Polypeptide conjugate for detecting internalization efficiency of antibody and antibody-derived molecule, and use thereof

Abstract

Provided are a polypeptide conjugate for detecting the internalization efficiency of an antibody and an antibody-derived molecule, and a use thereof. By specifically recognizing an antibody via a 3C protein, the labeling and detection efficiency in the evaluation of antibody internalization can be improved, while the actual internalization characteristics of the antibody are more accurately reflected. The invention enables efficient detection of the internalization capability of an antibody or an antibody in an antibody conjugate, and enables prediction of the killing capability of the antibody conjugate against target cells.

Description

A peptide conjugate for detecting the endocytosis efficiency of antibodies and antibody-derived molecules and its applications. Technical Field

[0001] This disclosure pertains to the field of biomedicine and relates to an antibody endocytosis detection platform based on 3C proteins and its use in assessing antibody endocytosis capacity. Background Technology

[0002] Antibody-drug conjugates (ADCs) consist of an antibody, a linker, and a small-molecule cytotoxic drug. They represent a rapidly developing next-generation group of anti-tumor drugs, with 15 ADCs currently approved for clinical trials. ADCs selectively deliver small-molecule drugs to tumor cells for more precise therapeutic effects. Specific recognition of target antigens and antibody endocytosis are crucial for achieving cell-targeted delivery. The evaluation of antibody endocytosis is an important step in ADC development.

[0003] Directly conjugating antibodies to toxins to form ADC molecules for kill assays is undoubtedly the most ideal method, but due to the complex conjugation process, it is difficult to achieve medium- to high-throughput labeling and screening. In contrast, indirect labeling uses non-chemical conjugation methods (such as secondary antibodies or peptides that recognize IgG) to label antibodies, which is simpler to operate and significantly improves throughput.

[0004] Common indirect labeling methods for detecting endocytosis include flow cytometry, Mab-Zap, and DT3C. Flow cytometry uses fluorescent secondary antibodies to label the target antibody and calculates the endocytosis rate by detecting the extracellular or intracellular fluorescence signal after endocytosis. However, this method is cumbersome and cannot reflect long-term endocytosis characteristics. Mab-ZAP and DT3C have excessively large molecules (210 kDa and 140 kDa, respectively), forming very large molecules after antibody labeling. This may inhibit antibody-target binding and endocytosis, resulting in a narrow detection window and making it difficult to accurately compare antibody endocytosis efficiency and cell-killing effects. Furthermore, because different cells have varying sensitivities to DT toxin, the method cannot fully reflect the cell-killing effect of the final ADC drug. Summary of the Invention

[0005] In the evaluation of antibody endocytosis, how to improve labeling and detection efficiency while reflecting more accurate antibody endocytosis characteristics is a direction worth exploring and developing.

[0006] In view of this, the present disclosure provides an antibody endocytosis detection platform based on 3C protein and its use in evaluating antibody endocytosis capacity. The platform is based on the antibody-specific recognition of the C1, C2 and C3 domains (3C) of streptococcal protein G. By conjugating small molecule toxins or pH-sensitive dyes to the 3C domains, the endocytosis activity of antibodies can be detected rapidly and cost-effectively.

[0007] In a first aspect, this disclosure provides a polypeptide conjugate or a pharmaceutically acceptable salt thereof, said polypeptide conjugate having the general formula 3C p -(LD) n The structure shown in (I) is, in which,

[0008] 3C p It is a 3C protein;

[0009] L represents a chemical bond or connecting subunit;

[0010] D represents a small molecule drug or tracer with biological activity;

[0011] n is a real number selected from 1 to 25.

[0012] In some embodiments, the 3C protein comprises the C1, C2, and / or C3 domains of streptococcal protein G or reactive derivatives thereof.

[0013] In some embodiments, the 3C protein has the general formula 3C-L. a -(G) x The sequence shown in (II) is, where,

[0014] 3C is a 3C peptide having the amino acid sequence shown in SEQ ID NO:22, or an amino acid sequence having at least 70% identity with it, or an amino acid sequence having at most 3 insertion, deletion or substitution mutations; preferably, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; the at most 3 mutations are preferably at most 3, 2, 1 or 0 mutations.

[0015] L a As is common in the art, in some embodiments the length of the connector is about 1 to 30 amino acids; in some embodiments, exemplary connectors include glycine-serine polymers (including, for example, (GS)). j (GGGGS) j (GGGS) j (SG) j (GSGGS) j (where j is a natural number ≥ 1), glycine-alanine polymer, alanine-serine polymer, proline linker, α-helical linker, cyclic linker, aromatic amino acid linker, etc.

[0016] G represents a reactive amino acid, a reactive peptide, or other reactive group. In some embodiments, exemplary reactive groups include amino (-NH2), carboxyl (-COOH), thiol (-SH), carbonyl (-CHO), maleimide, NHS ester, diazacyclic, sorting enzyme recognition motif, glutamine transferase recognition motif, formylglycine synthase recognition motif, farnesyltransferase recognition motif, isopentenyltransferase recognition motif, asparagine peptide ligase recognition motif, or an inserted non-natural amino acid, etc.

[0017] x is a real number selected from 1 to 10.

[0018] In some specific implementations, the L a It is GGGGS (SEQ ID NO:23).

[0019] In some specific implementations, G is a reactive amino acid, such as cysteine ​​(Cys) or lysine (Lys).

[0020] In some specific implementations, x is selected from 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0.

[0021] In some specific implementations, x is selected from 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0.

[0022] In some specific implementations, x is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0023] In some specific embodiments, the 3C protein has the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence having at least 70% identity with it, or an amino acid sequence with at most 3 insertion, deletion, or substitution mutations; preferably, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; the at most 3 mutations are preferably at most 3, 2, 1, or 0 mutations.

[0024] In some specific embodiments, the 3C protein has the amino acid sequence shown in SEQ ID NO:21.

[0025] In some implementations, L is a connection subunit, and the connection subunit is... Where m1 is selected from integers 2 to 8, L 1The peptide residues are selected from 1 to 8 amino acids, and the peptide residues are further optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, and 4-7 membered heterocyclic groups. 2 Selected from The a-terminus of the linker is covalently linked to the 3C protein, and the b-terminus is covalently linked to a biologically active small molecule drug or tracer D.

[0026] In some implementations, the L 1 Selected from peptide residues indicated by Val-Cit or Gly-Gly-Phe-Gly.

[0027] In some implementations, m1 is 5.

[0028] In some implementations, the connection subunit is Its a-terminus is covalently linked to the 3C protein, and its b-terminus is covalently linked to a biologically active small molecule drug or tracer D.

[0029] In some implementations, the aforementioned has general formula 3C p -(LD) n The polypeptide conjugate, wherein D is a small molecule drug with biological activity.

[0030] In some specific embodiments, the bioactive small molecule drug is a cytotoxic drug, and optionally, the cytotoxic drug includes a chemotherapeutic drug or an antibiotic.

[0031] In some specific embodiments, the cytotoxic drug includes a tubulin inhibitor, a DNA damaging agent, or a topoisomerase inhibitor; preferably, the tubulin inhibitor includes dolastatin, auristatin, maytansine, tubulolysins, and cryptomycins; the DNA damaging agent includes PBD drugs; and the topoisomerase inhibitor includes camptothecin drugs.

[0032] In some specific embodiments, the cytotoxic drug is selected from MMAE or a compound of formula (DI), said compound (DI) having the following structure:

[0033] in,

[0034] R1 R 2 The atoms connected to them together form a 5-6 membered heterocycle, which contains one or two oxygen atoms as ring atoms, and the 5-6 membered heterocycle may be optionally replaced by one or more deuterium atoms;

[0035] R 4 Selected from H or C1-C3 alkyl groups;

[0036] R 5 Selected from H, halogens, CN, OH, NH2, or C1-C3 alkyl groups;

[0037] R 6 Selected from H or C1-C3 alkyl groups;

[0038] R 7 The group is selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted by one or more groups selected from deuterium, halogen, CN, =O, OH, NH2 or C1-C3 alkyl.

[0039] In some specific implementations, the R 1 R 2 The atoms connected to them together form

[0040] In some specific implementations, R 4 Selected from H.

[0041] In some specific implementations, R 5 It is selected from H, halogen, CN, OH, NH2 or C1-C3 alkyl.

[0042] In some specific implementations, R 5 Selected from H.

[0043] In some specific implementations, R 6 Selected from H.

[0044] In some specific implementations, R 7 Selected from cyclopropyl.

[0045] In some specific embodiments, the compound represented by formula (DI) is selected from one of the following compounds:

[0046] In some implementations, D is a tracer.

[0047] In some implementations, the tracer includes fluorescent labels, chemiluminescent labels, and photosensitizers.

[0048] In some specific embodiments, the tracer is a fluorescent label, and optionally, the fluorescent label is a fluorescent dye; optionally, the fluorescent dye includes one or more of the following: pHAb Thiol Reactive Dye (Promega, G9835), pHAb Amine Reactive Dye (Promega, G9841), pHrodo Green iFL STP ester dye (Thermo Fisher Scientific, P36011), pHrodo Red iFL STP ester dye (Thermo Fisher Scientific, P36012), pHrodo Deep Red TFP ester (Thermo Fisher Scientific, P35358).

[0049] In some specific implementations, the aforementioned having general formula 3C p -(LD) n The polypeptide conjugate, wherein n is selected from real numbers from 1 to 25.

[0050] In some specific implementations, n is selected from 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6. 8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14. 9, 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17. 4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19. 9, 20.0, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21.0, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22.0, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23.0, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24.0, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, or 25.0.

[0051] In some embodiments, the polypeptide conjugate or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0052] Among them 3C p and n as defined above.

[0053] In a second aspect, this disclosure provides a complex or a pharmaceutically acceptable salt thereof, said complex having the general formula A·[3C] p -(LD) n ] m The structure shown in (III) is, in which,

[0054] A is an antibody or its antigen-binding fragment that specifically binds to the target antigen;

[0055] 3C p It is a 3C protein;

[0056] L represents a chemical bond or connecting subunit;

[0057] D represents a small molecule drug or tracer with biological activity;

[0058] n is a real number selected from 1 to 25;

[0059] m is a real number selected from 1 to 4.

[0060] In some implementations, the target antigens include CD33, CD30, HER2, HER3, CD22, CD79b, Nectin-4, BCMA, EGFR, CD19, Tissue Factor, FRα, c-Met, CDH6, LIV-1, LYPD3, FGFR2b, DLL3, CLDN18.2, TROP2, Muc-1, PD-L1, ROR1, MSLN, ENPP3, PSMA, B7H3, CDH3, CDH17, LRRC15, STEAP1, CEACAM6, CEACAM-5, p95HER2, CD16, ROR2, CD70, CD5, CD20, VEGF, Claudin6, CD74, TOPO2, EpCAM, CD25, CD123, CD228, FLT3, CD174, and CD16. 6. One or more of the following: CD326, CD71, SEZ-6, MUC-17, MUC-16, CRIPTO, ETBR, TIM1, TIM3, AFP, NaPi-2, FAP, SLITRK6, KIT / CD117, SLAMF7, GPNMB, AXL, PTK7, 5T4 / TPBG, PRLR, EFNA4, NOTCH3, CD142, CA6, GPR20, EphA2, FGFR3, FGFR4, GCC, IntegrinαV, CAIX, GD3, LAMP1, CD56, CD37, CD47, CD138, CD352, etc.

[0061] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and / or the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1-3 and / or the LCDR1-3 are selected from combinations thereof:

[0062] (1) The HCDR1-3 has HCDR1, HCDR2 and HCDR3 with VH as shown in SEQ ID NO:1; or / and the LCDR1-3 has LCDR1, LCDR2 and LCDR3 with VL as shown in SEQ ID NO:2;

[0063] (2) The HCDR1-3 has HCDR1, HCDR2 and HCDR3 with VH as shown in SEQ ID NO:5; or / and the LCDR1-3 has LCDR1, LCDR2 and LCDR3 with VL as shown in SEQ ID NO:6;

[0064] (3) The HCDR1-3 has HCDR1, HCDR2 and HCDR3 with VH as shown in SEQ ID NO:13; or / and the LCDR1-3 has LCDR1, LCDR2 and LCDR3 with VL as shown in SEQ ID NO:14;

[0065] (4) The HCDR1-3 have HCDR1, HCDR2, and HCDR3 with VH as shown in SEQ ID NO:16; or / and the LCDR1-3 have LCDR1, LCDR2, and LCDR3 with VL as shown in SEQ ID NO:17; or,

[0066] The HCDR1-3 or / and the LCDR1-3 have an amino acid sequence that is at least 70% identical to each CDR in any of the groups (1)-(4), or an amino acid sequence with at most 3 insertion, deletion or substitution mutations; preferably, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; the at most 3 mutations are preferably at most 3, 2, 1 or 0 mutations.

[0067] In some specific implementations, the HCDR1-3 and / or the LCDR1-3 are selected from the following combinations:

[0068] (1) The HCDR1-3 has an amino acid sequence as shown in SEQ ID NO:24-26; or / and the LCDR1-3 has an amino acid sequence as shown in SEQ ID NO:27-29;

[0069] (2) The HCDR1-3 has an amino acid sequence as shown in SEQ ID NO:7-9; or / and the LCDR1-3 has an amino acid sequence as shown in SEQ ID NO:10-12;

[0070] (3) The HCDR1-3 has an amino acid sequence as shown in SEQ ID NO:30-32; or / and the LCDR1-3 has an amino acid sequence as shown in SEQ ID NO:33-35;

[0071] (4) The HCDR1-3 has the amino acid sequence shown in SEQ ID NO:36-38; or / and the LCDR1-3 has the amino acid sequence shown in SEQ ID NO:39-41; or,

[0072] The HCDR1-3 or / and the LCDR1-3 have an amino acid sequence that is at least 70% identical to each CDR in any of the groups (1)-(4), or an amino acid sequence with at most 3 insertion, deletion or substitution mutations; preferably, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; the at most 3 mutations are preferably at most 3, 2, 1 or 0 mutations.

[0073] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region and / or the light chain variable region is selected from the following:

[0074] (1) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:1, or / and the light chain variable region is the amino acid sequence shown in SEQ ID NO:2;

[0075] (2) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:5, or / and the light chain variable region is the amino acid sequence shown in SEQ ID NO:6;

[0076] (3) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:13, or / and the light chain variable region is the amino acid sequence shown in SEQ ID NO:14;

[0077] (4) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:16, or / and the light chain variable region is the amino acid sequence shown in SEQ ID NO:17; or,

[0078] The heavy chain variable region and / or the light chain variable region have an amino acid sequence that is at least 70% identical to the heavy chain variable region and / or the light chain variable region in any of the groups (1)-(4) above, or have an amino acid sequence with at most 15 insertion, deletion or substitution mutations; preferably, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; the at most 15 mutations are at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.

[0079] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain constant region CH sequence and / or a light chain constant region CL sequence. Optionally, the heavy chain constant region CH and / or the light chain constant region CL are selected from complete constant region sequences or fragments thereof, the constant region fragment including CH1, a hinge region, CH2, CH3, or Fc. Optionally, CH is selected from human or mouse IgG1, IgG2, IgG3, or IgG4 constant regions, and CL is selected from human or mouse kappa constant regions or lambda constant regions. Optionally, the antibody or antigen-binding fragment includes both a complete heavy chain and a light chain.

[0080] In some specific embodiments, the heavy chain includes the heavy chain variable region VH and the heavy chain constant region, the heavy chain constant region having an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:15.

[0081] In some specific embodiments, the light chain includes the light chain variable region VL and the light chain constant region, the light chain constant region having an amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 18.

[0082] The heavy chain variable region and / or the light chain variable region have at least 70% amino acid sequence identity with any of the aforementioned sets of heavy chain variable regions and / or light chain variable regions, or have at most 15 insertion, deletion, or substitution mutations in their amino acid sequences; preferably, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; the at most 15 mutations are at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 mutations.

[0083] In some implementations, the antibody or antigen-binding fragment is:

[0084] (1) Chimeric antibodies or fragments thereof;

[0085] (2) Humanized antibodies or fragments thereof; and / or,

[0086] (3) Fully human antibodies or fragments thereof;

[0087] Preferably, the antibody or antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, full-length antibodies, antibody fragments, naked antibodies, conjugated antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody, or single-domain antibodies.

[0088] In some embodiments, the antigen-binding fragment is selected from one or more of F(ab')2, Fab', Fab, Fv, scFv, bispecific antibodies, nanobodies, and antibody minimum recognition units.

[0089] In some embodiments, the 3C protein is the 3C protein described in the first aspect of this disclosure.

[0090] In some embodiments, the connecting subunit is the connecting subunit described in the first aspect of this disclosure.

[0091] In some embodiments, the bioactive small molecule drug or tracer is the bioactive small molecule drug or tracer described in the first aspect of this disclosure.

[0092] In some implementations, n is as defined in the first aspect of this disclosure.

[0093] In some specific implementations, m is selected from real numbers from 1 to 4.

[0094] In some specific implementations, m is selected from 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0.

[0095] In a third aspect, this disclosure provides the aforementioned polypeptide conjugate 3C. p -(LD) n The preparation method of (I) mainly includes the following steps: linking a bioactive small molecule drug or tracer D to the 3C protein:

[0096] (1) Preparation of 3C protein:

[0097] 1) The nucleic acid sequence encoding the 3C protein was recombined into the expression vector pTT5 containing the signal peptide to obtain a recombinant plasmid expressing the 3C protein;

[0098] 2) Add the expression vector and transfection reagent to the culture medium, mix well, and let stand. Then add the mixture to the host cells and culture on a shaker. 16-22 hours after transfection, add protein-free feed and glucose. On days 5-6 after transfection, collect the cell expression supernatant.

[0099] 3) The cell expression supernatant was purified using a Ni column and a molecular sieve column to obtain the 3C protein;

[0100] (2) A small molecule drug or tracer D with biological activity is coupled with the 3C protein or its reactive derivative prepared in step (1) to obtain the polypeptide conjugate shown in formula (I) in the first aspect of this disclosure, wherein L is a chemical bond or linker unit.

[0101] (3) Remove unreacted small molecule drugs or tracer D.

[0102] In some embodiments, the 3C protein is the 3C protein described in the first aspect of this disclosure.

[0103] In some embodiments, the bioactive small molecule drug or tracer is the bioactive small molecule drug or tracer described in the first aspect of this disclosure.

[0104] In some embodiments, the molar equivalent of the small molecule drug or tracer D in step (2) is 2-12 times that of the 3C protein; optionally, the molar equivalent of the small molecule drug or tracer D is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12 times that of the 3C protein.

[0105] In some specific embodiments, the method of linking a biologically active small molecule drug or tracer D to the 3C protein or its reactive derivative is a method of reducing the 3C protein to convert it into a reactive derivative.

[0106] In some embodiments, the bioactive small molecule drug or tracer D is coupled to the 3C protein or its reactive derivative prepared in step (1) via a linker unit.

[0107] In some embodiments, the bioactive small molecule drug or tracer D is directly coupled to the 3C protein or its reactive derivative prepared in step (1).

[0108] In some specific implementations, the coupling reaction includes primary amino group reaction, thiol group reaction, and amino acid side chain reaction of modified proteins and peptides.

[0109] In some specific embodiments, the host cell is a eukaryotic or prokaryotic cell; optionally, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli, and / or Bacillus subtilis; preferably, the host cell is selected from Expi293 or CHO cells; more preferably, the host cell lacks fucosyltransferase, such as FUT8.

[0110] In a fourth aspect, this disclosure provides a method for evaluating or predicting the endocytic capacity of a target antibody or target antibody conjugate, which mainly includes the following steps:

[0111] (1) Co-incubating the target antibody or target antibody conjugate with the polypeptide conjugate shown in formula (I) of the first aspect of this disclosure to obtain a conjugate having the general formula A·[3C] of the second aspect of this disclosure. p -(LD) n ] m (III) The complex of the structure shown or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt thereof;

[0112] (2) Contact the complex or its pharmaceutically acceptable salt prepared in step (1) with the target cells and incubate them together;

[0113] (3) Detect the target cell viability inhibition rate / target cell killing rate or intracellular fluorescence after endocytosis;

[0114] Among them, the target cell viability inhibition rate / target cell killing rate or intracellular fluorescence after endocytosis indicates the endocytosis capacity of the antibody or antibody-drug conjugate.

[0115] Wherein, D in the polypeptide conjugate shown in formula (I) is:

[0116] (1) Small molecule drugs with biological activity; or

[0117] (2) The target antibody or the target antibody conjugate is suitable for the tracer.

[0118] In some specific implementations, m is selected from real numbers from 1 to 4.

[0119] In some specific implementations, m is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0.

[0120] In some embodiments, when the target antibody or target antibody conjugate is co-incubated with the polypeptide conjugate represented by formula (I) in the first aspect of this disclosure to form a complex or a pharmaceutically acceptable salt thereof, the molar concentration of the polypeptide conjugate is 1 to 8 times that of the target antibody or target antibody conjugate; optionally, the molar concentration of the polypeptide conjugate is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 times that of the target antibody or target antibody conjugate.

[0121] In some embodiments, the method for evaluating or predicting the endocytic capacity of a target antibody or target antibody conjugate further includes fitting an IC50 value. 50 The steps, etc.

[0122] In a fifth aspect, this disclosure provides a method for predicting the target cell killing ability of a target antibody conjugate, which mainly includes the following steps:

[0123] (1) Using the small molecule drug D in the target antibody conjugate, prepare the 3C as shown in formula (I) of the first aspect of this disclosure according to the method described in the third aspect of this disclosure. p -(LD) n (I) shows the polypeptide conjugate;

[0124] (2) The antibody in the target antibody conjugate is co-incubated with the polypeptide conjugate prepared in step (1) to obtain the polypeptide conjugate having the general formula A·[3C] in the second aspect of this disclosure. p -(LD) n ] m The complex of the structure shown or a pharmaceutically acceptable salt thereof;

[0125] (3) The general formula A·[3C p -(LD) n ] m The indicated complex or its pharmaceutically acceptable salt is contacted with and co-incubated with the target cells;

[0126] (4) Detect the target cell viability inhibition rate / target cell killing rate;

[0127] Among them, the target cell killing ability of the target antibody conjugate is indicated by the ratio of target cell viability inhibition rate to target cell killing rate.

[0128] In some embodiments, the bioactive small molecule drug is the bioactive small molecule drug described in the first aspect of this disclosure.

[0129] In some embodiments, when the antibody is co-incubated with the polypeptide conjugate of formula (I) to form a complex or a pharmaceutically acceptable salt thereof, the molar concentration of the polypeptide conjugate is 1 to 8 times that of the antibody; optionally, the molar concentration of the polypeptide conjugate is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 times that of the antibody.

[0130] In some implementations, the prediction of the target cell killing ability of the target antibody conjugate further includes fitting IC50. 50 The steps, etc.

[0131] In the fourth and fifth aspects above, the antibody-drug conjugate is selected from any one of antibody-drug conjugates (ADC), antibody fragment-drug conjugates (FDC), antibody-oligonucleotide conjugates (AOC), antibody-radionoid conjugates (ARC), or immunostimulatory antibody-drug conjugates (ISAC).

[0132] In the fourth and fifth aspects mentioned above, the target cells are tumor cells or cancer cells.

[0133] In some specific implementations, the tumor and / or carcinoma includes solid tumors or hematomas.

[0134] In some specific embodiments, the tumor and / or cancer may be selected from one or more of the following tumors or cancers, including but not limited to: lung cancer, pancreatic cancer, liver cancer, hepatocellular carcinoma, breast cancer, colorectal cancer, stomach cancer, esophageal cancer, nasopharyngeal carcinoma, kidney cancer, cervical cancer, prostate cancer, bladder cancer, uterine cancer, melanoma, head and neck cancer, bile duct cancer, thyroid cancer, ovarian cancer, glioblastoma, sarcoma, leukemia, lymphoma, or myeloma, etc.

[0135] In a sixth aspect, this disclosure provides an isolated nucleic acid molecule that encodes the aforementioned 3C protein.

[0136] In some embodiments, this disclosure provides an expression vector comprising the nucleic acid molecules described above.

[0137] In some embodiments, this disclosure provides isolated host cells comprising the nucleic acid molecules described above or the expression vectors described above; optionally, the host cells are eukaryotic or prokaryotic cells; preferably, the host cells are derived from mammalian cells, yeast cells, insect cells, Escherichia coli, and / or Bacillus subtilis; more preferably, the host cells are selected from Expi293 or CHO cells; even more preferably, the host cells lack fucosyltransferase, such as FUT8.

[0138] Verification has shown that this method has broad applicability to targets and overcomes the shortcomings of existing antibody endocytosis detection methods, such as low efficiency and small experimental window.

[0139] Terminology Definitions and Explanations

[0140] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0141] Furthermore, unless otherwise stated herein, the terms used in this disclosure have the following meanings: the definitions of groups and terms recorded in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in the examples, etc., can be arbitrarily combined and combined with each other. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0142] Furthermore, unless otherwise stated, singular terms herein shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, unless otherwise expressly indicated, the singular forms “a” and “this” include plural indicators.

[0143] The terms “comprising,” “including,” and “having” are used interchangeably herein to indicate the inclusiveness of a solution, meaning that the solution may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for solutions “composed of.” For example, “a composition comprising A and B” should be understood to include compositions consisting of A and B, as well as compositions containing other components besides A and B, both falling within the scope of the aforementioned “a composition.”

[0144] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0145] The terms "3C," "3C polypeptide," or "3C peptide" in this article refer to the C1, C2, and / or C3 domains of streptococcal protein G or their reactive derivatives. Streptococcal protein G (SPG) is a protein present in the streptococcal cell wall that binds to immunoglobulin G. The SPG gene structure can be mainly divided into four parts: the N-terminal signal peptide region, the albumin-binding region, the immunoglobulin (antibody)-binding region, and the C-terminal protein anchoring region, which includes the W and M regions. The antibody-binding domain contains three highly homologous IgG-binding regions (C1, C2, and C3 domains).

[0146] The “3C”, “3C polypeptide” or “3C peptide” disclosed herein also include variants, tandem variants or corresponding reactive derivatives of the C1, C2 or / and C3 domains of streptococcal protein G.

[0147] In some embodiments, a coupling group may be modified at the C-terminus of the 3C peptide to form a 3C protein or a reactive derivative thereof.

[0148] In some embodiments, the 3C protein has a structure such as 3C-L. a -(G) x The sequence shown; in some specific embodiments, the 3C has the sequence shown in SEQ ID NO:22, or a sequence having at least 70% identity with it, or a sequence with at most 3 insertion, deletion, or substitution mutations; preferably, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; the at most 3 mutations are preferably at most 3, 2, 1, or 0 mutations. Wherein, L a The linkers are common in this field, such as GGGS, (G4S)3, etc.; G is a reactive amino acid or reactive group, such as Cys, Lys, NH2 or sorted peptides, etc.; x is a real number selected from 1 to 10.

[0149] In this article, "3C", "3C polypeptide" or "3C peptide" can be used interchangeably.

[0150] The terms "specific binding," "immunobinding," and "immunobinding properties" used in this article refer to a type of non-covalent interaction formed between an immunoglobulin molecule and an antigen to which the immunoglobulin has specificity. This type of interaction involves antigen-binding molecules (e.g., antibodies) typically binding to antigens with high affinity and substantially the same antigens, but not to unrelated antigens. The strength or affinity of an immunobinding interaction is typically reflected by the equilibrium dissociation constant (KD), where a lower KD indicates higher affinity. The immunobinding properties of a selected peptide can be quantitatively determined using methods well-known in the art. For example, with antibodies, high affinity typically refers to an affinity of approximately 10. -8 M or lower, approximately 1×10 -9 M or lower, approximately 1×10 -10 M or lower, 1×10 -11 M or lower KD. KD is calculated as follows: KD = Kd / Ka, where Kd represents the dissociation rate and Ka represents the binding rate. The equilibrium dissociation constant KD can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.

[0151] The term "antigen-binding molecule" is used in the broadest sense in this document to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen-binding molecules include, but are not limited to, antibodies or antibody mimics. "Antibody mimic" refers to an organic compound or binding domain that can specifically bind to an antigen but is independent of the antibody structure. Exemplarily, antibody mimics include, but are not limited to, affibody, affitin, affilin, designed ankylosing spondylamine repeat (DARPin), aptamers, or Kunitz-type domain peptides.

[0152] The term "antibody" as used in the broadest sense herein refers to a polypeptide or combination of polypeptides containing sufficient sequence from the variable region of the immunoglobulin heavy chain and / or sufficient sequence from the variable region of the immunoglobulin light chain to specifically bind to an antigen. The term "antibody" as used herein encompasses various forms and structures, provided they exhibit the desired antigen-binding activity. The terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably herein and refer to a structure that is substantially similar to that of a natural antibody. The term "antibody" as used herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, complete antibodies, fragments of complete antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0153] The terms "antibody fragment" or "antigen-binding fragment" are used interchangeably herein to refer to a portion of an antibody that does not possess the full structure of a complete antibody, but only contains a portion or a variant of the complete antibody, which has the ability to bind to the same antigen recognized by the complete antibody. "Antibody fragment" or "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions like an antibody by binding to a specific antigen and forming a complex. For example, antibody fragments include isolated fragments consisting of light chain variable regions, "Fv" fragments consisting of heavy and light chain variable regions, recombinant single-chain polypeptide molecules (scFv) in which the light and heavy chain variable regions are linked by peptide linkers, and the smallest recognizing unit consisting of amino acid residues mimicking hypervariable regions. "Antigen-binding fragment" or "antibody fragment" as used herein includes, but is not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabody, and single-domain antibodies.

[0154] The term "antibody" in this article also includes alternative protein scaffolds or artificial scaffolds having a transplantable complementarity-determining region (CDR) or a CDR derivative. Such scaffolds include antibody-derived scaffolds (which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art for transplanting CDRs, including but not limited to tendinins, fibronectin, peptide aptamers, etc.

[0155] The term "antibody" in this article includes typical "quadruple-chain antibodies," which belong to immunoglobulins composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to-C-terminal direction. Optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to-C-terminal direction. Heavy chains are linked to each other and to each other with disulfide bonds, forming a "Y"-shaped structure. Because the amino acid composition and sequence of the immunoglobulin heavy chain constant region differ, their antigenicity also differs. Based on this, the "immunoglobulins" in this article can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can possess either a κ or λ chain.

[0156] The term "antibody" in this article also includes antibodies that do not contain light chains, such as heavy-chain antibodies (HCAbs) produced by camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe), and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (IgNARs) found in cartilaginous fish such as sharks.

[0157] Papain digestion of the intact antibody produces two identical antigen-binding fragments, called “Fab” fragments, each containing variable domains for both the heavy and light chains, as well as a constant domain for the light chain and a first constant domain (CH1) for the heavy chain. Thus, the term “Fab fragment” as used herein refers to the antibody fragment containing the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. The Fab’ fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab’-SH is the Fab’ fragment in which the cysteine ​​residues in the constant domain carry a free thiol group. Pepsin treatment produces the F(ab’)2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0158] The term "Fd" in this paper refers to an antibody composed of VH and CH1 domains. The term "Fv" refers to an antibody fragment composed of a single-arm VL and VH domain. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. Fv fragments possess the same binding properties and similar three-dimensional binding characteristics as Fab fragments. The VH and VL chains of an Fv fragment are linked together through non-covalent interactions. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can recognize and bind to antigens, although its affinity may be lower than that of a complete binding site.

[0159] The term "scFv" (single-chain variable fragment) in this document refers to a single polypeptide chain containing VL and VH domains linked together by a linker. Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof may also be used. Other linkers that can be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between the VH and VL of scFv, forming a disulfide-linked Fv (dsFv).

[0160] The term "diabody" in this article refers to a single polypeptide chain in which the VH and VL domains are expressed, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites.

[0161] The term "heavy chain antibody" in this article refers to an antibody that lacks a light chain, as is commonly used in antibody therapy. This term specifically includes, but is not limited to, homodimeric antibodies that contain a VH antigen-binding domain and constant CH2 and CH3 domains in the absence of a CH1 domain.

[0162] The terms "single domain antibody" (sdAb), "VHH," and "nanobody" used in this article have the same meaning and are used interchangeably. They refer to the cloning of the variable region of an antibody heavy chain to construct a single-domain antibody consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region. Single-domain antibodies can be derived from camel heavy chain antibodies or chondrogenic fish IgNARs.

[0163] The term "humanized antibody" in this article refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), while all or part of the non-CDR region (e.g., the FR and / or constant regions within the variable region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and the ability to strengthen the immune response.

[0164] The “antibody” in this article can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0165] The term "monoclonal antibody" as used herein refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that, apart from possible variants (e.g., containing naturally occurring mutations or generated during the manufacturing process of the formulation, such variants are typically present in small amounts), the individual antibodies comprising this population are identical and / or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. The modifier "monoclonal" herein should not be construed as requiring the production of the antibody or antigen-binding molecule by any particular method. For example, monoclonal antibodies can be produced using a variety of techniques, including (but not limited to) hybridoma techniques, recombinant DNA methods, phage library display techniques, methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and other methods known in the art.

[0166] The term "natural antibody" in this article refers to antibodies produced and paired by the immune system of multicellular organisms. The term "engineered antibody" in this article refers to non-natural antibodies obtained through techniques such as genetic engineering and antibody engineering. For example, "engineered antibodies" include chimeric antibodies, humanized antibodies, antibody fragments (such as scFv, sdAb, etc.), bispecific antibodies, etc.

[0167] The term "variable region" in this article refers to the region in the antibody heavy or light chain involved in antibody binding to the antigen. "Heavy chain variable region" is interchangeable with "VH" and "HCVR," and "light chain variable region" is interchangeable with "VL" and "LCVR." The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain is sufficient to confer antigen binding specificity. The term "complementarity-determining region" is interchangeable with "CDR" in this article, and usually refers to the hypervariable region (HVR) of the heavy chain variable region (VH) or light chain variable region (VL). This region is called the complementarity-determining region because it can form precise complementarity with the antigen epitope in its spatial structure. The heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR. The terms "framework region" and "FR region" are interchangeable, referring to the amino acid residues in the antibody heavy or light chain variable region other than the CDR. Typically, the variable region of an antibody consists of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0168] The term "CDR" in this paper may be labeled and defined in a manner known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. The tools and websites used include, but are not limited to, the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abysis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). The CDR in this paper includes overlaps and subsets of amino acid residues defined in different ways.

[0169] The term "heavy chain constant region" in this document refers to the carboxyl-terminal portion of the antibody heavy chain, which does not directly participate in antibody-antigen binding but exhibits effector functions, such as interaction with the Fc receptor, and has a more conserved amino acid sequence relative to the variable domains of the antibody. A "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to the natural antibody constant region, while the latter comprises only a portion of the full-length heavy chain constant region. Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" may be selected from the CH1, Fc, or CH3 domains.

[0170] The term "light chain constant region" in this article refers to the carboxyl terminus of the antibody light chain, which does not directly participate in the binding of the antibody to the antigen. The light chain constant region can be selected from the constant κ domain or the constant λ domain.

[0171] The term "Fc" in this document refers to the carboxyl-terminal portion of an antibody obtained by papain hydrolysis of an intact antibody, typically containing the CH3 and CH2 domains of the antibody. Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary slightly, the Fc region of the human IgG heavy chain is generally defined as extending from the amino acid residue at Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the Kabat numbering system) of the Fc region can be removed, for example, during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain; therefore, the Fc region may or may not include the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447).

[0172] The term "mutation" in this article includes gene mutations and amino acid mutations. Gene mutations refer to deletions, insertions, inversions, or substitutions of heterologous nucleic acids, which may lead to changes in the amino acid sequence of the corresponding protein product. Amino acid mutations, also known as nonsynonymous single nucleotide mutations, are caused by changes in a few single bases, resulting in changes in the amino acid sequence of the protein product. Changes in amino acids can affect protein stability, interactions, and enzyme activity, thereby leading to disease.

[0173] The term "amino acid (aa)" in this article refers to the basic building blocks of proteins, giving them their specific molecular structure and biochemical activity. In chemistry, amino acids are organic compounds containing both an amino group (-NH₂) and a carboxyl group (-COOH). Based on the position of the amino group attached to a carbon atom in a carboxylic acid, amino acids are classified as α, β, γ, δ, etc.: in α-amino acids, the amino and carboxyl groups are attached to the same carbon atom; in β-amino acids, they are attached to adjacent carbon atoms, and so on. In biology, the term "amino acid" usually specifically refers to α-amino acids, where the amino and carboxyl groups are directly attached to the same -CH- structure, with the general formula H₂NCHRCOOH (where R represents an organic substituent). For example, the 20 common amino acids include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartate, histidine, asparagine, glutamate, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, and proline.

[0174] The term "Val-Cit" in this article refers to the valine-citrulline residue.

[0175] The term "amino acid substitution" in this article refers to the removal of at least one amino acid residue from the natural or starting sequence, and the insertion of different amino acids at the same position. Substitutions can be single, where only one amino acid in the molecule is substituted, or they can be multiple, where two or more amino acids in the same molecule are substituted.

[0176] The term "conservative amino acid substitution" in this document refers to the substitution of an amino acid normally present in a sequence with a different amino acid having similar size, charge, or polarity. Examples of conservative substitution include replacing a nonpolar (hydrophobic) residue such as isoleucine, valine, and leucine with another nonpolar residue. Similarly, examples of conservative substitution include replacing a polar (hydrophilic) residue with another residue, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, replacing a basic residue such as lysine, arginine, or histidine with another residue, or replacing an acidic residue such as aspartic acid or glutamic acid with another acidic residue, are additional examples of conservative substitution. Examples of nonconservative substitution include replacing a nonpolar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine with a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or replacing a polar residue with a nonpolar residue.

[0177] The term "mutant" in this document refers to a "variant" of the protein or peptide that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid identity with the amino acid sequence of the protein or peptide.

[0178] The term "nucleic acid" in this document includes any compound and / or substance comprising a polymer containing nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5′ to 3′. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers containing mixtures of two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbones or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for the direct expression of the antibodies disclosed herein in vitro and / or in vivo, such as in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA carrier and / or the expression of the encoded molecule, thereby allowing the mRNA to be injected into a subject to generate antibodies in vivo.

[0179] The term "identity" in this document can be calculated as follows: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.

[0180] Taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap, the percentage of identity between the two sequences varies with the common positions shared by the sequences.

[0181] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at www.gcg.com) in the GAP program integrated into the GCG software package can be used to determine the percentage of identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. As another example, the GAP program in the GCG software package (available at www.gcg.com) can be used to determine the percentage of identity between two nucleotide sequences using an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The particularly preferred set of parameters (and unless otherwise specified, a set of parameters to be used) is a Blossum62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a shift vacancy penalty of 5.

[0182] The PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4 can be used to determine the percentage of identity between two amino acid sequences or nucleotide sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0).

[0183] Additionally or alternatively, the nucleic acid and protein sequences described in this invention can be further used as "query sequences" to perform searches against public databases to, for example, identify sequences of other family members or related sequences. For example, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J.Mol.Biol.215:403-10. BLAST nucleotide searches can be performed using the NBLAST program. BLAST protein searches can be performed using the XBLAST program to obtain amino acid sequences homologous to the protein molecules of this invention. To obtain alignment results with gaps for comparative purposes, gap BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res.25:3389-3402. When using BLAST and gap BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0184] The term "antibody conjugate" as used herein refers to a conjugate formed by linking an antibody or its antigen-binding fragment to a biologically active payload via a stable linker unit. The linker can be a covalent bond or a non-covalent interaction such as electrostatic force. The linker can be any type of linker known in the art. The payload can be any type of payload known in the art, such as a biologically active drug, oligonucleotide, siRNA, radionuclide, immune agonist, etc.

[0185] For example, the antibody-drug conjugates described herein may be antibody-drug conjugates (ADC), antibody fragment-drug conjugates (FDC), antibody-oligonucleotide conjugates (AOC), antibody-radionoid conjugates (ARC), or immunostimulatory antibody-drug conjugates (ISAC).

[0186] The term "small molecule drug" in this document refers to a drug with a molecular weight of less than 2000 Daltons and a well-defined chemical structure and mechanism of action. The biologically active small molecule drugs described in this disclosure exemplarily include cytotoxic drugs, such as chemotherapeutic agents or antibiotics. In some embodiments, the cytotoxic drug is selected from tubulin inhibitors, DNA damaging agents, or topoisomerase inhibitors. The tubulin inhibitors include, but are not limited to, dolastatin, auristatin, maytansine, tubulolysins, and cryptomycins; the DNA damaging agents include, but are not limited to, PBD drugs; and the topoisomerase inhibitors include, but are not limited to, camptothecin drugs.

[0187] The term "DAR" or "drug-antibody ratio" in this document refers to the average number of small molecule cytotoxic drugs attached to each protein molecule (such as 3C protein or antibody). In the antibody-drug conjugates disclosed herein, DAR is defined by the variable "n", which can be either an integer or a decimal.

[0188] The term "endocytosis" or "internalization" in this document refers to the process by which the cell membrane, through the contraction of actin proteins, invaginates large molecules that cannot be permeated by the cell membrane, encapsulating extracellular solid particles or liquids and forming small pools that enter the cell. In this document, "large molecules" specifically refers to antibody-antigen conjugates. Endocytosis can be classified into clathrin-dependent endocytosis, caverin-dependent endocytosis, or clathrin / cavitin-independent endocytosis, depending on its dependence on the clathrin. After entering the cell, antibody-antigen conjugates enter compartments such as early endosomes, late endosomes, circulating endosomes, and lysosomes, and are eventually recycled back to the cell membrane surface or hydrolyzed, playing a specific biological role. In this disclosure, "endocytosis" and "internalization" are used interchangeably.

[0189] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. The expression vectors of this disclosure contain polynucleotide sequences and additional sequence elements, for example, for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Some vectors that can be used to express the antibodies and antibody fragments of this disclosure include plasmids containing regulatory sequences (e.g., promoter and enhancer regions) that guide gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to guide efficient transcription of the gene carried on the expression vector. The expression vectors of this disclosure may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or norsocrine.

[0190] The term "host cell" in this article refers to a cell in which foreign nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0191] The general formula described in this article is 3C. p In the -(LD)n polypeptide conjugate, L represents a chemical bond or a linker unit. When L is a chemical bond, the 3C protein and D are directly covalently linked; when L is a linker unit, the 3C protein and D are linked through a linker unit.

[0192] In this article, "n is a real number between 1 and 3" means that n is any real number greater than or equal to 1 and less than or equal to 3.

[0193] In this article, "m is a real number from 1 to 4" means that m is any real number greater than or equal to 1 and less than or equal to 4.

[0194] In this article Indicates the connection site.

[0195] The diagrammatic representation of racemic or enantiomerically pure compounds in this article is derived from Maehr, J. Chem. Ed. 1985, 62:114-120. Unless otherwise specified, wedge bonds and virtual wedge bonds are used. The absolute configuration of a solid center is represented by black solid bonds and imaginary bonds. It indicates the relative configuration of a stereocenter (such as the cis-trans configuration of alicyclic compounds).

[0196] The term "stereoisomer" refers to isomers that are produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0197] The compounds disclosed herein may have asymmetric atoms such as carbon, sulfur, nitrogen, and phosphorus atoms, or asymmetric double bonds, and therefore may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E- and Z-type geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof or other mixtures, such as mixtures enriched with enantiomers or diastereomers. All such isomers and mixtures thereof are within the scope of the definition of the compounds disclosed herein. Alkyl groups or other substituents may contain additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms. All such isomers involved in all substituents, and mixtures thereof, are also included within the scope of the definition of the compounds disclosed herein. The compounds containing asymmetric atoms disclosed herein can be isolated in optically active pure form or in racemic form. The optically active pure form can be separated from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0198] The term "substituted" refers to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted; oxo substitution does not occur on aromatic groups.

[0199] The terms “optional” or “optionally” mean that the event or condition subsequently described may or may not occur, including both the occurrence and non-occurrence of said event or condition. For example, “optionally” substituted with a halogen means that the ethyl group can be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0200] The term "alkyl" refers to the general formula CnH. 2n+1 The alkyl group can be straight-chain or branched. The term "C1-C6 alkyl" should be understood to mean a straight-chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl group includes, but is 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, etc.; the term "C1-C3 alkyl" refers to an alkyl group containing 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl.

[0201] The “C1-C6 alkyl” mentioned in this article may further include “C1-C3 alkyl”.

[0202] The term "cycloalkyl" refers to a fully saturated carbon ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. The term "C3-C6 cycloalkyl" should be understood to mean a saturated monocyclic, fused, spirocyclic, or bridged ring having 3 to 6 carbon atoms, and specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0203] The term "heterocyclic group" refers to a fully saturated or partially saturated monocyclic, fused, spirocyclic, or bridged ring group containing 1-5 heteroatoms or heteroatom groups (i.e., atomic groups containing heteroatoms). These "heteroatoms or heteroatom groups" include, but are not limited to, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH-, or -NHC(=O)NH-, etc. The term "4-7 membered heterocyclic group" refers to a heterocyclic group with 4, 5, 6, or 7 ring atoms, containing 1-3 independently selected heteroatoms or heteroatom groups as described above. The term "5-6 membered heterocyclic group" refers to a heterocyclic group with 5 or 6 ring atoms, and whose ring atoms contain 1-3 independent heteroatoms or heterogroups selected from those described above. Examples of 4-membered heterocyclic groups include, but are not limited to, azirrocyclobutane and oxacyclobutane; examples of 5-membered heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoalkyl, pyrazolyl, pyrrolinyl, 4,5-dihydrooxazolyl, or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazine, trithiaalkyl, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; and examples of 7-membered heterocyclic groups include, but are not limited to, diazacycloheptane. "4-7 membered heterocyclic group" can encompass the ranges of "4-7 membered heterocyclic alkyl," "5-6 membered heterocyclic group," and "5-6 membered heterocyclic alkyl."

[0204] The term "halogen" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0205] This disclosure also includes compounds of this disclosure that are identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 31P, 32P, 35S, 18F, 123I, 125I, and 36Cl, respectively.

[0206] Certain isotopically labeled compounds of this disclosure (e.g., labeled with 3H and 14C) can be used in the analysis of compound and / or substrate tissue distribution. Tritium (i.e., 3H) and carbon-14 (i.e., 14C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron emission isotopes, such as 15O, 13N, 11C, and 18F, can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of this disclosure can generally be prepared by replacing unlabeled reagents with isotopically labeled reagents using a procedure similar to those disclosed in the schemes and / or examples below.

[0207] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments disclosed herein.

[0208] The chemical reactions in the specific embodiments of this disclosure are carried out in a suitable solvent, which must be suitable for the chemical changes of this disclosure and the reagents and materials required therefor. In order to obtain the compounds of this disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0209] An important consideration in synthetic route planning in this field is the selection of appropriate protecting groups for reactive functional groups (such as amino and carboxyl groups in this disclosure). For example, see Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in this disclosure are incorporated herein by reference in their entirety.

[0210] The term "tumor" or "tumor" in this document refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The term "cancer" in this document refers to or describes a physiological condition in mammals characterized by unregulated cell growth. This definition includes both benign and malignant cancers. The terms "cancer" and "tumor" are not mutually exclusive when used herein. Exemplarily, tumors or cancers described in this disclosure include solid tumors or hematologic malignancies; in some embodiments, exemplary tumors or cancers include, but are not limited to, one or more of the following: lung cancer, pancreatic cancer, liver cancer, hepatocellular carcinoma, breast cancer, colorectal cancer, gastric cancer, esophageal cancer, nasopharyngeal carcinoma, kidney cancer, cervical cancer, prostate cancer, bladder cancer, uterine cancer, melanoma, head and neck cancer, bile duct cancer, thyroid cancer, ovarian cancer, glioblastoma, sarcoma, leukemia, lymphoma, or myeloma. Attached Figure Description

[0211] Figure 1. Killing curves of SKBR3 cells by indirect and direct conjugated Trastuzumab toxins.

[0212] Figure 2. Killing curves of NCI-N87 cells by trastuzumab indirectly and directly conjugated toxins.

[0213] Figure 3. Killing curves of OVCAR3 cells by CDH6-Ab-01 indirectly and directly conjugated toxins.

[0214] Figure 4. Killing curves of SKBR3 cells by Lupartumab indirectly and directly conjugated toxins.

[0215] Figure 5. Killing curves of Caov3 cells by Lupartumab indirectly and directly conjugated toxins.

[0216] Figure 6. Killing curves of MCF-7 cells by Lupartumab indirectly and directly conjugated toxins.

[0217] Figure 7. 3C-pHdye applied to the detection of HER2 antibody internalization activity (SKBR3)

[0218] Figure 8. 3C-pHdye applied to the detection of CDH6 antibody internalization activity (OVCAR3)

[0219] Figure 9. 3C-pHdye applied to the detection of LIV-1 antibody internalization activity (OVCAR3)

[0220] Figure 10. 3C-pHdye applied to the detection of LIV-1 antibody internalization activity (NCI-H838)

[0221] Figure 11. Application of 3C-pHdye in the detection of LYPD3 antibody internalization activity (SKBR3)

[0222] Figure 12. Application of 3C-pHdye in the detection of LYPD3 antibody internalization activity (Caov3).

[0223] Figure 13. 3C-pHdye applied to the detection of LYPD3 antibody internalization activity (MCF-7) Detailed Implementation

[0224] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0225] The embodiments of the present invention are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0226] Example 1: Construction and Production of Monoclonal Antibodies and 3C Proteins

[0227] 1.1 Construction and production of anti-human HER2 monoclonal antibodies

[0228] The sequence of the anti-human HER2 monoclonal antibody (Trastuzumab) is shown in Table 1 below (antibody sequence from US5821337A). The heavy and light chain nucleotide sequences were cloned into the pTT5 vector (purchased from Ubisoft Biotechnology), and plasmids were prepared using established standard molecular biology methods. The expression vector and transfection reagent PEI (Polysciences, catalog number: 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021), mixed, and incubated for 15 min. This mixture was then added to Expi293F cells (Thermofisher, catalog number: A14527) and cultured in a shaker at 37°C with 5% CO2 at 120 rpm. On the second day after transfection, OPM-293ProFeed (Shanghai Aopomai, catalog number: F081918-001) and 6 g / L glucose (Sigma, catalog number: G7528) were added. On the sixth day after transfection, the cell expression supernatant was collected.

[0229] Table 1. Amino acid sequence information and CDR sequences of anti-human HER2 antibodies (based on Kabat partitioning)

[0230] 1.2 Construction and production of anti-human CDH6 antibody

[0231] The VH and VL sequences of the anti-human CDH6 antibody (CDH6-Ab-01, sequence source: WO2024165049A1) and the CDR sequences divided according to the Kabat form are shown in Table 2. The heavy / light chain constant regions are shown in SEQ ID NO: 3-4 in Table 1. Following the method in Example 1.1, plasmids encoding the VH and VL sequences of the antibody CDH6-Ab-01 were constructed and transfected into Expi293F cells to obtain the expression supernatant.

[0232] Table 2. Sequence information and CDR sequence of anti-human CDH6 antibody CDH6-Ab-01 (based on Kabat partitioning)

[0233] 1.3 Construction and production of anti-human LIV-1 monoclonal antibodies

[0234] The sequence of the anti-human LIV-1 monoclonal antibody (Ladiratuzumab) (antibody sequence from US20200165335A) is shown in Table 3 below. The nucleic acid sequences encoding the antibodies VH and VL were recombined into the expression vector pTT5 containing a signal peptide and a heavy chain constant region (SEQ ID NO:15) / light chain constant region sequence (SEQ ID NO:4) to obtain a recombinant plasmid expressing Ladiratuzumab. After sequencing verification, the plasmid was extracted.

[0235] Following the method in Example 1.1, the target plasmid was transfected into Expi293F cells to obtain the expression supernatant.

[0236] Table 3. Sequence information and CDR sequence of the anti-human LIV-1 antibody Ladiratuzumab (based on Kabat partitioning)

[0237] 1.4 Construction and production of anti-human LYPD3 monoclonal antibody

[0238] The sequence of the anti-human LYPD3 monoclonal antibody (Lupartumab) (antibody sequence from WO2011070088) is shown in Table 4 below. The nucleic acid sequences encoding the antibodies VH and VL were recombined into the expression vector pTT5 containing a signal peptide and a heavy chain constant region (SEQ ID NO:15) / light chain constant region sequence (SEQ ID NO:18) to obtain a recombinant plasmid expressing anti-FITC. Following the method in Example 1.1, the target plasmid was transfected into Expi293F cells to obtain the expression supernatant.

[0239] Table 4. Sequence and CDR sequence of the human LYPD3 monoclonal antibody Lupartumab (based on Kabat classification).

[0240] 1.5 Construction and production of isotype control anti-human FITC monoclonal antibody

[0241] The sequence of the anti-human FITC monoclonal antibody (anti-FITC-hIgG1) is shown in Table 5 below. The nucleic acid sequences encoding the antibodies VH and VL were recombined into the expression vector pTT5 containing a signal peptide and a heavy chain constant region (SEQ ID NO:3) / light chain constant region sequence (SEQ ID NO:4) to obtain a recombinant plasmid expressing anti-FITC. Following the method in Example 1.1, the target plasmid was transfected into Expi293F cells to obtain the expression supernatant.

[0242] Table 5. Sequence of anti-FITC-hIgG1 antibody against human FITC

[0243] 1.6 Construction and Production of 3C Protein

[0244] The 3C protein described in this disclosure has the following formula: 3C-L a -(G) x The structure is shown, where x is a real number selected from 1 to 10 as defined above. The sequence of an exemplary 3C protein is shown in Table 6 below. The nucleic acid sequence encoding the 3C protein is recombined into the expression vector pTT5 with a signal peptide to obtain a recombinant plasmid expressing the 3C protein. To facilitate purification, a purification tag, such as a his tag, can be added to the C-terminus of SEQ ID NO:21. Following the method in Example 1.1, the target plasmid is transfected into Expi293F cells to obtain the expression supernatant.

[0245] Table 6 3C protein / 3C peptide sequences

[0246] Example 2: Purification of Monoclonal Antibody and 3C Protein

[0247] 2.1 Purification of Monoclonal Antibodies

[0248] The corresponding antibodies were purified from the cell expression supernatants prepared in Examples 1.1-1.5 using Protein A affinity chromatography (GE, catalog number 17549802). The Protein A affinity column was washed with 3-5 column volumes of 6M guanidine hydrochloride, followed by 3-5 column volumes of pure water. The column was equilibrated with 3-5 column volumes of 1×PBS (pH 7.4) as a buffer. Cell supernatants were loaded for binding at a low flow rate, controlling the flow rate to maintain a retention time of approximately 1 min or longer. After binding, the column was washed with 3-5 column volumes of 1×PBS (pH 7.4) until the UV absorbance returned to baseline. Samples were eluted using elution buffer (0.02M citrate buffer, pH 3.5). Elution peaks were collected based on UV monitoring. The elution products were rapidly adjusted to pH 5-6 using 1M Tris-HCl (pH 8.0, Sinopharm, catalog number 30188336) for temporary storage. The elution products can be subjected to solution replacement using methods well-known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution replacement to the desired buffer system, or size exclusion chromatography (SUC) such as G-25 desalting column to replace the buffer system, or high-resolution SUC columns such as Superdex 200 to remove aggregate components from the elution products to improve sample purity. Proteins that meet the purity requirements after purification are aseptically filtered using a 0.22 μm filter (Millipore, catalog number SLGVR13SL), and after passing SEC-HPLC purity testing, aliquoted and stored at -80°C for later use.

[0249] 2.2 3C Protein Purification

[0250] The 3C protein was purified from the cell expression supernatant prepared in Example 1.6 using a Ni column (His Trap excel, Cytiva, 17371206). The column was first equilibrated with 3–5 column volumes of equilibration buffer (1X PBS phosphate buffer, pH 7.4), and then the clarified expression supernatant was loaded at a flow rate of 5 mL / min. After loading, the Ni column was washed with 3–5 column volumes of equilibration buffer, followed by washing with elution buffer (1×PBS phosphate buffer containing 5 mM imidazole, pH 7.4). After elution, the eluted protein was collected using elution buffer (1×PBS phosphate buffer containing 20 mM imidazole, pH 7.4).

[0251] Superdex molecular sieve column TM 75 Increase 10 / 300GL, Cytiva, 29148721) Equilibrate with equilibration buffer (1×PBS phosphate buffer, pH 7.4), concentrate the Ni-eluted sample to an appropriate volume and load it, collect the monomeric protein to obtain the purified final sample.

[0252] Example 3 Preparation of 3C peptide conjugates

[0253] 3.1 Preparation of 3C-LP1 Couplings

[0254] The structure of drug-linker 1 is shown below, and its preparation is based on patent document WO2023217227A1 (Example 39).

[0255] The protein solution (protein concentration 1-5 mg / ml) prepared in Example 2.2 was added with 12 molar equivalents of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720), and the mixture was reduced at 37°C for 3 h. 10% DMSO was added to the reduced protein solution, and 7 molar equivalents of drug-linker 1 were dissolved in DMSO and added to the reaction system. The reaction mixture was coupled at 25°C for 3 h. The reaction product was dialyzed into PBS to remove unreacted free toxins. The resulting 3C peptide conjugate was named 3C-LP1.

[0256] 3.2 Preparation of 3C-MMAE Couplings

[0257] The structure of drug-linker 2 is shown below. It was purchased from MedChemExpress (MCE) under catalog number HY-15575.

[0258] The protein solution prepared in Example 2.2 was added with 3.5 molar equivalents of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720), and the mixture was reduced at 37°C for 3 h. 10% DMSO was added to the reduced protein solution, and 3.5 molar equivalents of drug-linker 2 were dissolved in DMSO and added to the reaction system. The reaction mixture was coupled at 25°C for 17 h. The reaction product was dialyzed into PBS to remove unreacted free toxins. The resulting 3C polypeptide conjugate was named 3C-MMAE.

[0259] 3.3 Preparation of 3C-pHdye Couplings

[0260] The 3C protein solution prepared in Example 2.2 was added with 12 molar equivalents of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720), and the mixture was reduced at 37°C for 3 h. 10% DMSO was added to the reduced protein solution, and 7 molar equivalents of fluorescent dye (pHAb Thiol Reactive Dye, Promega, G9835) were dissolved in a 1:1 DMSO and H₂O solution and added to the reaction system. The reaction mixture was coupled at 25°C for 3 h. The reaction product was dialyzed into PBS to remove unreacted free dye. All experiments were conducted under light-protected conditions. The resulting 3C polypeptide conjugate was named 3C-pHdye.

[0261] 3.4 Purity and DAR value analysis of 3C peptide conjugates

[0262] The purity and DAR value of the 3C polypeptide conjugates prepared in Examples 3.1-3.3 were analyzed by SEC and LC-MS methods. The main method steps are as follows.

[0263] SEC Purity Analysis: The SEC-HPLC method was used to analyze the protein samples to characterize the molecular size uniformity of the recombinant proteins and determine their purity. The HPLC system used was an Agilent 1260, with a TSKgel G3000SWXL column from Tosoh Bioscience. The mobile phase was 200 mM phosphate buffer (pH 7.0) containing 10% isopropanol. The detection temperature was 25°C, the flow rate was 0.5 mL / min, and the detection wavelength was 280 nm. The SEC-HPLC data were analyzed using the manual integration method, and the protein purity was calculated using the area normalization method. The main peak was considered the monomer, the peaks before the main peak were called aggregates, and the peaks after the main peak were called fragments. The SEC purity of the samples was calculated (Table 7).

[0264] DAR value determination: The DAR value of ADC molecules was measured using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). First, the ADC molecules were treated with PNGase F (NEB#P0705L) to remove N-sugar modification, then treated with dithiothreitol (DTT, Sigma#646563) and incubated at 37°C for 1 h to reduce them to light and heavy chains. Analysis was then performed using a Thermo Vanquish UHPLC-Q Exactive Plus mass spectrometry system. The ADC molecules were injected into a Waters ACQUITY Protein BEH size-exclusion column. The mobile phase was an aqueous solution containing 0.1% formic acid, 0.05% TFA, and 25% acetonitrile. The flow rate was 0.2 mL / min, and the analysis time was 30 min. Subsequent analysis was performed using a Thermo Q Exactive Plus mass spectrometer. Using the mass spectrometry data analysis software Biopharma Finder 4.1, the molecular weight information of the light and heavy chain mass spectrometry peaks and the mass spectrometry response signals of each component were calculated through deconvolution processing using the Respect algorithm, thereby calculating the DAR value of the sample to be tested (Table 7).

[0265] Table 7. 3C peptide conjugates, their DAR values ​​and SEC purity

[0266] Example 4: Preparation and Analysis of Antibody Conjugates

[0267] The Trastuzumab, CDH6-Ab-01, and Lupartumab prepared in Example 1 were dialyzed to a 20 mM PB, 150 mM NaCl, 1 mM EDTA solution (pH 6.5). Eight times the volume of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific, 77720) was added to the antibody solution (antibody concentration 1-10 mg / ml), and the mixture was incubated at 4°C for 17 hours to reduce the antibody. 15 times the volume of drug-linker 1 was dissolved in DMSO and added to the reaction system, and the reaction mixture was coupled at 25°C for 6 hours. The reaction product was eluted using a Capto SP ImpRes cation exchanger (Cytiva) gradient to a pH 5.5 sodium acetate + NaCl solution to remove unreacted free small molecule toxins. Finally, the mixture was dialyzed overnight to a 559 buffer (10 mM sodium acetate, 9% Sucrose, pH 5.5). The purity and DAR value of the antibody conjugates were analyzed by SEC and LC-MS methods, and the results are shown in Table 8.

[0268] The Trastuzumab and Lupartumab prepared in Example 1 were dialyzed to a solution of 20 mM PB, 150 mM NaCl, and 1 mM EDTA (pH 6.5). 2.4-3.5 times the volume of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific, 77720) was added to the antibody solution (antibody concentration 1-10 mg / ml), and the mixture was incubated at 4°C for 17 hours to reduce the antibody. Ten molar equivalents of the drug-linker 2 compound (MCE, Cat.HY-15575) prepared in Example 3.2 (dissolved in DMSO to 20 mg / ml) were added to the reaction system, and the reaction mixture was coupled at 25°C for 4 hours. The reaction product was eluted using a Capto SP ImpRes cation exchanger (purchased from Cytiva) gradient to a pH 5.5 sodium acetate + NaCl solution to remove unreacted free small molecule toxins. Finally, the solution was changed to 559 buffer (10 mM sodium acetate, 9% Sucrose, pH 5.5) by overnight dialysis. The purity and DAR value of the antibody conjugate were analyzed using the method in Example 3.4, and the results are shown in Table 8.

[0269] Table 8. Structure, DAR value, and SEC purity of antibody-drug conjugates

[0270] Example 5: Application of 3C-LP1 and 3C-MMAE in antibody internalization detection

[0271] 5.1 Application of 3C-LP1 and 3C-MMAE in HER2 antibody internalization detection

[0272] The HER2-overexpressing human breast cancer cell line SKBR3 was purchased from ATCC and cultured in McCoy's 5A medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells in the logarithmic growth phase were digested with trypsin-EDTA (0.25%), and after counting, the cell density was adjusted and seeded into 96-well white clear-bottom cell culture plates (Cave Biotech, 062096) at a seeding condition of 4000 cells / 50 μL / well, and cultured overnight. The following day, the following mixtures were prepared using complete culture medium: the test antibody (prepared in Example 1) was mixed with 3C-LP1 (prepared in Example 3.1) or 3C-MMAE (prepared in Example 3.2) at a molar ratio of 1:3 to obtain COM-1 or COM-2; the test antibody was mixed with DT3C (a recombinant protein lacking the receptor-binding domain of diphtheria toxin (DT) and the C1, C2, and C3 domains of streptococcal protein G, purchased from Panchao Biotechnology) at a molar ratio of 1:4 to obtain COM-3. The test antibody was Trastuzumab. After incubating the above mixtures at room temperature for 30 minutes, the test antibody and 3C protein were allowed to form a complex, and the complex was serially diluted 3-fold. At the same time, Trastuzumab-ADC (Table 8, ADC-1; ADC-4) was prepared using complete culture medium and serially diluted 3-fold. Add the serially diluted samples (50 μL / well) to cell culture plates to achieve a starting and ending concentration of 3.33 nM for COM-1 / COM-2 / COM-3 or ADC. Gently mix and incubate for 5 days. Cell viability is assessed using a CellTiter-Meiluncell luminescence assay kit (Meilun Biotechnology, PWL111). Add 50 μL of assay solution to each well, gently shake, and incubate at room temperature for 10 minutes. Read the fluorescence values ​​using a microplate reader (PerkinElmer, Ensight-HH3400). Calculate the cell viability inhibition percentage (%) using the formula: Cell viability inhibition percentage (%) = 100 * (Control group reading - Experimental group reading) / Control group reading. The control group consists of untreated cells. Plot the antibody / ADC concentration on the x-axis and the inhibition percentage on the y-axis to obtain the IC50. 50 .

[0273] The HER2-overexpressing human gastric cancer cell line NCI-N87 was purchased from the Cell Bank of the Chinese Academy of Sciences. The complete culture medium was RPMI 1640 medium containing 10% fetal bovine serum. Following the above procedures, the starting and ending concentrations of COM-1 / COM-2 / COM-3 or ADC were maintained at 11.1 nM.

[0274] The complexes formed by the antibody and the 3C peptide conjugate are shown in the table below:

[0275] As shown in Figures 1 and 2 and Table 9, the indirectly coupled complexes COM-1-COM-3, as well as the directly coupled ADC-1 and ADC-4, all exhibited good specific killing effects against SKBR3 and NCI-N87 cells. The principle of this experiment is that after Trastuzumab forms a complex with 3C-LP1 or 3C-MMAE, it can be internalized by HER2-positive cells, similar to the killing mechanism of ADCs. After the 3C-MMAE or 3C-LP1 and antibody complex are internalized into lysosomes, the enzymes in the lysosomes specifically cleave the linkers on the complex or ADC and release toxins, thus exerting a cell-killing effect. The results showed that the indirectly coupled complexes COM-1-COM-3 and the directly coupled Trastuzumab ADCs (Table 8, ADC-1 and ADC-4) performed similarly in cell killing, indicating that 3C-LP1 or 3C-MMAE can be used to evaluate the internalization ability of antibodies.

[0276] Table 9. Maximum cell-killing rate and IC50 of Trastuzumab indirect and direct conjugated toxins 50 Note: In Figures 1A and 1B corresponding to Table 9, COM-3 comes from the same set of data. To show the effects of 3C polypeptide conjugates linked to different toxins, they are plotted separately; the same applies to Figures 2A and 2B.

[0277] 5.2 Application of 3C-LP1 in CDH6 antibody internalization detection

[0278] The CDH6-overexpressing human ovarian cancer cell line OVCAR3 was purchased from ATCC. The complete culture medium was RPMI 1640 containing 20% ​​fetal bovine serum and 10 μg / mL bovine insulin, and cultured at 37°C and 5% CO2. Cells in the logarithmic growth phase were digested with 0.02% EDTA, counted, and the cell density was adjusted before seeding into 96-well white clear-bottomed cell culture plates (Cave Biotech, 062096) at a seeding ratio of 2000 cells / 50 μL / well, and cultured overnight. The next day, the following mixtures were prepared using the complete culture medium: the test antibody was mixed with 3C-LP1 at a molar ratio of 1:3 to obtain COM-4; the test antibody was mixed with DT3C at a molar ratio of 1:4 to obtain COM-5. The test antibody was CDH6-Ab-01. After incubating the above mixtures at room temperature for 30 minutes, they were serially diluted 4-fold. Simultaneously, CDH6-ADC (Table 8, ADC-2) was prepared using the complete culture medium and serially diluted 4-fold. Add the serially diluted samples (50 μL / well) to cell culture plates to achieve a starting and ending concentration of COM-4 / COM-5 or ADC of 0.625 nM. Gently mix and continue culturing for 5 days. Cell viability is then assessed using the same method as in Example 5.1.

[0279] The complexes formed by the antibody and the 3C peptide conjugate are shown in the table below:

[0280] As shown in Figure 3 and Table 10, the indirectly coupled complexes COM-4 and COM-5, as well as the directly coupled ADC-2, all exhibited specific killing effects against OVCAR3. After incubation of CDH6-Ab-01 with 3C-LP1, 3C-LP1 could bind to the antibody to form a complex. This complex was internalized by CDH6-positive cells, exerting a cytotoxic effect, with an IC50 of 1:1. 50 The cell-killing effect of ADCs directly conjugated with toxins is closer to that of the classic DT3C method, which is superior to that of the ADC method. This result indicates that the method can be applied to predict the killing effect of antibody-conjugated ADC drugs in the early stage of antibody screening.

[0281] Table 10. Maximum cell-killing rate and IC50 of CDH6-Ab-01 indirectly and directly conjugated toxins. 50

[0282] 5.3 Application of 3C-LP1 / MMAE in LYPD3 Antibody Internalization Detection

[0283] The source and culture conditions of SKBR3, which highly expresses LYPD3, are the same as in Example 5.1. The human papillary ovarian adenocarcinoma Caov3 expressed in LYPD3 was purchased from Nanjing Kebai Biotechnology, and the complete culture medium was DMEM containing 10% fetal bovine serum. The human breast cancer cell line MCF-7 expressed in LYPD3 was purchased from ATCC, and the complete culture medium was DMEM containing 10% fetal bovine serum. Cells in the logarithmic growth phase were digested with 0.02% EDTA, and after counting, the cell density was adjusted and seeded into 96-well white transparent bottom cell culture plates (Cave Biotechnology, 062096) at a seeding condition of 3000 cells / 50 μL / well, and cultured overnight. The next day, the following mixtures were prepared using complete culture medium: the test antibody was mixed with 3C-LP1 or 3C-MMAE at a molar ratio of 1:3 to obtain COM-6 or COM-7; the test antibody was mixed with DT3C at a molar ratio of 1:4 to obtain COM-8. The test antibody was Lupartumab. After incubating the above mixture at room temperature for 30 minutes, perform a 4-fold serial dilution. Simultaneously, prepare Lupartumab-ADC (Table 8, ADC-3, ADC-5) using complete culture medium and perform a 4-fold serial dilution. Add 50 μL / well of the serially diluted samples to cell culture plates, ensuring the starting and ending concentrations of COM-6 / COM-7 / COM-8 or ADC are 100 nM. Gently mix and continue culturing for 5 days. Cell viability is assessed using the same method as in Example 5.1.

[0284] The complexes formed by the antibody and the 3C peptide conjugate are shown in the table below:

[0285] As shown in Figures 4-6 and Table 11, both the indirectly coupled COM-6 complex and the directly coupled ADC-3 showed IC50 on SKBR3, Caov3, and MCF-7 tumor cells. 50 It exhibits considerable specific killing effect; both the indirectly coupled COM-7 complex and the directly coupled ADC-5 complex show IC50 against SKBR3. 50 It exhibits considerable specificity in killing cells. However, under the same concentration treatment conditions, the COM-8 complex showed no killing window against any cells. This is presumably because the large molecular weight of DT3C creates steric hindrance, preventing the conjugate from being effectively internalized by cells, or because the cells may be insensitive to DT toxin.

[0286] The results showed that the DT3C method has certain limitations in the selection of cell types and targets when used for detecting antibody internalization ability. The 3C-LP1 / MMAE method disclosed in this paper exhibits cell-killing performance on antibody-labeled cells that is closer to the cell-killing ability of directly conjugated toxin ADCs, and can be used for a wider range of cell types, making it applicable to a broader range of targets, such as the internalization assessment of LYPD3 antibodies.

[0287] Table 11. Maximum cell-killing rate and IC50 of Lupartumab indirect and direct conjugated toxins 50 Note: NA represents non-lethal.

[0288] Example 6: Application of 3C-pHdye in the detection of antibody internalization activity

[0289] 6.1 Application of 3C-pHdye in HER2 antibody internalization detection

[0290] The HER2-overexpressing SKBR3 cells were sourced and cultured under the same conditions as in Example 5.1. Cells in the logarithmic growth phase were digested with trypsin-EDTA (0.25%), counted, and the cell density was adjusted before seeding into 96-well white transparent-bottom cell culture plates (Cave Biotech, 062096) at a seeding ratio of 20,000 cells / 50 μL / well, and cultured overnight. The next day, a mixture of test antibody and 3C-pHAb Thio was prepared using complete culture medium, with the test antibody concentration at 40 nM and the 3C-pHAb Thio concentration at 200 nM (i.e., a molar ratio of 1:5). The mixture was incubated at 37°C for 30 minutes, followed by storage at 4°C. The test antibody included Trastuzumab and the negative control Anti-FITC-hIgG1.

[0291] The complexes formed by the antibody and 3C-pHdye are shown in the table below:

[0292] The mixture was added to cell culture plates at different time points (0, 2, 4, 8, 24, and 28 hours after detection), 50 μL / well, gently mixed, and incubated at 37°C. After incubation at all time points, the supernatant was aspirated with a pipette, and the cells were washed twice with 200 μL / well PBS, followed by 100 μL / well PBS. Fluorescence intensity (excitation 532 nm, absorption 560 nm) was read using a microplate reader (PerkinElmer, Ensight-HH3400). A quantitative fluorescence data table and graph of antibody internalization were obtained by plotting time (hours) on the x-axis and fluorescence intensity on the y-axis (Table 12, Figure 7A). Simultaneously, cells were observed using the 560 nm fluorescence channel of a microscope (Nikon, DS-Qi2), and fluorescence imaging was performed at fixed exposure times (Figure 7B).

[0293] The principle of this experiment is that after Trastuzumab forms a complex with 3C-pHdye, it can be internalized by HER2-positive SKBR3 cells. Since pH-dye is a pH-sensitive dye, it only emits a strong fluorescent signal under acidic conditions, such as in lysosomes. Therefore, detecting the intensity of the fluorescent signal in cells can determine the amount of antibody entering the lysosomes, and thus infer the strength of the antibody's internalization ability. Based on this principle, the results of two detection methods showed that Trastuzumab labeled with 3C-pHdye emitted fluorescence after internalization. The detected fluorescence intensity increased with increasing incubation time, indicating a rapid internalization rate, reaching a plateau after 8 hours (the negative control, Anti-FITC-hIgG1, did not induce target-induced endocytosis in tumor cells, therefore no increase in fluorescence intensity over time was observed).

[0294] Table 12. Quantitative fluorescence data of 3C-pHdye applied to HER2 antibody internalization detection.

[0295] 6.2 Application of 3C-pHdye in the detection of CDH6 antibody internalization

[0296] The source and culture conditions of CDH6-overexpressing OVCAR3 were the same as in Example 5.2. Cell pretreatment and testing procedures were the same as in Example 6.1. The test antibodies included CDH6-Ab-01 and the negative control Anti-FITC-hIgG1.

[0297] The complexes formed by the antibody and 3C-pHdye are shown in the table below:

[0298] Plotting time (hours) on the x-axis and fluorescence intensity on the y-axis, a quantitative fluorescence data table and graph of antibody internalization were obtained (Table 13, Figure 8A). Simultaneously, cells were observed using the 560nm fluorescence channel of a microscope (Nikon, DS-Qi2), and fluorescence imaging was performed at a fixed exposure time (Figure 8B). The results of both detection methods showed that 3C-pHdye-labeled CDH6-Ab-01 emitted fluorescence after internalization, and the detected fluorescence intensity increased with increasing incubation time, but a plateau was not reached after 28 hours of continuous internalization.

[0299] Table 13. Quantitative fluorescence data of 3C-pHdye applied to the detection of CDH6 antibody internalization.

[0300] 6.3 Application of 3C-pHdye in LIV-1 Antibody Internalization Detection

[0301] The OVCAR3 cells expressing LIV-1 were sourced and cultured under the same conditions as in Example 5.2; human non-small cell lung cancer cells (NCI-H838) with high LIV-1 expression were purchased from Nanjing Kebai Biotechnology Co., Ltd., and the complete culture medium was RPMI 1640 medium containing 10% fetal bovine serum. Cell pretreatment and testing procedures were the same as in Example 6.1, with seeding conditions of 15,000 cells / 50 μL / well (OVCAR3) and 10,000 cells / 50 μL / well (NCI-H838). Test antibodies included Ladiratuzumab and the negative control Anti-FITC-hIgG1.

[0302] The complexes formed by the antibody and 3C-pHdye are shown in the table below:

[0303] Plotting time (hours) on the x-axis and fluorescence intensity on the y-axis, we obtained a quantitative fluorescence data table and graph for antibody internalization (Table 14; Figure 9, A; Figure 10, A). Simultaneously, we observed the cells using the 560nm fluorescence channel of a microscope (Nikon, DS-Qi2) and performed fluorescence imaging at fixed exposure times (Figure 9, B; Figure 10, B). The results of both detection methods showed that 3C-pHdye-labeled Ladiratuzumab emitted fluorescence after internalization, and the detected fluorescence intensity increased with increasing incubation time, but a plateau was not reached after 28 hours of continuous internalization.

[0304] Table 14. Quantitative fluorescence data of 3C-pHdye applied to LIV-1 antibody internalization detection.

[0305] 6.4 Application of 3C-pHdye in the detection of LYPD3 antibody internalization

[0306] The SKBR3 cells with high LYPD3 expression were sourced and cultured under the same conditions as in Example 5.1; the Caov3 and MCF-7 cells with moderate LYPD3 expression were sourced and cultured under the same conditions as in Example 5.3. Cell pretreatment and testing procedures were the same as in Example 6.1. The test antibodies included Lupartumab and the negative control Anti-FITC-hIgG1. Testing was conducted at 0, 2, 4, 8, 24, and 32 hours after detection.

[0307] The complexes formed by the antibody and 3C-pHdye are shown in the table below:

[0308] Plotting time (hours) on the x-axis and fluorescence intensity on the y-axis, we obtained a quantitative fluorescence data table and graph for antibody internalization (Table 15; Figure 11, A; Figure 12, A; Figure 13, A). Simultaneously, we observed the cells using the 560nm fluorescence channel of a microscope (Nikon, DS-Qi2) and performed fluorescence imaging at fixed exposure times (Figure 11, B; Figure 12, B; Figure 13, B). The results of both detection methods showed that 3C-pHdye-labeled Lupartumab emitted fluorescence after internalization, and the detected fluorescence intensity increased with increasing incubation time.

[0309] Table 15. Quantitative fluorescence data of 3C-pHdye applied to the detection of LYPD3 antibody internalization.

[0310] All teachings of the patents, publications and references cited in this article are incorporated herein by reference in their entirety.

[0311] While exemplary embodiments have been specifically shown and described, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of the embodiments covered by the appended claims.

Claims

1. A polypeptide conjugate or a pharmaceutically acceptable salt thereof, said polypeptide conjugate having 3C p -(LD) n The structure shown in (I) is, in which, 3C p It is a 3C protein; L represents a chemical bond or connecting subunit; D represents a small molecule drug or tracer with biological activity; n is a real number selected from 1 to 25.

2. The polypeptide conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the 3C protein comprises the C1, C2, and / or C3 domains of streptococcal protein G or a reactive derivative thereof; optionally, the 3C protein has the general formula 3C-L. a -(G) x The sequence shown in (II); where, 3C is a 3C peptide having the amino acid sequence shown in SEQ ID NO:22, or an amino acid sequence having at least 70% identity with it, or an amino acid sequence having at most 3 insertion, deletion, or substitution mutations; preferably, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; the at most 3 mutations are preferably at most 3, 2, 1, or 0 mutations; L a For the connector, GGGGS is preferred; G is a reactive amino acid, a reactive peptide, or other reactive group; optionally, G is a reactive amino acid; preferably, G is cysteine ​​(Cys) or lysine (Lys); x is a real number selected from 1 to 10; preferably, x is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

3. The polypeptide conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein, The 3C protein has the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence that is at least 70% identical to it, or an amino acid sequence with at most 3 insertion, deletion, or substitution mutations; preferably, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; the at most 3 mutations are preferably at most 3, 2, 1, or 0 mutations.

4. The polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein, When L is a connecting subunit, its structure is as follows: Wherein, m1 is selected from integers 2 to 8, L1 is selected from peptide residues consisting of 1 to 8 amino acids, and the peptide residues are further optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, and 4-7 membered heterocyclic groups, L 2 Selected from The a-end of the linker unit is covalently linked to the 3C protein, and the b-end is covalently linked to the bioactive small molecule drug or tracer D. Optionally, the L 1 Selected from peptide residues indicated by Val-Cit or Gly-Gly-Phe-Gly; Optionally, m1 is 5.

5. The polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein, The structure of the connecting subunit is as follows: Its a-terminus is covalently linked to the 3C protein, and its b-terminus is covalently linked to the biologically active small molecule drug or tracer D.

6. The polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, wherein, D is a small molecule drug with biological activity; optionally, D is a cytotoxic drug; optionally, the cytotoxic drug includes chemotherapeutic drugs or antibiotics; optionally, the cytotoxic drug includes tubulin inhibitors, DNA damaging agents, or topoisomerase inhibitors; preferably, the tubulin inhibitor includes dolastatin, auristatin, maytansine, tubulolysins, and cryptomycins; the DNA damaging agent includes PBD drugs; and the topoisomerase inhibitor includes camptothecin drugs.

7. The polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-6, wherein, The cytotoxic drug is selected from MMAE or compounds of formula (DI), wherein the compounds of formula (DI) have the following structure: in, R 1 R 2 The atoms connected to them together form a 5-6 membered heterocycle, which contains one or two oxygen atoms as ring atoms, and the 5-6 membered heterocycle may be optionally replaced by one or more deuterium atoms; R 4 Selected from H or C1-C3 alkyl groups; R 5 Selected from H, halogens, CN, OH, NH2, or C1-C3 alkyl groups; R 6 Selected from H or C1-C3 alkyl groups; R 7 The group is selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted by one or more groups selected from deuterium, halogen, CN, =O, OH, NH2 or C1-C3 alkyl.

8. The polypeptide conjugate according to claim 7, or a pharmaceutically acceptable salt thereof, wherein, The R 4 Selected from H; and / or The R 5 Selected from H, halogens, CN, OH, NH2, or C1-C3 alkyl groups; preferably, the R... 5 Selected from H; and / or The R 6 Selected from H; and / or The R 7 Selected from cyclopropyl.

9. The polypeptide conjugate or a pharmaceutically acceptable salt thereof according to claim 7 or 8, wherein, The compound represented by formula (DI) is selected from one of the following compounds:

10. The polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, wherein, D is a tracer; optionally, the tracer includes fluorescent labeling, chemiluminescent labeling, and photosensitizer; optionally, the fluorescent dye is selected from one or more of pHAb Thiol Reactive Dye (Promega, G9835), pHAb Amine Reactive Dye (Promega, G9841), pHrodo Green iFL STP ester dye (Thermo Fisher Scientific, P36011), pHrodo Red iFL STP ester dye (Thermo Fisher Scientific, P36012), pHrodo Deep Red TFP ester (Thermo Fisher Scientific, P35358).

11. The polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-10, wherein, The 'n' is selected from 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0, 15. 1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17. 6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.0, 20. 1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21.0, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22.0, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23.0, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24.0, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, or 25.

0.

12. A complex or a pharmaceutically acceptable salt thereof, said complex having A·[3C p -(LD) n ] m The structure shown in (III) is, in which, A represents an antibody or its antigen-binding fragment; 3C p -(LD) n The polypeptide conjugate of claims 1-11 or a pharmaceutically acceptable salt thereof; m is selected from real numbers from 1 to 4.

13. The complex according to claim 12 or a pharmaceutically acceptable salt thereof, wherein, The antibody or antigen-binding fragment includes: (1) a chimeric antibody or a fragment thereof; and / or (2) Humanized antibodies or fragments thereof; and / or, (3) Fully human antibodies or fragments thereof; Optionally, the antibody or antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, full-length antibodies, antibody fragments, naked antibodies, conjugated antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody, or single-domain antibodies; Preferably, the antigen-binding fragment is selected from one or more of F(ab')2, Fab', Fab, Fv, scFv, bispecific antibodies, nanobodies, and antibody minimum recognition units.

14. The complex according to claim 12 or 13, or a pharmaceutically acceptable salt thereof, wherein, The value of m is selected from 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.

0.

15. A method for preparing the polypeptide conjugate according to any one of claims 1-11, comprising the following steps: linking a biologically active small molecule drug or tracer D to a 3C protein or a reactive derivative thereof: (1) Preparation of the 3C protein or its reactive derivative as described in any one of claims 1-3: 1) The nucleic acid sequence encoding the 3C protein was recombined into the expression vector pTT5 containing the signal peptide to obtain a recombinant plasmid expressing the 3C protein; 2) Add the expression vector and transfection reagent to the culture medium, mix well, and let stand. Then add the mixture to the host cells and culture on a shaker. 16-22 hours after transfection, add protein-free feed and glucose. On days 5-6 after transfection, collect the cell expression supernatant. 3) The cell expression supernatant was purified using a Ni column and a molecular sieve column to obtain the 3C protein; (2) The bioactive small molecule drug or tracer D described in any one of claims 1-11 is coupled to the 3C protein or its reactive derivative prepared in step (1) to obtain the polypeptide conjugate of formula (I) as described in any one of claims 1-11, wherein L is the chemical bond or linker unit described in any one of claims 1-11; wherein, The molar equivalent of the small molecule drug or tracer D is 2-12 times that of the 3C protein; optionally, the molar equivalent of the small molecule drug or tracer D is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12 times that of the 3C protein. (3) Remove unreacted small molecule drugs or tracer D.

16. The preparation method according to claim 15, wherein, (1) The bioactive small molecule drug or tracer D is coupled to the 3C protein or its reactive derivative prepared in step (1) via a linker unit; or (2) The bioactive small molecule drug or tracer D is directly coupled to the 3C protein or its reactive derivative prepared in step (1); Optionally, the coupling includes primary amino group reactions, thiol group reactions, and amino acid side chain reactions of modified proteins and peptides.

17. The preparation method according to claim 15 or 16, wherein, The host cell is a eukaryotic or prokaryotic cell; optionally, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli, and / or Bacillus subtilis; preferably, the host cell is selected from Expi293 or CHO cells.

18. A method for evaluating or predicting the endocytic capacity of a target antibody or target antibody conjugate, comprising the following steps: (1) Co-incubate the target antibody or target antibody conjugate with the polypeptide conjugate according to any one of claims 1-11 to obtain a complex with the structure according to any one of claims 12-14 or a pharmaceutically acceptable salt thereof; (2) The complex or a pharmaceutically acceptable salt thereof prepared in step (1) is contacted with the target cells and co-incubated; (3) Detect the target cell viability inhibition rate / target cell killing rate or intracellular fluorescence after endocytosis; The endocytic capacity of the antibody or antibody conjugate is indicated by the detection results of step (3); in, In any of the polypeptide conjugates according to claims 1-11, D is: (1) Small molecule drugs with biological activity; or (2) The target antibody or the target antibody conjugate is suitable for the tracer.

19. The method of claim 18, wherein, When the target antibody or target antibody conjugate is co-incubated with the polypeptide conjugate shown in formula (I) to form a complex or a pharmaceutically acceptable salt thereof, the molar concentration of the polypeptide conjugate is 1 to 8 times that of the target antibody or target antibody conjugate; optionally, the molar concentration of the polypeptide conjugate is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 times that of the target antibody or target antibody conjugate.

20. A method for predicting the target cell killing ability of a target antibody conjugate, comprising the following steps: (1) Using the small molecule drug D in the target antibody conjugate, prepare 3C as described in any one of claims 15-17. p -(LD) n (I) The polypeptide conjugate shown in the structure; (2) The antibody in the target antibody conjugate is co-incubated with the polypeptide conjugate prepared in step (1) to obtain a complex with the structure of any one of claims 12-14 or a pharmaceutically acceptable salt thereof; (3) Contact the complex obtained in step (2) or its pharmaceutically acceptable salt with the target cells and incubate them together; (4) Detect the target cell viability inhibition rate / target cell killing rate; The detection results of step (4) indicate the target cell killing ability of the target antibody conjugate; Optionally, the bioactive small molecule drug D is the bioactive small molecule drug as described in any one of claims 1-11.

21. The method according to claim 20, wherein, When the antibody in the target antibody conjugate is co-incubated with the polypeptide conjugate to form a complex or a pharmaceutically acceptable salt thereof, the molar concentration of the polypeptide conjugate is 1-8 times that of the antibody in the target antibody conjugate; optionally, the molar concentration of the polypeptide conjugate is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 times that of the antibody in the target antibody conjugate.

22. The method of any one of claims 18-21, wherein, The antibody-drug conjugate is selected from any one of antibody-drug conjugates (ADC), antibody fragment-drug conjugates (FDC), antibody-oligonucleotide conjugates (AOC), antibody-radionoid conjugates (ARC), or immunostimulatory antibody-drug conjugates (ISAC).

23. The method of any one of claims 18-21, wherein, The target cells are tumor cells or cancer cells; optionally, the tumor and / or cancer includes solid tumors or hematologic malignancies; optionally, the tumor and / or cancer may be selected from one or more of the following tumors or cancers: lung cancer, pancreatic cancer, liver cancer, hepatocellular carcinoma, breast cancer, colorectal cancer, gastric cancer, esophageal cancer, nasopharyngeal carcinoma, kidney cancer, cervical cancer, prostate cancer, bladder cancer, uterine cancer, melanoma, head and neck cancer, bile duct cancer, thyroid cancer, ovarian cancer, glioblastoma, sarcoma, leukemia, lymphoma, or myeloma, etc.

24. An isolated nucleic acid molecule, wherein, The nucleic acid molecule encodes the 3C protein as described in claim 2 or 3.

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