min. After incubation, pass through a column and collect all cell suspensions that have passed through the sorting column to further remove CD25 from PBMCs. + Cells, to obtain PBMCs CD8-CD25- Cells. There is no order restriction in the use of (CD8) microbeads and (CD25) microbeads; (CD25) microbeads can also be used first to remove CD25 cells from PBMCs. + Cells, to obtain PBMCs CD25- After cell removal, (CD8) microbeads were used to further remove CD8+ from PBMCs to obtain PBMCs. CD8-CD25- cell.

[0290] 2) PBMC incubation: PBMCs CD8-CD25- Centrifuge cells at 400g for 5 min, discard the supernatant, and resuspend in complete culture medium (AIMV, Life Technologies) containing 6% inactivated human AB serum. Adjust PBMCs. CD8-CD25-Cell density up to 5x10 6 pcs / ml. PBMC CD8-CD25- Cells were added at a rate of 100 μl / well to 96-well transparent flat-bottom plates and incubated at 37°C in a 5% CO2 incubator for 2 hours.

[0291] 3) Co-culturing the test drug with PBMCs: Prepare appropriate concentrations of the positive control, negative control, and test drug using complete culture medium (containing 6% inactivated AB serum), and add 100 μl / well to each well of PBMCs already coated in step 2). CD8-CD25- Cells were incubated in 96-well clear flat-bottomed plates at 37°C with PBMCs. CD8-CD25- Cells were co-cultured at different time gradients.

[0292] 4) EdU (5-ethynyl-2'-deoxyuridine) labeling: PBMC CD8-CD25- After co-culturing cells with the test drug, preheated 5x EdU working solution (37°C) was added to achieve a final EdU concentration of 10 μM. Cells were then incubated at 37°C with 5% CO2 for 2 hours. After EdU labeling, cells were fixed with 4% paraformaldehyde and then permeabilized with PBS containing 0.3% Triton X-100. After permeabilization, 50 μl of Click reaction solution was added to each well, gently mixed, and incubated at room temperature in the dark for 30 minutes. The reaction solution was then removed, completing the EdU labeling process.

[0293] 5) Fluorescent antibody labeling: Add CD4 fluorescent antibody (5 μl / test, Invitrogen) to each well. TM ) and CD3 fluorescent antibody (5 μl / test, Invitrogen) TM The cells in the labeled wells were incubated at 4°C in the dark for 30 min. After incubation, the cells were washed twice with 1×PBS, centrifuged at 800g for 3 min, and the supernatant was discarded. The cells were then resuspended in 1×PBS for use in flow cytometry.

[0294] 6) Flow cytometry: Flow cytometry was used to analyze each sample, with at least 10,000 events / samples collected for each case. In PBMCs treated with the test drug, proliferating EdU cells were screened using flow cytometry. + CD4 + CD3 + Helper T cells (Th cells).

[0295] Example 1

[0296] Following the above method, the EdU kit was used to detect the test drugs KLH (keyhole limpet hemocyanin, purchased from Thermo Scientific), Etanercept (Enbrel, purchased from MCE, catalog number 185243-69-0), NJH395, and AC102-6-1-1 with PBMC. CD8-CD25- After co-culturing cells for 24–120 hours, CD4 + CD3 + The proportion of T cells.

[0297] As shown in Figure 1, when using the same donor PBMC in the presence of KLH CD8-CD25- CD4 counts were measured after cells were cultured for 24 hours, 48 ​​hours, and 72 hours. + CD3 + The proportion of T cell proliferation was high, with a high proportion of proliferating CD4 cells detected only after 72 hours of culture. + CD3 + T cells. KLH is a commonly used immunogenic carrier protein, and the results were consistent with those obtained after 72 hours.

[0298] When using the same donor PBMC in the presence of Etanercept CD8-CD25- Low proportions of proliferating CD4 were detected when cells were cultured for 24, 48, and 72 hours. + CD3 + T cells. This is consistent with the clinical incidence of ADA of Etanercept (a TNF inhibitor) ranging from 3.6% to 8.7%.

[0299] When using PBMCs from the same donor in the presence of NJH395 CD8-CD25- Cells were cultured for 24, 48, and 72 hours, but an extremely high proportion of proliferating CD4 was detected only after 72 hours of culture. + CD3 + T cells. This is consistent with the clinical incidence of ADA in NJH395 being 100% (14 / 14).

[0300] When using the same donor PBMC in the presence of AC102-6-1-1 CD8-CD25- Low proportions of CD4 were detected in cells cultured for 24, 48, and 72 hours at all three times. + CD3 + T cells.

[0301] As shown in Figure 2, when using a PBMC from another donor in the presence of KLH CD8-CD25- CD4 levels were measured when cells were cultured for 72, 96, and 120 hours. + CD3 + The proportion of T cell proliferation was low. Low proportions of proliferating CD4+ cells were detected after 96 and 120 hours of culture. + CD3 + T cells. This is inconsistent with the KLH immunogenicity results.

[0302] When using a PBMC from another donor in the presence of Etanercept CD8-CD25- CD4 levels were measured when cells were cultured for 72, 96, and 120 hours. + CD3 + The proportion of T cell proliferation was observed. Extremely low proportions of proliferating CD4+ cells were detected after 96 and 120 hours of culture. + CD3 + T cells; after 72 hours of culture, a low proportion of proliferating CD4 was detected. + CD3 + T cells.

[0303] When using a PBMC from another donor in the presence of NJH395 CD8-CD25- CD4 levels were measured when cells were cultured for 72, 96, and 120 hours. + CD3 + The proportion of T cell proliferation was observed. Extremely low proportions of proliferating CD4+ cells were detected after 96 and 120 hours of culture. + CD3 + T cells (not entirely consistent with clinical results of NJH395); after 72 hours of culture, a very high proportion of proliferating CD4 cells were detected. + CD3 + T cells.

[0304] When using a PBMC from another donor in the presence of AC102-6-1-1 CD8-CD25- CD4 levels were measured when cells were cultured for 72, 96, and 120 hours. + CD3 + The proportion of T cell proliferation was low. Low proportions of proliferating CD4 cells were detected after 72, 96, and 120 hours of culture. + CD3 + T cells.

[0305] summary

[0306] Approximately 72 hours after stimulation with the test drug, compared to 24 hours, 48 ​​hours, 96 hours, and 120 hours, CD4...+ CD3 + T cells proliferate more actively and can be used to evaluate the immunogenicity of the drug under test.

[0307] Example 2

[0308] To better evaluate the immunogenicity of antibody-immunostimulant conjugates and further optimize CD4 + CD3 + The proportion of T cells is an indicator. Various antibody-immunostimulant conjugates, commercially available or self-made drugs are used as immunostimulants in conjunction with PBMCs. CD8-CD25- After co-culturing for 72 hours, PBMCs were analyzed. CD8-CD25- CD4 proliferating in + CD3 + The proportion of T cells.

[0309] 1) Fresh PBMCs from 11 donors (including people of Asian and Caucasian descent) purchased from Shanghai Saili Biotechnology Co., Ltd. were used to prepare 5x10 6 / ml PBMC CD8-CD25- Cells were added at a volume of 100 μl / well to each of 96-well clear plates and incubated at 37°C in a 5% CO2 incubator for 2 hours. Subsequently, the test drug was added at a volume of 100 μl / well and cultured for 72 hours. Detection was performed according to the above experimental method.

[0310] As shown in Figure 3, for PBMCs from multiple donors, a low proportion of proliferating CD4 was detected under the blank culture conditions (in the absence of the test drug). + CD3 + T cells; in the presence of highly immunogenic KLH, different proportions of proliferating CD4 were detected in different donors. + CD3 + T cells; a low proportion of proliferating CD4 was detected in the presence of low-immunogenic Etanercept. + CD3 + T cells; a high proportion of proliferating CD4 cells were detected in the presence of highly immunogenic NJH395. + CD3 + T cells; low proportion of proliferating CD4 cells detected in the presence of the low-immunogenic AIAC drug BDC1001 analogue. + CD3 + T cells.

[0311] The above results indicate that CD4 + CD3 +T cell proliferation response is highly correlated with the incidence of acute drug-induced hypertension (ADA) with clinical antibody drugs and AIACs. This method can be used to predict the incidence of ADA with clinical antibody drugs and AIACs. A high proportion of proliferating CD4 cells was detected in the presence of the AIAC drug SBT6050, which lacks clear immunogenicity. + CD3 + T cells; a low proportion of proliferating CD4 cells were detected in the presence of AC102-6-1-1, which had not been clinically confirmed in patients. + CD3 + T cells.

[0312] 2) Considering that different donors have different genetic and immune activation backgrounds, it is necessary to further use the immune stimulation coefficient to compare data in order to predict or evaluate the immunogenicity of AIAC.

[0313] Specifically, the drug to be tested will be compared with PBMCs. CD8-CD25- After co-culturing for 72 hours, PBMCs were analyzed. CD8-CD25- CD4 proliferating in + CD3 + The proportion of T cells, divided by the corresponding donor's proliferating CD4 count under culture conditions in the absence of the test drug (Blank). + CD3 + The proportion of T cells is used to obtain the Stimulation Index (SI) of the corresponding drug in the corresponding donor.

[0314] The calculation results are shown in Figure 4. For all PBMCs of the test donors, the calculated SI was 1 under the culture condition in the absence of the test drug (Blank); the calculated SI was 1.59 to 8.93 in the presence of KLH; 0.21 to 2.42 in the presence of etanercept; 6.55 to 34.85 in the presence of NJH395; 0.42 to 3.08 in the presence of BDC1001 analogs; 2.74 to 13.34 in the presence of SBT6050; and 0.59 to 1.93 in the presence of AC102-6-1-1.

[0315] To better predict the risk of clinical ADA, a specific SI threshold for ADA occurrence needs to be defined. The SI threshold is defined as follows: at least 90% of the samples in the positive control group (e.g., KLH and NJH395) have a stimulation coefficient greater than the threshold, and at least 90% of the samples in the negative control group (e.g., escinnapathide and BDC analogs) have a stimulation coefficient less than or equal to the threshold. The SI threshold for this experiment was calculated to be 1.6 (as shown by the dashed line in Figure 4).

[0316] When the immunostimulation coefficient obtained by co-culturing the immune agonist conjugate is greater than 1.6, the immune agonist conjugate will induce drug antibody production with a high probability; when the immunostimulation coefficient obtained by co-culturing the immune agonist conjugate is less than or equal to the critical value of the immunostimulation coefficient, the immune agonist conjugate will induce or will not induce drug antibody production with a low probability.

[0317] Example 3

[0318] In Example 2, PBMC CD8-CD25- The co-culture time of cells with the immune agonist conjugate was 72 h. Based on the results, PBMCs were predicted to... CD8-CD25- Similar results can be achieved by co-culturing cells with immune agonist conjugates for 66-78 hours.

Claims

A method for evaluating the production of drug antibodies (ADA) induced by immune agonist conjugates, characterized in that, The method includes the following steps: 1) Collect PBMCs collected within 24 hours, remove CD8 and CD25 cells to obtain PBMCs. CD8-CD25- cell; 2) Incubate the PBMC CD8-CD25- Cells were incubated and then co-cultured with an immune agonist conjugate. 3) EdU (5-ethynyl-2'-deoxyuridine) was used for labeling, and proliferating CD4 cells were screened after labeling. + CD3 + T cells, or EdU + CD4 + CD3 + T cells; 4) Through the EdU + CD4 + CD3 + T cells account for a significant portion of total CD4+. + CD3 + The percentage of T cells was used to assess the probability of ADA production induced by immune agonist conjugates. The method as described in claim 1, characterized in that, The immune agonist conjugate comprises a target molecule, a linker, and an immune agonist; preferably, the target molecule is an antibody or its antigen-binding fragment. The method as described in claim 1 or 2, characterized in that, Regarding EdU in step 4) + CD4 + CD3 + T cells account for a significant portion of total CD4+. + CD3 + Data processing of T cell percentage: EdU obtained from co-culture of immune agonist conjugates. + CD4 + CD3 + T cells account for a significant portion of total CD4+. + CD3 + The percentage of T cells divided by EdU in the blank control group + CD4 + CD3 + T cells account for a significant portion of total CD4+. + CD3 + The percentage of T cells is used to obtain the immune stimulation coefficient. The method as described in claim 3, characterized in that, Multiple positive and negative controls with known clinical immunogenicity were set up to determine the critical value of the immunostimulation coefficient. When the immunostimulation coefficient obtained by co-culturing the immunostimulator conjugate is greater than the critical value of the immunostimulation coefficient, the immunostimulator conjugate will induce the production of drug antibodies with a high probability. When the immunostimulation coefficient obtained by co-culturing the immunostimulator conjugate is less than or equal to the critical value of the immunostimulation coefficient, the immunostimulator conjugate will induce or not induce the production of drug antibodies with a low probability. The method as described in claim 4, characterized in that, The positive control is a drug with a clinical ADA production probability of at least 90%, and the negative control is a drug with a clinical ADA production probability of less than 10%. The method as described in claim 4, characterized in that, The positive control is selected from one or more of the following groups: keyhole limpet hemocyanin carrier protein, structure (I-1) and structure (I-2); In the formula (I-1), A is trastuzumab and z is 4; In the formula (I-2), Ab is pertuzumab and z is 4. The method as described in claim 4, characterized in that, The negative control is selected from one or more of the following groups: etanercept, structure of formula (I-3) and structure of formula (I-4); In the formula (I-3), Ab is trastuzumab and z is 4; In formula (I-4), A is the modified trastuzumab, with GALPETGG (SEQ ID NO:1) introduced into the C-terminus of the trastuzumab light chain, wherein LPETGG (SEQ ID NO:2) is the recognition sequence of the ligase donor substrate, and GA is the spacer sequence; z is 2. The method as described in claim 4, characterized in that, The high probability is 90%-100%; and / or The low probability is 0-10%. The method as described in claim 4, characterized in that, The probability of ADA being generated clinically after administration of the aforementioned immune agonist conjugate is M / T; where M is the number of patients who received the medication with an immune stimulation coefficient greater than the critical value of the immune stimulation coefficient, and T is the total number of patients who received the medication. The method as described in claim 4, characterized in that, The critical value for the immune stimulation coefficient is 1.2-2.

5. The method as described in claim 3, characterized in that, In step 2), PBMC CD8-CD25- The co-culture time of cells with immune agonist conjugates is 66-78 hours. The method as described in claim 3, characterized in that, The immune agonist conjugate includes the structure of formula (II): Where n is an integer from 2 to 10; Lm2 is or mixtures thereof; x is selected from hydrogen, OH, NH2, an amino acid fragment containing 1-10 amino acids, or a nucleotide fragment containing 1-10 nucleotides; Lk is L 1 -L 2 -L 3 The combination; L 1 and L 3 Each is selected independently from: -CH2-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-; and C 1-4 Alkylene groups in combination with one or more of the following groups: -CH2-, -NH-, -C(O)-, -NHC(O)-, -C(O)NH-; L 2 Does not exist or C 7-34 Alkylene, wherein one or more of the -CH2- structures in the alkylene are optionally replaced by -O-, and the alkylene is optionally replaced by -NH-, -OH or -NH(CO)CH3; Y and W are either absent independently, or they are PABC or spacers containing 1-10 amino acids or combinations thereof; B2 is selected from the following groups or combinations thereof: -(CH2) k C(O)-、-(CH2) k C(O)-Val-Cit-PABC-、-(CH2) k C(O)-Val-Cit-PABC-(NH-CR 1 R 2 -C(O)) d -、-(CH2) k1 C(O)-NH-(C2H4-O) j -(CH2) k2 C(O)-Lys-、-(CH2) k C(O)-NH-(C2H4-O) j -、-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d -NH-(C2H4-O) j -、-(CH2) k C(O)-(NH-CR 1 R 2 -C(O)) d ; a and b are each independently 0 or 1; Each k, k1, and k2 is an integer from 0 to 10, preferably 0, 1, or 2; especially 1 or 2. d is an integer from 1 to 10, especially 1 or 2; j is an integer from 1 to 10, especially 1, 3 or 4; R 1 and R 2 Each is independently selected from: hydrogen, -OH, -NH2, -C 1-6 Alkyl, -OC 1-6 Alkyl, -NH-C 1- 6-alkyl, -C 1-6 Alkyl-NH2, -N(C) 1-6 alkyl)-C 1-6 Alkyl, -C 1-6 Alkyl-OC 1-6 Alkyl, -C 1-6 Alkyl-NH-C 1-6 Alkyl, -C(O)-C 1-6 Alkyl group, -NHC(O)-C 1-6 Alkyl, -C(O)-NH2, -C(O)NH-C 1-6 Alkyl, -C(O)N(C) 1-6 alkyl)-C 1-6 Alkyl group, -S(=O)2-C 1-6 Alkyl group, -NHS(=O)2-C 1-6 Alkyl group, -S(=O)2O-C 1- 6-alkyl, -S(=O)2NH-C 1-6 Alkyl groups and -S(=O)2N(C) 1-6 alkyl)-C 1-6 alkyl; PL is an immune agonist; z is an integer or non-integer from 1 to 20; A is a target molecule; preferably, the target molecule is an antibody or antigen-binding fragment; the target molecule is modified to contain a Sortase enzyme recognition sequence, and the target molecule is coupled to (Gly)n under the action of Sortase enzyme, where n is an integer from 2 to 10. The method as described in claim 3, characterized in that, The targets for the targeted molecule recognition are selected from one or more of the following groups: CD19, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KIT, CD123, CD138, CD142, CD174, CD227 / MUC1, CD352, CLDN18.2, DLL3, ErbB2 / HER2, CN33, GPNMB, ENPP3, Nectin-4, EGF. RvIII, SLC44A4 / AGS-5, mesothelin, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, cadherin 6, FGFR2, FGFR3, CA6, CanAg, integrin αV, TDGF1, Ephrin A4, Trop2, PTK7, NOTCH3, C4.4A, FLT3, ROR1, ROR2, and ROR1 / 2. A kit for evaluating the production of ADA induced by immune agonist conjugates, characterized in that, The kit includes PBMC cells collected within 24 hours and an EdU detection reagent. The kit as described in claim 14, characterized in that, The immune agonist conjugate is an antibody-immunoagonist conjugate. The kit as described in claim 14, characterized in that, The kit also includes a positive control and a negative control. The kit as described in claim 16, characterized in that, The positive control is a drug with a clinical ADA production probability of at least 90%, and the negative control is a drug with a clinical ADA production probability of less than 10%. The kit as described in claim 16, characterized in that, The positive control is selected from one or more of the following groups: keyhole limpet hemocyanin carrier protein, structure (I-1) and structure (I-2); In the formula (I-1), A is trastuzumab and z is 4; In the formula (I-2), Ab is pertuzumab and z is 4. The kit as described in claim 16, characterized in that, The negative control is selected from one or more of the following groups: etanercept, structure of formula (I-3) and structure of formula (I-4); In the formula (I-3), Ab is trastuzumab and z is 4; In formula (I-4), A is the modified trastuzumab, with GALPETGG (SEQ ID NO:1) introduced into the C-terminus of the trastuzumab light chain, wherein LPETGG (SEQ ID NO:2) is the recognition sequence of the ligase donor substrate, and GA is the spacer sequence; z is 2.

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