Camptothecin derivative-specific binding antibodies and uses thereof

Abstract

The present disclosure relates to antibodies or antigen-binding fragments thereof that specifically bind to camptothecin derivatives and uses thereof.

Description

[0001] Priority Statement

[0002] This application claims the benefit of PCT application No. PCT / CN2024 / 078239, filed on February 23, 2024. The entire contents of the foregoing application are incorporated herein by reference. Technical Field

[0003] This disclosure relates to antibodies that specifically bind to camptothecin derivatives and their uses. Background Technology

[0004] In the field of cancer treatment, the application of small molecule compounds with anticancer properties is often limited due to their toxicity to normal tissues. This problem can be effectively alleviated by conjugating these compounds with monoclonal antibodies, thereby developing antibody-drug conjugates (ADCs).

[0005] Camptothecin derivatives have shown promising applications in the field of antibody-drug conjugate (ADC) therapy. For example, Daiichi Sankyo invented an ADC drug, DS-8201a, with camptothecin DXd as the warhead molecule, which exhibited good anti-tumor effects. AstraZeneca announced a novel DNA topoisomerase 1 inhibitor-antibody drug conjugate (Design and Preclinical Evaluation of a Novel B7-H4-Directed Antibody-Drug Conjugate, AZD8205, Alone and in Combination with the PARP1-Selective Inhibitor AZD5305, Clin Cancer Res 2022: OF1-OF16.), in which the connector toxin AZ'0133 showed good in vivo efficacy. ADCs are often heterogeneous and require comprehensive evaluation of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies. Therefore, there is an urgent need in modern technology to develop reagents and methods for detecting drugs containing camptothecin derivatives, such as antibody drugs conjugated with camptothecin derivatives. Summary of the Invention

[0006] This disclosure relates to antibodies that specifically bind to camptothecin derivatives, their antigen-binding fragments, and their uses.

[0007] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to a camptothecin derivative, the antibody or antigen-binding fragment thereof comprising: The heavy chain variable region (VH) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein VH CDR1 contains an amino acid sequence with at least 80% identity to a selected VH CDR1 amino acid sequence, VH CDR2 contains an amino acid sequence with at least 80% identity to a selected VH CDR2 amino acid sequence, and VH CDR3 contains an amino acid sequence with at least 80% identity to a selected VH CDR3 amino acid sequence; and The system includes light chain variable regions (VLs) comprising CDR1, 2, and 3, wherein VL CDR1 contains an amino acid sequence having at least 80% identity with a selected VL CDR1 amino acid sequence, VL CDR2 contains an amino acid sequence having at least 80% identity with a selected VL CDR2 amino acid sequence, and VL CDR3 contains an amino acid sequence having at least 80% identity with a selected VL CDR3 amino acid sequence. The selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1, 2, and 3, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 31, 32, and 33, respectively; (2) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4, 5, and 6, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 34, 35, and 36, respectively. (3) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7, 8, and 9, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 37, 38, and 39, respectively; (4) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10, 11, and 12, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 37, 38, and 39, respectively. (5) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13, 14, and 15, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 40, 41, and 42, respectively. (6) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16, 17, and 18, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 31, 32, and 33, respectively. (7) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 19, 20, and 21, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 34, 35, and 36, respectively; (8) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 22, 23, and 24, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 37, 38, and 39, respectively; (9) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 25, 26, and 27, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 37, 38, and 39, respectively; and (10) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 28, 29, and 30, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 40, 41, and 42, respectively.

[0008] In some embodiments, according to the Kabat definition, VH includes CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 1, 2, and 3, respectively, and VL includes CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 31, 32, and 33, respectively.

[0009] In some embodiments, according to the Kabat definition, VH includes CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 4, 5, and 6, respectively, and VL includes CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 34, 35, and 36, respectively.

[0010] In some embodiments, according to the Kabat definition, VH includes CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 7, 8, and 9, respectively, and VL includes CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 37, 38, and 39, respectively.

[0011] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 10, 11, and 12, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 37, 38, and 39, respectively.

[0012] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 13, 14, and 15, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 40, 41, and 42, respectively.

[0013] In some embodiments, according to Chothia's definition, VH includes CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 16, 17, and 18, respectively, and VL includes CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 31, 32, and 33, respectively.

[0014] In some embodiments, according to Chothia's definition, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 34, 35, and 36, respectively.

[0015] In some embodiments, according to Chothia's definition, VH comprises CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 22, 23, and 24, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 37, 38, and 39, respectively.

[0016] In some embodiments, according to Chothia's definition, VH includes CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 25, 26, and 27, respectively, and VL includes CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 37, 38, and 39, respectively.

[0017] In some embodiments, according to Chothia's definition, VH includes CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 28, 29, and 30, respectively, and VL includes CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 40, 41, and 42, respectively.

[0018] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to a camptothecin derivative, the antibody or antigen-binding fragment comprising: a heavy chain variable region (VH) containing an amino acid sequence having at least 90% identity with a selected VH sequence; and a light chain variable region (VL) containing an amino acid sequence having at least 90% identity with a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 44; (2) The selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 46; (3) The selected VH sequence is SEQ ID NO: 47, and the selected VL sequence is SEQ ID NO: 48; (4) The selected VH sequence is SEQ ID NO: 49, and the selected VL sequence is SEQ ID NO: 50; and (5) The selected VH sequence is SEQ ID NO: 51, and the selected VL sequence is SEQ ID NO: 52.

[0019] In some implementations, VH includes the sequence of SEQ ID NO: 43, and VL includes the sequence of SEQ ID NO: 44.

[0020] In some implementations, VH includes the sequence of SEQ ID NO: 45, and VL includes the sequence of SEQ ID NO: 46.

[0021] In some implementations, VH includes the sequence of SEQ ID NO: 47, and VL includes the sequence of SEQ ID NO: 48.

[0022] In some implementations, VH includes the sequence of SEQ ID NO: 49, and VL includes the sequence of SEQ ID NO: 50.

[0023] In some implementations, VH includes the sequence of SEQ ID NO: 51, and VL includes the sequence of SEQ ID NO: 52.

[0024] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to a camptothecin derivative, the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3 identical to selected VH sequences; and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3 identical to selected VL sequences. The selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 44; (2) The selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 46; (3) The selected VH sequence is SEQ ID NO: 47, and the selected VL sequence is SEQ ID NO: 48; (4) The selected VH sequence is SEQ ID NO: 49, and the selected VL sequence is SEQ ID NO: 50; and (5) The selected VH sequence is SEQ ID NO: 51, and the selected VL sequence is SEQ ID NO: 52.

[0025] In one aspect, this disclosure relates to an antibody or an antigen-binding fragment thereof, which includes VH CDR1, 2, 3 and VL CDR1, 2, 3 of the antibodies or antigen-binding fragments thereof described herein.

[0026] In some embodiments, camptothecin derivatives are specifically bound when the VH pairs with the VL, or camptothecin derivatives are specifically bound when the VL pairs with the VH.

[0027] In some embodiments, the camptothecin derivative is a compound of general formula (I) or a salt, solvate, stereoisomer, or isotopic variant thereof: LG-LD (I), Where LG represents no ligand or no linker; L represents no linker or no linker; and D is a compound of the following structural formula (II) or a salt, solvate, stereoisomer or isotopic variant thereof: (II), R1 and R2 independently represent hydrogen atoms and hydroxyl groups, and may contain halogen atoms, nitro groups, or cyano groups. 1-6 alkyl group, C2-6 alkenyl group, C 1-6 alkoxy group, C 1-6 Aminoalkoxy groups, halogen atoms, nitro groups, cyano groups, mercapto groups, alkylthio groups, amino groups that may contain protecting groups, or C groups that may contain protecting groups or have C atoms at the amino position. 1-6 C of alkyl group 1-6 The aminoalkyl group may contain a protecting group or a C at the amino position. 1-6 C of alkyl group 1-6 Aminoalkylamino groups, which may contain C 1-6 Alkyl, C 1-6 C of heterocycles with alkoxy, amino, halogen, nitro, or cyano groups 1-6 Alkyl groups, which may contain C 1-6 Alkyl, C 1-6 The carbonyl group of a heterocyclic compound containing an alkoxy, amino, halogen, nitro, or cyano group, which may contain a C 1-6 Alkyl, C 1-6 C of a heterocyclic ring consisting of an alkoxy group, an amino group (which may contain a protecting group), a halogen group, a nitro group, a cyano group, or a protecting group. 1-6 The alkylamino group may contain a protecting group or a C-shaped group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The amino-heterocyclic group of the alkyl group may contain a protecting group or a C-shaped protecting group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The alkyl group may contain a heterocyclic amino group, or may contain a protecting group or a C-shaped group. 1-6 The carbamoyl group of the alkyl group; R3 represents C 1-6 alkyl groups; R4 represents C 1-6 Alkyl groups, which may contain protecting amino groups, quaternary trialkylammonium groups such as -N.sup.+(CH3)3, and C groups that may contain protecting C groups. 1-6 Alkylamino groups, which may contain protecting groups, are C14 groups. 1-6 An aminoalkyl group, which may contain a protecting group, is a C-shaped group. 1-6 alkylaminoalkyl group, sulfonic acid group or carboxyl group; Z represents oxygen atom, sulfur atom, CR5 R6, where R5 and R6 each represent hydrogen atom or carbon atom. 1-6 Alkyl, or N-R7, where R7 represents a hydrogen atom, C 1-6 Alkyl groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 Alkylaminoalkyl group, or protecting group of amino group; and m and n each represent 0, 1 or 2.

[0028] In some embodiments, the camptothecin derivative is a compound of general formula (I) or a salt, solvate, stereoisomer, or isotopic variant thereof: LG-LD (I), Where LG represents no ligand or no linker; L represents no linker or no linker; and D is a compound of the following structural formula (VI) or a salt, solvate, stereoisomer, or isotopic variant thereof:

[0029] (VI)

[0030] R1 and R2 independently represent hydrogen atoms and hydroxyl groups, and may contain halogen atoms, nitro groups, or cyano groups. 1-6 alkyl group, C 2-6 alkenyl group, C 1-6 alkoxy group, C 1-6 Aminoalkoxy groups, halogen atoms, nitro groups, cyano groups, mercapto groups, alkylthio groups, amino groups that may contain protecting groups, or C groups that may contain protecting groups or have C atoms at the amino position. 1-6 C of alkyl group 1-6 The aminoalkyl group may contain a protecting group or a C at the amino position. 1-6 C of alkyl group 1-6 Aminoalkylamino groups, which may contain C 1-6 Alkyl, C 1-6 C of heterocycles with alkoxy, amino, halogen, nitro, or cyano groups 1-6 Alkyl groups, which may contain C 1-6 Alkyl, C 1-6 The carbonyl group of a heterocyclic compound containing an alkoxy, amino, halogen, nitro, or cyano group, which may contain a C 1-6 Alkyl, C 1-6 C of a heterocyclic ring consisting of an alkoxy group, an amino group (which may contain a protecting group), a halogen group, a nitro group, a cyano group, or a protecting group. 1-6 The alkylamino group may contain a protecting group or a C-shaped group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The amino-heterocyclic group of the alkyl group may contain a protecting group or a C-shaped protecting group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The alkyl group may contain a heterocyclic amino group, or may contain a protecting group or a C-shaped group. 1-6 The carbamoyl group of the alkyl group; Alternatively, R1 and R2, together with the atoms they are attached to, form a 3-, 4-, 5-, or 6-membered heterocyclic alkyl group or a 3-, 4-, 5-, or 6-membered cycloalkyl group, wherein the 3-, 6-membered heterocyclic alkyl group or 3-, 6-membered cycloalkyl group is optionally substituted by one, two, three, or four substituents selected from hydrogen atoms, hydroxyl groups, and C atoms that may contain halogen atoms, nitro groups, or cyano groups.1-6 alkyl group, C 2-6 alkenyl group, C 1-6 alkoxy group, C 1-6 Aminoalkoxy groups, halogen atoms, nitro groups, cyano groups, mercapto groups, alkylthio groups, amino groups that may contain protecting groups, or C groups that may contain protecting groups or have C atoms at the amino position. 1-6 C of alkyl group 1-6 The aminoalkyl group may contain a protecting group or a C at the amino position. 1-6 C of alkyl group 1-6 Aminoalkylamino groups, which may contain C 1-6 Alkyl, C 1-6 C of heterocycles with alkoxy, amino, halogen, nitro, or cyano groups 1-6 Alkyl groups, which may contain C 1-6 Alkyl, C 1-6 The carbonyl group of a heterocyclic compound containing an alkoxy, amino, halogen, nitro, or cyano group, which may contain a C 1-6 Alkyl, C 1-6 C of a heterocyclic ring consisting of an alkoxy group, an amino group (which may contain a protecting group), a halogen group, a nitro group, a cyano group, or a protecting group. 1-6 The alkylamino group may contain a protecting group or a C-shaped group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The amino-heterocyclic group of the alkyl group may contain a protecting group or a C-shaped protecting group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The alkyl group may contain a heterocyclic amino group, or may contain a protecting group or a C-shaped group. 1-6 The carbamoyl group of the alkyl group; R3 represents C 1-6 alkyl groups; Z' represents hydrogen atom, deuterium atom, oxygen atom, sulfur atom, halogen atom, nitro group, cyano group, hydroxyl group, and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 6-10 Aryl, C 3-14 Cycloalkyl, 5-14-membered heteroaryl, 4-14-membered heterocycloalkyl, C 6-10 Aryl-C 1-4 Alkyl-, C 3-14 cycloalkyl-C 1-4 Alkyl-, (5-14-membered heteroaryl)-C 1-4 Alkyl-, (4-14 membered heterocyclic alkyl)-C 1-4 Alkyl-, OR8, C(O)R8, C(O)NR8R8, C(O)OR8, S(O)2R8 and S(O)2NR8R8, wherein Z' of C1-6 Alkyl, C 1-6 Haloalkyl, C 6-10 Aryl, C 3-14 Cycloalkyl, 5-14-membered heteroaryl, 4-14-membered heterocycloalkyl, C 6-10 Aryl-C 1-4 Alkyl-, C 3-14 cycloalkyl-C 1-4 Alkyl-, (5-14-membered heteroaryl)-C 1-4 Alkyl- and (4-14 membered heterocyclic alkyl)-C 1-4 Each alkyl group is optionally substituted by one, two, three, four, or five independently selected substituents, which are selected from hydrogen atoms, deuterium atoms, halogen atoms, nitro groups, cyano groups, hydroxyl groups, C6 groups, and C7 groups. 1-6 Alkyl groups, amino groups that may contain protecting groups, quaternary trialkylammonium groups such as -N.sup.+(CH3)3, and C groups that may contain protecting groups 1-6 Alkylamino groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 alkylaminoalkyl groups, sulfonic acid groups, or carboxyl groups; and R8 is selected from hydrogen atom, deuterium atom, halogen atom, nitro group, cyano group, hydroxyl group, and C. 1-6 Alkyl groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 Alkylaminoalkyl group, or a protecting group of amino group.

[0031] In some implementations, the antibody or its antigen-binding fragment is a single-chain variable fragment (scFv).

[0032] In some implementations, the antibody or its antigen-binding fragment is a mouse IgG1 antibody or its antigen-binding fragment.

[0033] In some implementations, the antibody or its antigen-binding fragment also contains a detectable marker.

[0034] In some implementations, the detectable label is an enzyme label, a biotin label, or a fluorescein label.

[0035] In one aspect, this disclosure relates to a nucleic acid comprising a polynucleotide encoding a polypeptide, the polypeptide comprising: (1) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 1, 2 and 3 respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 44. (2) An immunoglobulin light chain or a fragment thereof comprising a VL, the VL comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 31, 32 and 33, respectively, and wherein the VL binds a camptothecin derivative when paired with a VH comprising an amino acid sequence as shown in SEQ ID NO: 43; (3) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 4, 5 and 6, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 46. (4) An immunoglobulin light chain or a fragment thereof comprising a VL, the VL comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 34, 35 and 36, respectively, and wherein the VL binds a camptothecin derivative when paired with a VH comprising an amino acid sequence as shown in SEQ ID NO: 45. (5) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 7, 8 and 9, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 48. (6) An immunoglobulin light chain or a fragment thereof comprising a VL, the VL comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 37, 38 and 39, respectively, and wherein the VL binds a camptothecin derivative when paired with a VH comprising an amino acid sequence as shown in SEQ ID NO: 47. (7) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 10, 11 and 12, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 50; (8) An immunoglobulin light chain or a fragment thereof comprising a VL, the VL comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 37, 38 and 39, respectively, and wherein the VL binds a camptothecin derivative when paired with a VH comprising an amino acid sequence as shown in SEQ ID NO: 49. (9) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 13, 14 and 15, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 52; (10) An immunoglobulin heavy chain or a fragment thereof comprising a light chain variable region (VL), the VL comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 40, 41 and 42, respectively, and wherein the VL binds a camptothecin derivative when paired with a heavy chain variable region (VH) comprising an amino acid sequence as shown in SEQ ID NO: 51; (11) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 16, 17 and 18, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 44; (12) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 19, 20 and 21, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 46; (13) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 22, 23 and 24, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 48; (14) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 25, 26, and 27, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 50; or (15) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 28, 29 and 30, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 52.

[0036] In some embodiments, the nucleic acid includes a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof containing a VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 1, 2 and 3, respectively.

[0037] In some embodiments, the nucleic acid includes a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof containing a VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 16, 17 and 18, respectively.

[0038] In some embodiments, the nucleic acid includes a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof containing a VL, the VL comprising CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 31, 32 and 33, respectively.

[0039] In some embodiments, camptothecin derivatives are specifically bound when the VH pairs with the VL, or camptothecin derivatives are specifically bound when the VL pairs with the VH.

[0040] In some embodiments, the immunoglobulin heavy chain or a fragment thereof is the mouse immunoglobulin heavy chain or a fragment thereof (e.g., the mouse IgG1 heavy chain or a fragment thereof), and the immunoglobulin light chain or a fragment thereof is the mouse immunoglobulin light chain or a fragment thereof.

[0041] In some implementations, the nucleic acid encodes a single-stranded variable fragment (scFv). In some implementations, this nucleic acid is cDNA.

[0042] In one respect, this disclosure relates to a vector comprising one or more nucleic acids described herein.

[0043] In one aspect, this disclosure relates to a vector comprising the two nucleic acids described herein, wherein the vector encodes a VL region and a VH region, the VL region and the VH region together binding a camptothecin derivative.

[0044] In one aspect, this disclosure relates to a pair of vectors, each of which includes a nucleic acid as described herein, wherein the pair of vectors together encodes a VL region and a VH region, the VL region and VH region together binding a camptothecin derivative.

[0045] In one respect, this disclosure relates to a cell comprising the vectors or vector pairs described herein.

[0046] In some implementations, the cell is a CHO cell.

[0047] In one respect, this disclosure relates to a cell that includes one or more nucleic acids described herein.

[0048] In one respect, this disclosure relates to a cell comprising the two nucleic acids described herein.

[0049] In some implementations, the two nucleic acids together encode the VL region and the VH region, which together bind to a camptothecin derivative.

[0050] In one aspect, this disclosure relates to a method for generating an antibody or an antigen-binding fragment thereof, the method comprising: (a) The cells are cultured under conditions sufficient to induce the cells described herein to produce the antibody or the antigen-binding fragment; and (b) Collect the antibody or antigen-binding fragment produced by the cell.

[0051] In one aspect, this disclosure relates to a composition comprising an antibody or an antigen-binding fragment thereof as described herein. In some embodiments, the composition further comprises a buffer solution.

[0052] In one aspect, this disclosure relates to a kit comprising the antibody or antigen-binding fragment thereof described herein.

[0053] In some implementations, the kit is an enzyme-linked immunosorbent assay (ELISA) kit, a fluorescence immunoassay kit, or a chemiluminescent immunoassay kit.

[0054] In one aspect, this disclosure relates to a method for detecting a sample containing a camptothecin derivative, the method comprising contacting the sample with an antibody or antigen-binding fragment or composition thereof described herein.

[0055] In one aspect, this disclosure relates to a method for detecting samples containing camptothecin derivatives, the method comprising using a kit described herein.

[0056] In some implementations, the sample is a tissue sample, blood sample, serum sample, or plasma sample.

[0057] In some implementations, the samples are obtained from subjects who have already been given a therapeutic agent containing a camptothecin derivative.

[0058] In one aspect, this disclosure relates to a method for detecting samples containing camptothecin derivatives for non-diagnostic and non-therapeutic purposes.

[0059] As used herein, the term "antibody" refers to any antigen-binding molecule containing at least one (e.g., one, two, three, four, five, or six) complementarity-determining regions (CDRs) (e.g., any of the three CDRs from the immunoglobulin light chain or any of the three CDRs from the immunoglobulin heavy chain) and capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives such as bispecific antibodies, single-chain antibodies, biantibodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0060] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody that is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of the heavy chain or a variable domain of the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0061] As used herein, the term "human antibody" refers to an antibody encoded by an endogenous nucleic acid present in humans (e.g., a rearranged heavy or light chain human immunoglobulin locus). In some embodiments, human antibodies are collected from humans or generated in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are generated in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are generated in bacterial or yeast cells. In some embodiments, human antibodies are generated in transgenic non-human animals (e.g., cattle) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0062] As used herein, the term "chimeric antibody" refers to an antibody containing sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody containing a variable domain sequence (e.g., all or part of a light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and a constant domain of a human antibody. Other examples of chimeric antibodies are described herein and are known in the art.

[0063] As used herein, the term "humanized antibody" refers to a non-human antibody containing a minimum sequence derived from a non-human (e.g., mouse) immunoglobulin and a sequence derived from a human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (receptor antibody) wherein the hypervariable region (e.g., CDR) residues of the receptor antibody are replaced with hypervariable region (e.g., CDR) residues of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capability. In some embodiments, the Fv framework residues of a human immunoglobulin are replaced with corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, the humanized antibody may contain residues not found in the receptor antibody or donor antibody. These modifications can be made to further improve antibody performance. In some embodiments, the humanized antibody comprises at least one, and typically substantially all, of two variable domains, wherein all or substantially all of the hypervariable loops (CDRs) correspond to the hypervariable loops of a non-human (e.g., mouse) immunoglobulin, and all or substantially all of the framework regions are framework regions of a human immunoglobulin. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of human immunoglobulins. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for producing humanized antibodies are described herein.

[0064] As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of the heavy or light chains of mammalian immunoglobulins) capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0065] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe animals (human or non-human) for whom treatment is provided according to the method of the invention. The invention is contemplated for both veterinary and non-veterinary applications. Human patients can be adults or adolescents (e.g., persons under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. This includes, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domesticated, farm, and zoo animals.

[0066] As used herein, when referring to antibodies, the phrase "specific binding" means that, compared to other molecules, an antibody preferably interacts with its target molecule (e.g., camptothecin derivatives) because the interaction depends on the presence of a specific structure on the target molecule; in other words, the reagent recognizes and binds to molecules that include a specific structure, rather than all molecules as is typically the case. Antibodies that specifically bind to a target molecule can be called target-specific antibodies. For example, antibodies that specifically bind to camptothecin derivative molecules can be called camptothecin derivative-specific antibodies or anti-camptothecin derivative antibodies.

[0067] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and refer to an amino acid polymer of any length containing at least two amino acids.

[0068] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably and refer to a nucleotide polymer of any length consisting of at least two nucleotides, including but not limited to DNA, RNA, DNA / RNA hybrids, and their modifications.

[0069] As used herein, unless the contrary is explicitly stated, the singular forms “a”, “an”, and “the” include plural references.

[0070] As used herein, the terms “comprising” and “including” are intended to indicate the presence of the stated feature, whole, component or step, but do not exclude the presence or addition of one or more other features, wholes, components, steps or combinations thereof.

[0071] As used herein, the term “about” means approximately, within a certain range, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated value. Generally, the term “about” is used herein to modify values ​​that differ by 20%, typically 10%, more typically 5%, or even more typically 1% above or below a given value. Sometimes, such a range may fall within experimental error or may relate to the type of standard method used to measure and / or determine a given value or range.

[0072] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this invention, chemical elements are identified according to the CAS version of the periodic table of elements as shown on the inner cover of the 75th edition of the *Handbook of Chemistry and Physics*, and specific functional groups are generally defined as described therein. Furthermore, the general principles of organic chemistry, as well as descriptions of specific functional groups and reactivity, are found in the following references: *Organic Chemistry*, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's *Advanced Organic Chemistry*, 5 th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, ComprehensiveOrganic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modem Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0073] When listing a range of values, it is intended to cover every value within that range and its subranges. For example, "C 1-6 "Aims to cover C1, C2, C3, C4, C5, C6, C 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C4-6 C 4-5 and C 5-6 .

[0074] As used herein, the term "alkyl" whether used as part of another term or independently refers to an acyclic, straight-chain or branched saturated hydrocarbon group that may optionally be independently substituted (i.e., unsubstituted or substituted) by one or more substituents as described below. The term "C i-j "Alkyl" refers to an alkyl group having i to j carbon atoms. In some embodiments, the alkyl group contains 1 to 12 carbon atoms. In some embodiments, the alkyl group contains 1 to 11 carbon atoms. In some embodiments, the alkyl group contains 1 to 11 carbon atoms, 1 to 10 carbon atoms, 1 to 9 carbon atoms, 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Non-limiting examples of alkyl groups include methyl; ethyl; n-propyl and isopropyl; n-butyl, sec-butyl, isobutyl, and tert-butyl; neopentyl, etc. Where the valence allows, the alkyl group may optionally be composed of one, two, three, or (in the presence of a valence group) In the case of an alkyl group with two or more carbon atoms, four or more substituents independently selected from the following: alkoxy, acyloxy, amino, aryl, aryloxy, azide, cycloalkyl, cycloalkoxy, haloyl, heterocyclic, heteroaryl, heterocyclic alkyl, heteroarylalkyl, heterocyclic oxy, heteroaryloxy, hydroxyl, nitro, thio, silyl, cyano, =O, =S, and =NR', wherein R' is H, alkyl, aryl, or heterocyclic. In some embodiments, the alkyl group may optionally be substituted with a haloyl, amino, hydroxyl, methoxy, nitro, cyano, etc. Each substituent itself may be unsubstituted, or, where the valence allows, substituted with an unsubstituted substituent as defined herein for each respective group.

[0075] As used herein, the term "alkenyl," whether used as part of another term or independently, refers to a straight-chain or branched hydrocarbon group having at least one carbon-carbon double bond, which may optionally be independently substituted (i.e., unsubstituted or substituted) by one or more substituents described herein, and includes groups having "cis" and "trans" orientations. In some embodiments, the alkenyl group contains 2 to 12 carbon atoms. In some embodiments, the alkenyl group contains 2 to 11 carbon atoms. In some embodiments, the alkenyl group contains 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. In some embodiments, the alkenyl group contains 2 carbon atoms. Non-limiting examples of alkenyl groups include ethylidene (or vinyl), propenyl, butenyl, pentenyl, 1-methyl-2-buten-1-yl, 5-hexenyl, etc. The optionally substituted alkenyl group is the optionally substituted alkenyl group as described herein with respect to alkyl groups.

[0076] As used herein, the term "alkoxy" whether used as part of another term or independently refers to a chemical substituent of the formula -OR, where R is an alkyl group, specifically C. 1-12 Alkyl, C 1-10 Alkyl, C 1-6 Alkyl groups, etc. The alkoxy group may be unsubstituted or substituted. Optionally substituted alkoxy groups are those that are optionally substituted as defined herein with respect to alkyl groups.

[0077] As used herein, the term "halogen" or "halogenated group" refers to fluoride, chloride, bromide, and iodide ions, especially fluoride and chloride ions, and more particularly fluoride ions.

[0078] As used herein, unless otherwise stated, the term “heteroatom” means nitrogen (N), oxygen (O), and sulfur (S), and may include any oxidized form of nitrogen and sulfur, as well as any quaternized form of basic nitrogen.

[0079] As used herein, the terms “heterocyclic group” or “heterocycle”, whether used as part of another term or independently, refer to a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having fused or bridged 4-, 5-, 6-, 7-, or 8-membered rings, which, unless otherwise specified, contains one, two, three, or four heteroatoms independently selected from nitrogen, oxygen, and sulfur. Heterocyclic groups can be 3- to 12-membered, for example, 3- to 10-membered, 4- to 12-membered, 4- to 10-membered, 5- to 12-membered, 5- to 10-membered, or 5- to 8-membered. Heterocyclic groups can be aromatic or non-aromatic. Aromatic heterocyclic groups are heteroaryl groups as described herein. Non-aromatic 5-membered heterocyclic groups have zero or one double bond, non-aromatic 6- and 7-membered heterocyclic groups have zero to two double bonds, and non-aromatic 8-membered heterocyclic groups have zero to two double bonds and / or zero or one carbon-carbon triple bond. Unless otherwise specified, heterocyclic groups have 1 to 16 carbon atoms. Some heterocyclic groups may have up to 9 carbon atoms. Non-aromatic heterocyclic groups include pyrrolinyl, pyrrolylalkyl, pyrazolinyl, pyrazolylalkyl, imidazolinyl, imidazolinyl, piperidinyl, homopiperidinyl, piperazinyl, pyridazinyl, oxazolyl, isoxazolyl, morpholinyl, thiomorpholinyl, thiazolinyl, isothiazolyl, thiazolinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyranyl, dihydropyranyl, dithiazolinyl, etc.

[0080] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The methods and materials used in this invention are described herein; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0081] Other features and advantages of the invention will become clear from the detailed description, accompanying drawings, and claims below. Attached Figure Description

[0082] Figure 1 The CDR sequences of anticamptothecin derivative antibodies, defined by Kabat numbering, are listed.

[0083] Figure 2 The CDR sequences of anticamptothecin derivative antibodies, defined by Chothia numbers, are listed.

[0084] Figure 3 The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-camptothecin derivative antibody are listed.

[0085] Figure 4 Several amino acid sequences discussed in this disclosure are listed.

[0086] Figures 5A to 5D This demonstrates the assay of trastuzumab analogues using ELISA with 1D12 as the detection antibody. Figure 5A ), T-CPT2 ( Figure 5B ), T-VA-AZ0132 ( Figure 5C ) and T-GGFG-Dxd ( Figure 5D When the concentration of antibody is specified, a standard curve is used with antibody concentration (ng / mL) as the X-axis and calibrated absorbance value (Delta represents OD450nm to OD630nm) as the Y-axis.

[0087] Figures 6A to 6D This demonstrates the determination of trastuzumab analogues by ELISA using anti-Dxd mIgG antibody as the detection antibody. Figure 6A ), T-CPT2 ( Figure 6B ), T-VA-AZ0132 ( Figure 6C ) and T-GGFG-Dxd ( Figure 6D When the concentration of antibody is specified, a standard curve is used with antibody concentration (ng / mL) as the X-axis and calibrated absorbance value (Delta represents OD450nm to OD630nm) as the Y-axis.

[0088] Figure 7 The standard curves are shown, using antibody concentration (ng / mL) as the X-axis and optical density value as the Y-axis, when 1D12 is used as the detection antibody to determine the concentrations of T-CPT2, T-VA-AZ0132 and T-GGFG-Dxd by ELISA.

[0089] Figure 8 The standard curves are shown, using toxin molecule concentration (nM) as the X-axis and optical density value as the Y-axis, when using 1D12 to detect ISO-GGFG-Dxd, with different concentrations of Dxd, eczema, CTP2, AZ0132, SN38 or MMAE as competing molecules.

[0090] Figure 9 Immunohistochemical (IHC) results of trastuzumab in vivo in the NUGC-4 xenograft mouse model are shown. Biotin-1D12 was used as the detection antibody. Detailed Implementation

[0091] This disclosure provides examples of antibodies that bind to camptothecin derivatives and their antigen-binding fragments.

[0092] Camptothecin derivatives

[0093] As used herein, the term "camptothecin derivative" refers to a compound or its salts, solvates, stereoisomers, or isotopic variants that include the structural moiety represented by formula (II). These are derived from the natural product camptothecin. For example, it can be a compound represented by formula (II), or a conjugate of a compound represented by formula (II) with a ligand via a linker.

[0094] (II)

[0095] In some embodiments, the camptothecin derivative is a compound of general formula (I) or a salt, solvate, stereoisomer, or isotopic variant thereof: LG-LD (I), Where LG represents no ligand or no linker; L represents no linker or no linker; and D is a compound of the following structural formula (II) or a salt, solvate, stereoisomer, or isotopic variant thereof. R1 and R2 independently represent hydrogen atoms and hydroxyl groups, and may contain halogen atoms, nitro groups, or cyano groups. 1-6 alkyl group, C 2-6 alkenyl group, C 1-6 alkoxy group, C 1-6 Aminoalkoxy groups, halogen atoms, nitro groups, cyano groups, mercapto groups, alkylthio groups, amino groups that may contain protecting groups, or C groups that may contain protecting groups or have C atoms at the amino position. 1-6 C of alkyl group 1-6 The aminoalkyl group may contain a protecting group or a C at the amino position. 1-6 C of alkyl group 1-6 Aminoalkylamino groups, which may contain C 1-6 Alkyl, C 1-6 C of heterocycles with alkoxy, amino, halogen, nitro, or cyano groups 1-6 Alkyl groups, which may contain C 1-6 Alkyl, C 1-6 The carbonyl group of a heterocyclic compound containing an alkoxy, amino, halogen, nitro, or cyano group, which may contain a C 1-6 Alkyl, C 1-6 C of a heterocyclic ring consisting of an alkoxy group, an amino group (which may contain a protecting group), a halogen group, a nitro group, a cyano group, or a protecting group. 1-6 The alkylamino group may contain a protecting group or a C-shaped group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The amino-heterocyclic group of the alkyl group may contain a protecting group or a C-shaped protecting group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The alkyl group may contain a heterocyclic amino group, or may contain a protecting group or a C-shaped group. 1-6 The carbamoyl group of the alkyl group; R3 represents C1-6 alkyl groups; R4 represents C 1-6 Alkyl groups, which may contain protecting amino groups, quaternary trialkylammonium groups such as -N.sup.+(CH3)3, and C groups that may contain protecting C groups. 1-6 Alkylamino groups, which may contain protecting groups, are C14 groups. 1-6 An aminoalkyl group, which may contain a protecting group, is a C-shaped group. 1-6 alkylaminoalkyl group, sulfonic acid group or carboxyl group; Z represents oxygen atom, sulfur atom, CR5 R6, where R5 and R6 each represent hydrogen atom or carbon atom. 1-6 Alkyl, or N-R7, where R7 represents a hydrogen atom, C 1-6 Alkyl groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 Alkylaminoalkyl group, or protecting group of amino group; and m and n each represent 0, 1 or 2.

[0096] In some implementations, D in equation (I) is represented by the following structural equation (III): (III) Where R1 and R2 represent hydrogen atoms and C atoms, respectively. 1-3 Alkyl, C 2-3 alkenyl, hydroxymethyl, hydroxyl, C 1-3 Alkoxy, halogen, nitro, amino, C 1-3 Alkylamino, cyano-C 1-3 Alkyl, aminomethyl, etc.

[0097] R3 represents an ethyl group.

[0098] R4 represents C 1-3 Alkyl group, hydrogen atom, amino group (which may contain protecting groups), C 1-6 Alkylamino, amino-C 1-6 Alkyl or C 1-6 Alkylamino-C 1-6 Alkyl groups. Particularly preferred are hydrogen atoms, amino groups, methylamino groups, dimethylamino groups, aminomethyl groups, ethylamino groups, diethylamino groups, aminoethyl groups, methylaminomethyl groups, dimethylaminomethyl groups, hydroxyethylamino groups, etc.

[0099] Z represents oxygen atom, sulfur atom, CR5 R6, where R5 and R6 each represent hydrogen atom or carbon atom. 1-6 alkyl.

[0100] In some implementations, D in equation (I) is represented by the following structural formula (IV): (Ⅳ), Examples of preferred combinations of R4, R1, and R2 include those that make: R4 is H, NH2, NHCH3, or N(CH3)2, and R1 or R2 is H, CH3 or F.

[0101] The most preferred combination is where R4 is H or NH2, R1 is 4-methyl and R2 is 5-fluoro, or both R1 and R2 are H.

[0102] In some embodiments, D in formula (I) is a compound selected from the following compounds or compounds used as payloads in ADCs: (V-1) (V-2) (V-3) (V-4) (V-5) and (V-6)

[0103] In some embodiments, the camptothecin derivative may be a compound represented by structural formula (VI), or a conjugate of a compound represented by structural formula (VI) with a ligand via a linker.

[0104]

[0105] (VI)

[0106] In some embodiments, the camptothecin derivative is a compound of general formula (I) or a salt, solvate, stereoisomer, or isotopic variant thereof: LG-LD (I), Where LG indicates no ligand or no linker; L indicates no linker or no linker; and D is a compound of the following structural formula (VI) or a salt, solvate, stereoisomer, or isotopic variant thereof. R1 and R2 independently represent hydrogen atoms and hydroxyl groups, and may contain halogen atoms, nitro groups, or cyano groups. 1-6 alkyl group, C 2-6 alkenyl group, C 1-6 alkoxy group, C 1-6 Aminoalkoxy groups, halogen atoms, nitro groups, cyano groups, mercapto groups, alkylthio groups, amino groups that may contain protecting groups, or C groups that may contain protecting groups or have C atoms at the amino position. 1-6 C of alkyl group 1-6The aminoalkyl group may contain a protecting group or a C at the amino position. 1-6 C of alkyl group 1-6 Aminoalkylamino groups, which may contain C 1-6 Alkyl, C 1-6 C of heterocycles with alkoxy, amino, halogen, nitro, or cyano groups 1-6 Alkyl groups, which may contain C 1-6 Alkyl, C 1-6 The carbonyl group of a heterocyclic compound containing an alkoxy, amino, halogen, nitro, or cyano group, which may contain a C 1-6 Alkyl, C 1-6 C of a heterocyclic ring consisting of an alkoxy group, an amino group (which may contain a protecting group), a halogen group, a nitro group, a cyano group, or a protecting group. 1-6 The alkylamino group may contain a protecting group or a C-shaped group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The amino-heterocyclic group of the alkyl group may contain a protecting group or a C-shaped protecting group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The alkyl group may contain a heterocyclic amino group, or may contain a protecting group or a C-shaped group. 1-6 The carbamoyl group of the alkyl group; Alternatively, R1 and R2, together with the atoms they are attached to, form a 3-, 4-, 5-, or 6-membered heterocyclic alkyl group or a 3-, 4-, 5-, or 6-membered cycloalkyl group, wherein the 3-, 6-membered heterocyclic alkyl group or 3-, 6-membered cycloalkyl group is optionally substituted by one, two, three, or four substituents selected from hydrogen atoms, hydroxyl groups, and C atoms that may contain halogen atoms, nitro groups, or cyano groups. 1-6 alkyl group, C 2-6 alkenyl group, C 1-6 alkoxy group, C 1-6 Aminoalkoxy groups, halogen atoms, nitro groups, cyano groups, mercapto groups, alkylthio groups, amino groups that may contain protecting groups, or C groups that may contain protecting groups or have C atoms at the amino position. 1-6 C of alkyl group 1-6 The aminoalkyl group may contain a protecting group or a C at the amino position. 1-6 C of alkyl group 1-6 Aminoalkylamino groups, which may contain C 1-6 Alkyl, C 1-6 C of heterocycles with alkoxy, amino, halogen, nitro, or cyano groups 1-6 Alkyl groups, which may contain C 1-6 Alkyl, C 1-6 The carbonyl group of a heterocyclic compound containing an alkoxy, amino, halogen, nitro, or cyano group, which may contain a C 1-6 Alkyl, C 1-6C of a heterocyclic ring consisting of an alkoxy group, an amino group (which may contain a protecting group), a halogen group, a nitro group, a cyano group, or a protecting group. 1-6 The alkylamino group may contain a protecting group or a C-shaped group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The amino-heterocyclic group of the alkyl group may contain a protecting group or a C-shaped protecting group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The alkyl group may contain a heterocyclic amino group, or may contain a protecting group or a C-shaped group. 1-6 The carbamoyl group of the alkyl group; R3 represents C 1-6 alkyl groups; Z' represents hydrogen atom, deuterium atom, oxygen atom, sulfur atom, halogen atom, nitro group, cyano group, hydroxyl group, and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 6-10 Aryl, C 3-14 Cycloalkyl, 5-14-membered heteroaryl, 4-14-membered heterocycloalkyl, C 6-10 Aryl-C 1-4 Alkyl-, C 3-14 cycloalkyl-C 1-4 Alkyl-, (5-14-membered heteroaryl)-C 1-4 Alkyl-, (4-14 membered heterocyclic alkyl)-C 1-4 Alkyl-, OR8, C(O)R8, C(O)NR8R8, C(O)OR8, S(O)2R8 and S(O)2NR8R8, wherein Z' of C 1-6 Alkyl, C 1-6 Haloalkyl, C 6-10 Aryl, C 3-14 Cycloalkyl, 5-14-membered heteroaryl, 4-14-membered heterocycloalkyl, C 6-10 Aryl-C 1-4 Alkyl-, C 3-14 cycloalkyl-C 1-4 Alkyl-, (5-14-membered heteroaryl)-C 1-4 Alkyl- and (4-14 membered heterocyclic alkyl)-C 1-4 Each alkyl group is optionally substituted by one, two, three, four, or five independently selected substituents, which are selected from hydrogen atoms, deuterium atoms, halogen atoms, nitro groups, cyano groups, hydroxyl groups, C6 groups, and C7 groups. 1-6 Alkyl groups, amino groups that may contain protecting groups, quaternary trialkylammonium groups such as -N.sup.+(CH3)3, and C groups that may contain protecting groups 1-6 Alkylamino groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups1-6 alkylaminoalkyl groups, sulfonic acid groups, or carboxyl groups; and R8 is selected from hydrogen atom, deuterium atom, halogen atom, nitro group, cyano group, hydroxyl group, and C. 1-6 Alkyl groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 Alkylaminoalkyl group, or a protecting group of amino group.

[0107] In some implementations, D in equation (I) is represented by the following structural formula (VII).

[0108] (VII)

[0109] Where R1 and R2 represent hydrogen atoms and C atoms, respectively. 1-3 Alkyl, C 2-3 alkenyl, hydroxymethyl, hydroxyl, C 1-3 Alkoxy, halogen, nitro, amino, C 1-3 Alkylamino, cyano-C 1-3 Alkyl, aminomethyl, etc.; Alternatively, R1 and R2 together with the atoms they are attached to form a 5- or 6-membered heterocyclic alkyl group, wherein the 5- or 6-membered heterocyclic alkyl group is optionally substituted by one, two, three, or four substituents selected from hydrogen atoms, hydroxyl groups, C atoms, and C atoms. 1-6 Alkyl groups, amino groups that may contain protecting groups, C 1-6 Alkylamino group, amino-C 1-6 alkyl groups or C 1-6 Alkylamino-C 1-6 Alkyl groups.

[0110] R3 represents C 1-3 Alkyl group. In some embodiments, R3 is ethyl.

[0111] Z' represents hydrogen atom, deuterium atom, halogen atom, hydroxyl group, and C. 1-6 Alkyl, C 3-14 cycloalkyl, C 6-10 Aryl-C 1-4 Alkyl-, C 3-14 cycloalkyl-C 1-4 Alkyl-, (5-14-membered heteroaryl)-C 1-4 Alkyl-, (4-14 membered heterocyclic alkyl)-C 1-4 Alkyl-, where Z' is C 1-6 Alkyl, C 3-14 cycloalkyl, C 6-10Aryl-C 1-4 Alkyl-, C 3-14 cycloalkyl-C 1-4 Alkyl-, (5-14-membered heteroaryl)-C 1-4 Alkyl- and (4-14 membered heterocyclic alkyl)-C 1-4 Each alkyl group is optionally substituted by one, two, three, four, or five independently selected substituents, which are selected from hydrogen atoms, deuterium atoms, halogen atoms, nitro groups, cyano groups, hydroxyl groups, C6 groups, and C7 groups. 1-6 Alkyl groups, amino groups that may contain protecting groups, quaternary trialkylammonium groups such as -N.sup.+(CH3)3, and C groups that may contain protecting groups 1-6 Alkylamino groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 Alkylaminoalkyl group, sulfonic acid group or carboxyl group.

[0112] In some implementations, Z' represents a hydrogen atom, C 1-6 Alkyl, C 3-14 cycloalkyl and C 3-14 cycloalkyl-C 1-4 Alkyl-, where Z' is C 1-6 Alkyl, C 3-14 cycloalkyl and C 3-14 cycloalkyl-C 1-4 Each alkyl group is optionally substituted by one, two, three, four, or five independently selected substituents, which are selected from hydrogen atoms, amino groups that may contain protecting groups, quaternary trialkylammonium groups such as -N.sup.+(CH3)3, and C groups that may contain protecting groups. 1-6 Alkylamino groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 Alkylaminoalkyl group, sulfonic acid group or carboxyl group.

[0113] In some implementations, Z' represents a hydrogen atom.

[0114] In some implementations, Z' represents C 1-6 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, and n-butyl. In some embodiments, Z' represents ethyl.

[0115] In some implementations, Z''s C 1-6 The alkyl group is optionally substituted by one, two, three, four, or five independently selected substituents, which are selected from hydrogen atoms, deuterium atoms, hydroxyl groups, C atoms, and C2 groups. 1-6 Alkyl groups, amino groups that may contain protecting groups, and C groups that may contain protecting groups 1-6Alkylamino group. In some embodiments, the amino group present at Z' and C... 1-6 The protecting groups in the alkylamino groups include methyl, ethyl, n-propyl, isopropyl, methoxy, methoxy, ethoxy, propoxy, sulfonyl groups optionally substituted with one or more methyl groups, one or more ethyl groups, one or more n-propyl groups, or one or more isopropyl groups, and phosphine groups optionally substituted with one or more methyl groups, one or more ethyl groups, one or more n-propyl groups, or one or more isopropyl groups.

[0116] In some implementations, Z' represents C 3-14 Cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, bicyclo[1.1.1]pent-1-yl, bicyclo[2.1.0]pent-1-yl. In some embodiments, Z''s C 3-14 The cycloalkyl group is optionally substituted by one, two, three, four, or five independently selected substituents, which are selected from hydrogen atoms, deuterium atoms, hydroxyl groups, C atoms, and C2 groups. 1-6 Alkyl groups, amino groups that may contain protecting groups, and C groups that may contain protecting groups 1-6 Alkylamino group. In some embodiments, the amino group present at Z' and C... 1-6 The protecting groups in the alkylamino groups include methyl, ethyl, n-propyl, isopropyl, methoxy, methoxy, ethoxy, propoxy, sulfonyl groups optionally substituted with one or more methyl groups, one or more ethyl groups, one or more n-propyl groups, or one or more isopropyl groups, and phosphine groups optionally substituted with one or more methyl groups, one or more ethyl groups, one or more n-propyl groups, or one or more isopropyl groups.

[0117] In some embodiments, D in formula (I) is a compound selected from the following compounds or compounds used as payloads in ADCs: (VIII-1) (VIII-2) (VIII-3) (VIII-4) (VIII-5) (VIII-6) (VIII-7) (VIII-8) (VIII-9) (VIII-10) As used herein, the term "ligand" refers to a compound that recognizes and binds to an antigen or receptor associated with a target cell. Ligands can be cell-binding agents. The "cell-binding agents" of this invention can be of any kind currently known or becoming known, including peptides and non-peptides that bind to cells or cellular components (e.g., receptors, proteins, DNA, RNA, etc.). Typically, these can be antibodies (such as polyclonal and monoclonal antibodies, especially monoclonal antibodies) or fragments thereof, lymphokines, hormones, growth factors, vitamins (such as folic acid, which can bind to their cell surface receptors, such as the folic acid receptor), nutrient transport molecules (such as transferrin), probodies, nanobodies, or any other cell-binding molecules or substances.

[0118] In some embodiments, the cell binder is an antibody, a single-chain antibody, an antibody fragment that specifically binds to target cells, a monoclonal antibody, a single-chain monoclonal antibody, a monoclonal antibody fragment that specifically binds to target cells, a chimeric antibody, a chimeric antibody fragment that specifically binds to target cells, a domain antibody (e.g., sdAb), or a domain antibody fragment that specifically binds to target cells. In some embodiments, the cell binder is a humanized antibody, a humanized single-chain antibody, or a humanized antibody fragment. In some embodiments, the cell binder is a surface-reconstructed antibody, a surface-reconstructed single-chain antibody, or a surface-reconstructed antibody fragment. In some embodiments, the cell binder is an antibody or its antigen-binding portion (including antibody derivatives). The cell binder may bind ligands on target cells, such as cell surface ligands, including cell surface receptors. In some embodiments, the cell binder binds to target cells selected from: tumor cells, virus-infected cells, microbial-infected cells, parasite-infected cells, autoimmune cells, activated cells, bone marrow cells, activated T cells, B cells, or melanocytes.In some implementations, the cell binder binds to the expression of 5T4, ADAM-9, ALK, AMHRII, ASCT2, Axl, B7-H3, BCMA, C4.4a, CA6, CA9, CanAg, CD123, CD138, CD142, CD166, CD184, CD19, CD20, CD205, CD22, CD248, CD25, CD3, CD30, CD33, CD352, CD37, CD38, CD40L, CD44v6, CD45, CD46, CD48, CD51, CD56, and C. D7, CD70, CD71, CD74, CD79b, CDH6, CEACAM5, CEACAM6, cKIT, CLDN18.2, CLDN6, CLL-1, c-MET, Epidermal Growth Factor, CSP-1, CXCR5, DLK-1, DLL3, DPEP3, Anti-adhesion factor, EFNA4, EGFR, EGFRviii, ENPP3, EpCAM, EphA2, EphA3, ETBR, FGFR2, FGFR3, FLT3, FOLR-α, FSH, GCC, GD2, GD3, Globo H, GPC-1, GPC3, gpNMB, HER-2, HER-3, HLA-DR, HSP90, IGF-1R, IL-13R, IL1RAP, IL7R, Interleukin-4 receptor (IL4R), KAAG-1, LAMP-1, Lewis Y antigen, LGALS3BP, LGR5, LH / hCG, LHRH, LIV-1, LRP-1, LRRC15, Ly6E, MAGE, Mesothelin (MSLN), MET, MHC Cellular cells containing one or more of the following: Class I chain-associated proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2b, cohesin-4, NOTCH3, OAcGD2, OX001L, p-cadherin, PD-L1, phosphatidylserine (PS), polymorphic epithelial mucin (PEM), prolactin receptor (PRLR), PSMA, PTK7, RNF43, ROR1, ROR2, SAIL, SLAMF7, SLC44A4, SLITRK6, SSTR2, STEAP-1, STING, STn, TIM-1, TM4SF1, TNF-α, TRA, TROP-2, tumor-associated glycoprotein 72 (TAG-72), tumor-specific epitope of mucin-1 (TA-MUC1), CD5, TIM-3, UPK2, or UPK1b antigen.In some implementations, the cell binder is a cysteine-engineered antibody or its antigen-binding fragment that specifically binds to 5T4, ADAM-9, ALK, AMHRII, ASCT2, Axl, B7-H3, BCMA, C4.4a, CA6, CA9, CanAg, CD123, CD138, CD142, CD166, CD184, CD19, CD20, CD205, CD22, CD248, CD25, CD3, CD30, CD33, CD352, CD37, CD38, CD40L, CD44v6, CD45, CD46, and CD48. CD51, CD56, CD7, CD70, CD71, CD74, CD79b, CDH6, CEACAM5, CEACAM6, cKIT, CLDN18.2, CLDN6, CLL-1, c-MET, Epidermal Growth Factor, CSP-1, CXCR5, DLK-1, DLL3, DPEP3, Anti-adhesion Factor, EFNA4, EGFR, EGFRviii, ENPP3, EpCAM, EphA2, EphA3, ETBR, FGFR2, FGFR3, FLT3, FOLR-α, FSH, GCC, GD2, GD3, Globo H, GPC-1, GPC3, gpNMB, HER-2, HER-3, HLA-DR, HSP90, IGF-1R, IL-13R, IL1RAP, IL7R, Interleukin-4 receptor (IL4R), KAAG-1, LAMP-1, Lewis Y antigen, LGALS3BP, LGR5, LH / hCG, LHRH, LIV-1, LRP-1, LRRC15, Ly6E, MAGE, Mesothelin (MSLN), MET, MHC Cellular cells containing one or more of the following: Class I chain-associated proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2b, cohesin-4, NOTCH3, OAcGD2, OX001L, p-cadherin, PD-L1, phosphatidylserine (PS), polymorphic epithelial mucin (PEM), prolactin receptor (PRLR), PSMA, PTK7, RNF43, ROR1, ROR2, SAIL, SLAMF7, SLC44A4, SLITRK6, SSTR2, STEAP-1, STING, STn, TIM-1, TM4SF1, TNF-α, TRA, TROP-2, tumor-associated glycoprotein 72 (TAG-72), tumor-specific epitope of mucin-1 (TA-MUC1), CD5, TIM-3, UPK2, or UPK1b antigen.

[0119] As used herein, the term "connector" or "linker compound" refers to a compound that can be linked together to form a ligand-drug conjugate by reacting, for example, with groups of a ligand compound and a therapeutic agent compound, respectively, through a conjugation reaction. The "connector" of this invention can be of any kind currently known or becoming known.

[0120] Antibody and antigen binding fragment

[0121] This disclosure provides antibodies against camptothecin derivatives and their antigen-binding fragments. Typically, antibodies (also known as immunoglobulins) consist of two classes of polypeptide chains: a light chain and a heavy chain. The non-limiting antibody of this disclosure can be a complete tetraimmunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be any isotype (including IgM, IgG, IgE, IgA, or IgD) or subisotype (including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.). The light chain can be a κ light chain or a λ light chain. The antibody may comprise two identical copies of the light chain and two identical copies of the heavy chain. Each contains a variable domain (or variable region, V...). H The heavy chains, consisting of multiple constant domains (or constant regions), are linked together by disulfide bonds within their constant domains to form the "backbone" of the antibody. Each chain contains a variable domain (or variable region, V...). L A light chain and a constant structural domain (or constant region) are each bonded to a heavy chain via disulfide bonds. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bonded. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conservative framework regions (FRs).

[0122] These hypervariable regions (called complementarity-determining regions (CDRs)) form loops that comprise the main antigen-binding surface of the antibody. The four framework regions are predominantly in a beta-sheet conformation, while the CDRs form loops that connect the beta-sheet structures and, in some cases, form part of them. The CDRs in each chain are held tightly together by the framework regions and, together with CDRs from other chains, contribute to the formation of the antigen-binding region.

[0123] Identifying the CDR region of an antibody by analyzing its amino acid sequence is a well-known method, and many definitions of CDRs are commonly used. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop region. These methods and definitions are described in the following references, such as Martin, "Protein sequence and structure analysis of antibody variable domains", Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains", Molecularimmunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., BiophysChem. 68(1-3): 9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2): 269-94 (Jan. 1998); Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology, 7:64 (2007). All of the above references are incorporated herein by reference in their entirety.

[0124] In some implementations, antibodies are complete immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, but their constant regions differ, particularly the hinge and upper CH2 domain. The sequences and differences of IgG subclasses are known in the art and described in the following references, such as Vidarsson et al., "IgG subclasses and allotypes: from structure to effector functions", Frontiers in immunology 5 (2014); Irani et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases", Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk (ed.), "The human IgG subclasses: molecular analysis of structure, function and regulation", Elsevier, 2016. The full text of the above references is incorporated herein by reference.

[0125] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, camels). The antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies including an immunoglobulin-binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to a portion of the antibody that retains the specific binding activity of the intact antibody; that is, any portion of the antibody capable of specifically binding to an epitope on a target molecule of the intact antibody. It includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, the antibody or its antigen-binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, biantibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and any polypeptide including an antibody-binding domain or a binding domain homologous to it. Non-limiting examples of antigen-binding domains include, for example, heavy chain and / or light chain CDRs of an intact antibody, variable regions of heavy chain and / or light chain of an intact antibody, full-length heavy chain or light chain of an intact antibody, or a single CDR from the heavy chain or light chain of an intact antibody.

[0126] In some implementations, scFv has a heavy chain variable structure domain and a light chain variable structure domain.

[0127] Anti-camptothecin derivative antibodies and their antigen-binding fragments

[0128] This disclosure provides antibodies that specifically bind to camptothecin derivatives with high affinity, but not to non-camptothecin derivatives at detectable levels. These antibodies are particularly useful as reagents for detecting camptothecin derivatives in preclinical pharmacological studies of therapeutic agents containing camptothecin derivatives in cynomolgus monkeys or another non-human primate. The antibodies of this invention can also be used in preclinical immunohistochemical studies.

[0129] The antibodies of this invention can bind to the six ring structures of the core structure of camptothecin derivatives (rings A, B, C, D, E, and F of compounds represented by the following structural formula (II)). These advantages include higher binding affinity and specificity, as well as more diverse epitope recognition.

[0130] (II)

[0131] The antibodies of this invention can also bind to the five ring structures of the core structure of camptothecin derivatives (rings B, C, D, E, and F of compounds represented by the following structural formula (II)). These advantages include higher binding affinity and specificity, as well as more diverse epitope recognition.

[0132]

[0133] (VII)

[0134] This disclosure provides antibodies that specifically bind to camptothecin derivatives and their antigen-binding fragments. The antibodies and antigen-binding fragments described herein are capable of binding to camptothecin derivatives.

[0135] This disclosure provides, for example, anticamptothecin derivative antibodies 1D12, 1B4, 1B7, 1C2 and 1E4, their chimeric antibodies, and their human or humanized antibodies.

[0136] The CDR sequences of 1D12 and 1D12-derived antibodies include CDRs of the heavy chain variable domain (SEQ ID NO: 1-3) and light chain variable domains (SEQ ID NO: 31-33) as defined by Kabat numbers. CDRs can also be defined by the Chothia system. According to Chothia numbers, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 16-18, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 31-33.

[0137] The amino acid sequence of the heavy chain variable region of 1D12 is shown in SEQ ID NO: 43. The amino acid sequence of the light chain variable region of 1D12 is shown in SEQ ID NO: 44.

[0138] The CDR sequences of 1B4 and 1B4-derived antibodies include CDRs of the heavy chain variable domain (SEQ ID NO: 4-6) and light chain variable domains (SEQ ID NO: 34-36) as defined by Kabat numbers. CDRs can also be defined by the Chothia system. According to Chothia numbers, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 19-21, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 34-36.

[0139] The amino acid sequence of the heavy chain variable region of 1B4 is shown in SEQ ID NO: 45. The amino acid sequence of the light chain variable region of 1B4 is shown in SEQ ID NO: 46.

[0140] The CDR sequences of 1B7 and 1B7-derived antibodies include CDRs of the heavy chain variable domain (SEQ ID NO: 7-9) and light chain variable domains (SEQ ID NO: 37-39) as defined by Kabat numbers. CDRs can also be defined by the Chothia system. According to Chothia numbers, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 22-24, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 37-39.

[0141] The amino acid sequence of the heavy chain variable region of 1B7 is shown in SEQ ID NO: 47. The amino acid sequence of the light chain variable region of 1B7 is shown in SEQ ID NO: 48.

[0142] The CDR sequences of 1C2 and 1C2-derived antibodies include CDRs of the heavy chain variable domain (SEQ ID NO: 10-12) and light chain variable domains (SEQ ID NO: 37-39) as defined by Kabat numbers. CDRs can also be defined by the Chothia system. According to Chothia numbers, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 25-27, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 37-39.

[0143] The amino acid sequence of the heavy chain variable region of 1C2 is shown in SEQ ID NO: 49. The amino acid sequence of the light chain variable region of 1C2 is shown in SEQ ID NO: 50.

[0144] The CDR sequences of 1E4 and 1E4-derived antibodies include CDRs of the heavy chain variable domain (SEQ ID NO: 13-15) and light chain variable domains (SEQ ID NO: 40-42) as defined by Kabat numbers. CDRs can also be defined by the Chothia system. According to Chothia numbers, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 28-30, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 40-42.

[0145] The amino acid sequence of the heavy chain variable region of 1E4 is shown in SEQ ID NO: 51. The amino acid sequence of the light chain variable region of 1E4 is shown in SEQ ID NO: 52.

[0146] The amino acid sequences of the heavy chain variable region and the light chain variable region of the modified antibody are also provided. In some embodiments, the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43, 45, 47, 49, or 51. In some embodiments, the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44, 46, 48, 50, or 52. The heavy chain variable region sequence may pair with the corresponding light chain variable region sequence, and they together bind to a camptothecin derivative.

[0147] Humanization percentage refers to the percentage of identity of the heavy or light chain variable region sequence compared to human antibody sequences in the International Immunogenetic Information System (IMGT) database. Top hit means that the heavy or light chain variable region sequence is more closely related to a specific species than to other species. For example, top hit with humans means that the sequence is more closely related to humans than to other species. (The text then repeats itself, so the translation will only include the first instance.) Macaca fascicularis The term "optimal alignment" means that the sequence has the same percentage of identity as human and cynomolgus monkey sequences, and that this percentage of identity is the highest compared to sequences from other species. In some embodiments, the humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. Detailed descriptions of how to determine the humanization percentage and how to determine the optimal alignment are known in the art and are described, for example, in Jones et al., "The INNs and outs of antibody nonproprietarynames", MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, the entire text of which is incorporated herein by reference. High humanization percentages generally have several advantages, such as greater safety and efficacy in humans, greater likelihood of being tolerated by human subjects, and / or a lower likelihood of side effects. In some implementations, the variable region is fully human, for example, derived from human heavy chain immunoglobulin locus sequences (e.g., recombinations of human IGHV, human IGHD, and human IGHJ genes), and / or human κ chain immunoglobulin locus sequences (e.g., recombinations of human IGKV and human IGKJ genes).

[0148] In addition, in some embodiments, the antibody or its antigen-binding fragment described herein may also contain one, two or three heavy chain variable regions (CDRs) selected from the following: SEQ ID NO: 1-3, SEQ ID NO: 4-6, SEQ ID NO: 7-9, SEQ ID NO: 10-12, SEQ ID NO: 13-15, SEQ ID NO: 16-18, SEQ ID NO: 19-21, SEQ ID NO: 22-24, SEQ ID NO: 25-27 and SEQ ID NO: 28-30; and / or one, two or three light chain variable regions (CDRs) selected from the following: SEQ ID NO: 31-33, SEQ ID NO: 34-36, SEQ ID NO: 37-39 and SEQ ID NO: 40-42.

[0149] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 comprises or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VH CDR1 amino acid sequence, CDR2 comprises or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VH CDR2 amino acid sequence, and CDR3 comprises or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VH CDR3 amino acid sequence. In some embodiments, the antibody may have a light chain variable region (VL) comprising CDR1, 2, and 3, wherein CDR1 comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VL CDR1 amino acid sequence, CDR2 comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VL CDR2 amino acid sequence, and CDR3 comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VLCDR3 amino acid sequence. The selected VHCDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are shown in [illustration]. Figure 1 (Kabat CDR) and Figure 2 (Chothia CDR)

[0150] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored 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 as the corresponding position in the second sequence. Considering the number of vacancies that need to be introduced to achieve optimal alignment of the two sequences and the length of each vacancy, the percentage of identity between the two sequences is a function of the number of common positions shared by the sequences. For example, sequence comparison and determination of the percentage of identity between two sequences can be accomplished using a Blossum 62 scoring matrix, where the vacancy penalty is 12, the vacancy extension penalty is 4, and the frameshift vacancy penalty is 5.

[0151] This disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides, wherein the polypeptides comprise immunoglobulin heavy chains or immunoglobulin light chains. Immunoglobulin heavy chains or immunoglobulin light chains comprise, for example... Figure 1 or Figure 2 The CDR shown, or having the following characteristics Figure 3 The sequence is shown. When a polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to a camptothecin derivative.

[0152] Anticamptothecin derivative antibodies and their antigen-binding fragments can also be antibody or antibody fragments and antibody variants (including derivatives and conjugates) of multispecific (e.g., bispecific) antibodies or antibody fragments. Other antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multispecific (multimer, e.g., bispecific) antibodies, human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly prepared antibodies (i.e., intracellular antibodies), and their antigen-binding fragments. Antibodies or their antigen-binding fragments can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0153] The provided method is suitable as long as the antibody fragment retains the desired affinity and specificity of the full-length antibody. Therefore, an antibody fragment binding to a camptothecin derivative will retain the ability to bind to the camptothecin derivative. An Fv fragment is an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a tightly bound heavy-chain variable domain and a light-chain variable domain, which can be covalently bound, such as scFv. In this configuration, it is the interaction of the three CDRs of each variable domain that defines the antigen-binding site on the surface of the VH-VL dimer. In general, the six CDRs or a subset thereof collectively confer antibody antigen-binding specificity. However, even a single variable domain (or half an Fv containing only three CDRs specific to a particular antigen) has the ability to recognize and bind antigens, but the affinity is generally lower than that of the entire binding site.

[0154] A single-chain Fv (scFv), or antibody fragment, comprises the VH and VL domains (or regions) of an antibody, wherein these domains are contained within a single polypeptide chain. Typically, scFv polypeptides also include polypeptide linkers between the VH and VL domains, allowing the scFv to form the desired structure for antigen binding.

[0155] The Fab fragment contains a variable and a constant domain of the light chain and a variable and a first constant domain (CH1) of the heavy chain. The F(ab')2 antibody fragment comprises a pair of Fab fragments, which are typically covalently linked near their carboxyl terms by a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known in the art.

[0156] The anti-camptothecin derivative antibody or antigen-binding moiety of this disclosure can be derivatized or linked to another molecule (e.g., another peptide or protein). Typically, the antibody or its moiety is derivatized such that camptothecin derivative binding is not adversely affected by derivatization or labeling. For example, the antibody or antibody moiety of this disclosure can be functionally linked (through chemical coupling, gene fusion, non-covalent binding, or other means) to one or more other molecular entities, such as another antibody or a detectable marker or tag. Examples include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C 15 N、 35 S), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors, phycoerythrin, or Alexa Fluor® dyes), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotin groups, predetermined peptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains, epitope tags), and magnetic reagents such as gadolinium chelates. In some embodiments, the labels are linked by spacer arms of various lengths to reduce potential steric hindrance.

[0157] Antibody characteristics

[0158] This disclosure provides antibodies that specifically bind to camptothecin derivatives with high affinity.

[0159] Common techniques for measuring the affinity of antibodies for camptothecin derivatives include, for example, ELISA, RIA, BLI (biomembrane interference), and surface plasmon resonance (SPR).

[0160] In some specific implementations, the antibody (or its antigen-binding fragment) is delivered in less than 0.1 seconds. -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than 0.00001s -1 Less than 0.000001s -1 or less than 0.0000001s -1 The dissociation rate (koff) is specifically bound to the camptothecin derivative. In some embodiments, the dissociation rate (koff) is greater than 0.01 s. -1 Greater than 0.001s -1 Greater than 0.0001s -1 Greater than 0.00001s -1 Greater than 0.000001s -1 Greater than 0.0000001s -1or greater than 0.00000001s -1 .

[0161] In some implementations, the kinetic binding rate (kon) is greater than 1 × 10⁻⁶. 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some implementations, the kinetic binding rate (kon) is less than 1 × 10⁻⁶. 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0162] The affinity can be derived from the quotient of the kinetic rate constant (KD = koff / kon). In some implementations, KD is less than 1 × 10⁻⁶. -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M, less than 1×10 -10 M, less than 1×10 -11 M, less than 1×10 -12 M, less than 1×10 -13 M, less than 1×10 -14 M. In some implementations, KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some implementations, KD is greater than 1 × 10⁻⁶. - 7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10 -11 M, greater than 1×10 -12 M, greater than 1×10 - 13 M, greater than 1×10 -14 M.

[0163] In some embodiments, thermal stability is determined. In some embodiments, the antibody or antigen-binding fragment as described herein may have a Tm greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some implementations, Tm is less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0164] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is a human IgG1 antibody, optionally having a SI mutation, LALA mutation, N297A mutation, YTE mutation, and / or FLAA mutation. In some embodiments, the antibody is a human IgG4 antibody, optionally having a SI mutation, LALA mutation, N297A mutation, YTE mutation, and / or FLAA mutation.

[0165] In some embodiments, the antibody or antigen-binding fragment does not have a functional Fc region. For example, the antibody or antigen-binding fragment is a Fab, Fab', F(ab')2, or Fv fragment. In some embodiments, the Fc region has LALA mutations (L234A and L235A mutations according to EU numbers) or LALA-PG mutations (L234A, L235A, P329G mutations according to EU numbers). In some embodiments, the Fc region has FLAA mutations (F234A and L235A according to EU numbers). In some embodiments, the Fc region has N297A mutations according to EU numbers. In some embodiments, the Fc region has YTE mutations (M252Y, S254T, and T256E according to EU numbers).

[0166] Methods for preparing antibodies against camptothecin derivatives

[0167] Camptothecin derivatives conjugated to keyhole cyanin (KLH) or ovalbumin (OVA) can be used as immunogens to generate antibodies using standard techniques for the preparation of polyclonal and monoclonal antibodies. Polyclonal antibodies can be generated in animals through multiple injections (e.g., subcutaneous or intraperitoneal) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein may be conjugated to an agent that is immunogenic in the species to be immunized. Animals may be injected with the antigenic peptide or protein more than once (e.g., twice, three times, or four times).

[0168] Immunogens are commonly used to prepare antibodies by immunizing suitable subjects (e.g., humans or transgenic animals expressing at least one human immunoglobulin locus). Suitable immunogenic formulations may contain, for example, conjugates of camptothecin derivatives with KLH or OVA. The formulation may also include adjuvants (such as Freund's complete or incomplete adjuvants) or similar immunostimulants.

[0169] Antibody titers in immunized subjects can be monitored over time using standard techniques, such as enzyme-linked immunosorbent assay (ELISA), using immobilized camptothecin derivative peptides or peptides. If necessary, antibody molecules can be isolated from mammals (e.g., from blood) and further purified using well-known techniques (such as protein A or protein G chromatography) to obtain IgG fractions. At an appropriate time following immunization, for example when the specific antibody titer is highest, antibody-producing cells can be obtained from the subject and used to monitor antibody titers using standard techniques, such as those originally developed by Kohler et al. Nature Hybridoma technology and human B-cell hybridoma technology (Kozbor et al., 256:495-497, 1975) as described in the article 256:495-497, 1975 Immunol. Today Monoclonal antibodies can be prepared using hybridoma techniques such as EBV-hybridoma (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) or trioma technique. The techniques for generating hybridomas are well-known (see Current Protocols in Immunology, 1994, edited by Coligan et al., John Wiley & Sons, Inc., New York, NY). Hybridoma cells that produce monoclonal antibodies are detected by screening for antibodies binding to camptothecin derivatives in the hybridoma culture supernatant, for example, using a standard ELISA assay.

[0170] Variants of the antibody or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human, humanized, or chimeric antibody or its antigen-binding fragment described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence constituting the antigen-binding domain of the antibody or antigen-binding domain. Within this group of variants, some antibody or antigen-binding fragments will have increased affinity for a target (e.g., camptothecin derivatives). Any combination of deletions, insertions, and / or combinations can be made to obtain an antibody or its antigen-binding fragment with increased binding affinity to the target. Introducing amino acid changes into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as altering (e.g., increasing or decreasing) the number of glycosylation sites, altering the type of glycosylation sites (e.g., altering the amino acid sequence so that different sugars are linked by enzymes present in the cell), or introducing new glycosylation sites.

[0171] The antibodies disclosed herein may be derived from any species of animal, including mammals. Non-limiting examples of natural antibodies include those derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camels (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits, including genetically engineered rodents to produce human antibodies).

[0172] Human and humanized antibodies include antibodies having variable and constant regions derived from human immunoglobulin sequences (or having the same amino acid sequence as antibodies derived from human immunoglobulin sequences). Human antibodies may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or somatic mutations in vivo), such as in CDR.

[0173] Humanized antibodies are typically formed by grafting a non-human CDR onto a human framework (FR). Therefore, a humanized antibody has one or more amino acid sequences introduced from a non-human source. These non-human amino acid residues are often referred to as “imported” residues and are typically derived from the “imported” variable domain. Humanization is essentially achieved by, for example, replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. These methods are described, for example, by Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), and Verhoeyen et al., Science, 239:1534-1536 (1988); all of these references are incorporated herein by reference in their entirety. Thus, a “humanized” antibody is a chimeric antibody in which a small portion of the intact human V domain is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are often mouse antibodies, in which some CDR residues and some FR residues are replaced by residues from similar sites in human antibodies.

[0174] The selection of human VH and VL domains for the preparation of humanized antibodies is crucial for reducing immunogenicity. Following a so-called "best-fit" method, mouse antibody V domain sequences were screened against an entire library of known human domain sequences. The human sequence that best approximates the mouse sequence was then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)).

[0175] More importantly, antibody humanization preserves high specificity and affinity for antigens, as well as other advantageous biological properties. To achieve this, humanized antibodies can be prepared using methods that analyze parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available to illustrate and visualize the possible three-dimensional conformations of selected candidate immunoglobulin sequences. Examination of these visualizations allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues affecting the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the receptor and the introduced sequence to obtain desired antibody characteristics, such as increased affinity for the target antigen.

[0176] Typically, amino acid sequence variants of human, humanized, or chimeric anticamptothecin derivative antibodies will contain an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence present in the light or heavy chain of the original antibody.

[0177] In some implementations, Balb / c mice are used to generate antibodies.

[0178] Identity or homology relative to the original sequence is typically defined as the percentage of amino acid residues in the candidate sequence that are identical to those in human, humanized, or chimeric anticamptothecin derivative antibodies or fragments, after aligning the candidate sequence with the original sequence and introducing vacancies where necessary to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity.

[0179] Further modifications can be made to antibodies or antigen-binding fragments against camptothecin derivatives. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in that region. The resulting homodimeric antibodies can have any increased in vitro and / or in vivo half-life. Homodimeric antibodies with increased in vitro and / or in vivo half-life can also be prepared using heterobifunctional cross-linking agents, such as those described by Wolff et al. (…). Cancer Res., As described in 53:2560-2565, 1993. Alternatively, the antibody can be engineered to have a double Fc region (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0180] In some embodiments, antibodies against camptothecin derivatives or their antigen-binding fragments can be covalently modified. These covalent modifications can be performed via chemical or enzymatic synthesis, or via enzymatic or chemical cleavage. Other types of covalent modifications to antibodies or antibody fragments can be introduced into the molecule by reacting the target amino acid residues of the antibody or fragment with an organic derivatizer capable of reacting with selected side chain or N-terminal or C-terminal residues.

[0181] Recombinant vector

[0182] This disclosure also provides recombinant vectors (e.g., expression vectors) comprising isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), host cells into which these recombinant vectors are introduced (i.e., vectors that contain polynucleotides and / or include polynucleotides), and recombinant antibody polypeptides or fragments thereof generated by recombinant techniques.

[0183] As used herein, a “vector” is any construct capable of delivering one or more target polynucleotides to a host cell when introduced into that host cell. An “expression vector” is capable of delivering one or more target polynucleotides and expressing them as encoded polypeptides in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the target polynucleotide is positioned for expression in the vector by operatively linking to regulatory elements (such as promoters, enhancers, and / or poly-A tails) within or near the integration site of the target polynucleotide in or flanking the genome of the target polynucleotide, such that the target polynucleotide will be translated in the host cell into which the expression vector has been introduced.

[0184] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-glucan), transformation, transfection and infection, and / or transduction (e.g., with recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0185] In some embodiments, the use of a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses) to introduce the polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) may involve the use of a non-pathogenic (defective), replicative virus, or a replication-defective virus may be used. In the latter case, viral replication typically occurs only in complementary viral packaging cells. Suitable systems are disclosed in the following literature, such as Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NYAcad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Patent Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89 / 01973; U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science. 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for integrating DNA into such expression systems are well known to those skilled in the art. DNA can also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA can be increased by coating DNA onto biodegradable beads that are efficiently transported into cells.

[0186] For expression, a DNA insert comprising a polynucleotide encoding an antibody or polypeptide disclosed herein may be operatively linked to a suitable promoter (e.g., a heterologous promoter), such as the phage λPL promoter, E. coli (…). E. coliThe expression construct may include promoters such as lac, trp, and tac, early and late SV40 promoters, and promoters of retroviral LTRs. Other suitable promoters are known to those skilled in the art. The expression construct may also contain transcription initiation and termination sites, and ribosome binding sites for translation within the transcription region. The coding portion of the mature transcript expressed by the construct may include a translation initiation codon at the beginning of the polypeptide to be translated and a stop codon (UAA, UGA, or UAG) at an appropriate position at the end of the polypeptide to be translated.

[0187] As described above, the expression vector may include at least one optional marker. Such markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culture in *E. coli* and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as *E. coli*, *Streptomyces*, and *Salmonella typhimurium* cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 and *S. fall armyworm* Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Suitable culture media and conditions for the host cells described herein are known in the art.

[0188] Non-restrictive vectors for use with bacteria include pQE70, pQE60, and pQE-9 from Qiagen; pBS, Phagescript, Bluescript, pNH8A, pNH16a, pNH18A, and pNH46A from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 from Pharmacia. Non-restrictive eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene; and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

[0189] Suitable non-restrictive bacterial promoters include the *E. coli* lacI and lacZ promoters, T3 and T7 promoters, gpt promoters, λPR and PL promoters, and trp promoters. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, promoters of retroviral LTRs (such as the Roussarcoma virus (RSV) promoter), and metallothionein promoters (such as the mouse metallothionein-I promoter).

[0190] In brewer's yeast ( Saccharomyces cerevisiaeIn this study, many vectors containing constitutive or inducible promoters can be used, such as α-factor, alcohol oxidase, and PGH. For reviews, see: Ausubel et al. (1989), Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al. , Methods Enzymol., 153: 516-544 (1997).

[0191] Introducing constructs into host cells can be achieved through calcium phosphate transfection, DEAE-glucan-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. These methods are described in many standard laboratory manuals, such as Davis et al.'s Basic Methods in Molecular Biology (1986), the full text of which is incorporated herein by reference.

[0192] Transcription of DNA encoding the disclosed antibodies in higher eukaryotes can be increased by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, typically about 10-300 bp in length, that increase the transcriptional activity of the promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located lateside at base pairs 100-270 of the origin of replication, the cytomegalovirus early promoter enhancer, and polyomavirus and adenovirus enhancers located lateside at the origin of replication.

[0193] To induce the translated protein to be secreted into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. This signal can be an endogenous or heterologous signal of the polypeptide.

[0194] Peptides (e.g., antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusion proteins) or those tagged with histidine, and can include not only secretion signals but also additional heterologous functional regions. For example, regions of additional amino acids (especially charged amino acids) can be added to the N-terminus of the peptide to improve stability and durability in host cells, during purification, or during subsequent processing and storage. Furthermore, peptide moieties can be added to the peptide to facilitate purification. These regions can be removed prior to the final preparation of the peptide. Adding peptide moieties to peptides to induce secretion or excretion, improve stability, and facilitate purification are conventional techniques well known in the art.

[0195] Application of antibodies

[0196] The antibodies or antigen-binding fragments disclosed herein can be used to detect samples containing camptothecin derivatives.

[0197] The disclosed anti-camptothecin derivative antibody and its antigen-binding fragment can be used to detect and / or measure the level of camptothecin derivatives in a sample.

[0198] The term "sample" may include, but is not limited to, biological samples (such as blood, tissues, and cells), environmental samples (such as soil or water), chemical mixtures, or synthetic compounds. Samples may be in their raw state or may have undergone some form of processing, such as purification, concentration, dilution, or extraction. The purpose of examining a sample is to detect and potentially quantify the presence of camptothecin derivatives using antibodies or their antigen-binding fragments.

[0199] In some embodiments, the antibody-antigen binding fragment can be used to detect and / or measure the level of camptothecin derivatives in samples from animals (e.g., non-human primates such as cynomolgus monkeys or rhesus monkeys). In some embodiments, the antibody-antigen binding fragment can be used to detect and / or measure the level of camptothecin derivatives in samples from humans. Suitable detection and measurement methods include immunological methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and immunohistochemistry. In some embodiments, the antibody-antigen binding portion can be used to detect and / or measure the level of camptothecin derivatives in samples from humans for preclinical or clinical immunohistochemical (IHC) studies.

[0200] The antibodies or antigen-binding fragments disclosed herein can be used to prepare kits for detecting samples containing camptothecin derivatives.

[0201] In some embodiments, the kit may also include a device for determining the reactivity of an antibody or antigen-binding fragment to a camptothecin derivative in a sample. For example, the kit may contain reagents, detectable labels, and / or containers for measuring the specific binding of the antibody or antigen-binding fragment of the present invention to a camptothecin derivative.

[0202] In some implementations, the kit also contains a buffer solution.

[0203] This document also provides compositions comprising at least one (e.g., one, two, three, or four) of the antibody or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibody or antigen-binding fragments described herein may be present in any combination in the composition. The composition may be formulated in any manner known in the art.

[0204] The methods described herein include detecting samples containing camptothecin derivatives, and these methods include contacting the sample with an antibody or an antigen-binding fragment thereof or a composition containing camptothecin derivatives.

[0205] In some implementations, the sample is a tissue sample, blood sample, serum sample, or plasma sample.

[0206] In some implementations, the samples are obtained from subjects who have already been given a therapeutic agent containing a camptothecin derivative.

[0207] Example

[0208] The present invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0209] Example 1. Generation of antibodies that specifically bind to camptothecin derivatives

[0210] Keyhole cyanin (KLH) conjugated with compound CPT2 (Formula V-2) and / or ovalbumin (OVA) conjugated with compound CPT2 were emulsified with adjuvants and then used to immunize Balb / c mice. Antibody immune responses were monitored by antigen-specific immunoassay.

[0211] When the desired immune response is achieved, antigen-specific immune cells are isolated from immunized mice to further obtain antibodies that specifically bind to camptothecin derivatives or to obtain the light and heavy chain variable region sequences of these antibodies. For example, single-cell techniques (e.g., using Beacon) are employed. ® A photofluidic system (Berkeley Lights) was used to screen and identify plasma cells that secrete antigen-specific monoclonal antibodies, and reverse transcription and PCR sequencing were used to obtain the variable region sequence of the antibody. The obtained variable region sequence was cloned into a vector containing a sequence encoding the mouse IgG1 constant region for antibody expression. The binding affinity of the expressed antibody to camptothecin derivatives was verified using ELISA or BLI.

[0212] Exemplary antibodies obtained include 1D12, 1B4, 1B7, 1C2, and 1E4. Figure 1 The CDR sequence defined by Kabat is shown. Figure 2 The CDR sequence as defined by Chothia is shown. The heavy and light chain variable regions of these antibodies are shown. Figure 3 middle.

[0213] Example 2. Binding affinity of antibody specifically to camptothecin derivatives

[0214] Binding affinity for BSA and BSA-CPT2

[0215] Use equipped with Octet ®AMC Biosensors’ ForteBio Octet system validates the affinity of antibodies for bovine serum albumin (BSA, Solarbio Life Science, Cat#: A8020) or BSA-CPT2 (obtained by chemically linking BSA to the compound CPT2) using biomembrane interference (BLI).

[0216] By Octet ® The AMC biosensor captures antibody supernatant samples at 1000 rpm for 200 seconds. This is followed by injection of 200 nM BSA or BSA-CPT2 at 1000 rpm for 180 seconds. Dissociation is monitored for 600 seconds. After the final titration injection, the chip is regenerated with glycine (pH 1.7, 1000 rpm / min, 30 seconds).

[0217] By fitting the data as a whole to a 1:1 Langmuir fusion model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994). Methods Enzymology 6. 99-110) Simultaneously use OctecAnalysis Studio 12.2.2.26 to obtain the kinetic binding rate (kon) and dissociation rate (koff). Affinity is derived from the quotient of the kinetic rate constant (KD = koff / kon).

[0218] As will be understood by those skilled in the art, the same method was used to adjust the appropriate parameters (e.g., antibody concentration) for each test antibody. The results of the antibody tests are shown in Table 1.

[0219] Table 1

[0220] The results showed that antibodies 1D12, 1B4, 1B7, 1C2, and 1E4 could bind to BSA-CPT2, but not to BSA.

[0221] Binding affinity with anti-EGFR / TROP2 bispecific antibodies, anti-HER3 antibodies, and their ADCs

[0222] The binding affinity of the tested antibodies to the anti-EGFR / TROP2 bispecific antibody, the anti-HER3 antibody, or their ADCs was verified using the same experimental procedures as described above. The results are shown in the table below.

[0223] T-6F7-E-6C4 is an anti-EGFR / TROP2 IgG1 bispecific antibody possessing an anti-TROP2 antigen-binding domain (T-6F7, VH: SEQ ID NO: 54, VL: SEQ ID NO: 53) and an anti-EGFR antigen-binding domain (E-6C4, VH: SEQ ID NO: 55, VL: SEQ ID NO: 53). 3E1 is an anti-HER3 IgG1 monoclonal antibody, with its heavy and light chain sequences shown in SEQ ID NO: 56 and SEQ ID NO: 53, respectively. T-6F7-E-6C4 and 3E1 are conjugated to compound CPT-2 via a CPT-L linker (shown below) to obtain ADCs with a drug-antibody ratio (DAR) of approximately 8, named T-6F7-E-6C4-CPT2 and 3E1-CPT2, respectively.

[0224]

[0225] CPT-L

[0226] Table 2

[0227] The results showed that antibodies 1D12, 1B4, 1B7, 1C2 and 1E4 could bind to T-6F7-E-6C4-CPT2 and 3E1-CPT2, but not to T-6F7-E-6C4 and 3E1.

[0228] Binding affinity for antibodies targeting different targets or ADCs with different payloads

[0229] The affinity of the tested antibody for antibodies targeting different targets or ADCs with different payloads was verified using the same experimental procedures as previously described. The results are shown in the table below.

[0230] Trastuzumab is a humanized monoclonal antibody targeting human HER2, and its heavy and light chain sequences are shown in SEQ ID NO:57-58. By linking the VH and VL sequences of trastuzumab to the constant region of IgG1, trastuzumab analogs are obtained. These trastuzumab analogs are further conjugated to compound CPT-2 via the CPT-L linker to obtain an ADC with a DAR of approximately 8, named T-CPT2.

[0231] Goxatozumab (Trodelvy) TM Goxatozumab is an antibody-drug conjugate derived from Immunomedics (now Gilead) that contains a humanized anti-Trop2 monoclonal antibody and an SN-38 payload. The heavy and light chain sequences of the antibody moiety in goxatozumab are shown in SEQ ID NO: 59-60.

[0232] Compounds VA-AZ0132 (Mal-PEG8-amide-Val-Ala-(4-NH2)-Exatecan, MedChemExpress, Cat#: HY-145399) and GGFG-Dxd (Deruxtecan, MedChemExpress, Cat#: HY-13631E) were each conjugated with trastuzumab analogs with a DAR of approximately 8, resulting in ADCs T-VA-AZ0132 and T-GGFG-Dxd, respectively. The formulas for T-VA-AZ0132 and T-GGFG-Dxd are shown below (n is approximately 8).

[0233]

[0234] Table 3

[0235] The results showed that antibodies 1D12, 1B4, 1B7, 1C2 and 1E4 could bind to T-6F7-E-6C4-CPT2, T-VA-AZ0132 and T-GGFG-Dxd, but not to T-6F7-E-6C4, gosatutuzumab, trastuzumab analogs or sacitrus analogs.

[0236] The above binding affinity experiments show that 1D12, 1B4, 1B7, 1C2, and 1E4 all exhibit specific binding with high affinity to camptothecin derivatives (such as compounds CPT2, AZ0132, and Dxd).

[0237] Example 3. Detection of samples containing camptothecin derivatives using 1D12 and anti-Dxd mIgG antibodies.

[0238] In this experiment, the concentrations of T-CPT2, T-VA-AZ0132, T-GGFG-Dxd and trastuzumab analogues were determined using 1D12 and anti-Dxd mIgG antibody (Abmart, Cat#: ADC-Ab001).

[0239] Specifically, 1D12 or anti-Dxd mIgG antibody was diluted to a final concentration of 2000 ng / mL and added to each well of a 96-well plate (ELISA plate) at 100 μL / well, then incubated overnight at 2°C–8°C. Afterward, the plate was washed four times with PBS-T buffer (PBS supplemented with Tween™ 20). Unbound areas were blocked with 2% BSA (bovine serum albumin) at 37°C for 2 hours. The plate was then washed four times with PBS-T buffer. A series of diluted samples (T-CPT2, T-VA-AZ0132, T-GGFG-Dxd, or trastuzumab analogue) containing 1% BSA were added to each well and incubated at 37°C for 1 hour. After washing the plate four times with PBS-T buffer, anti-κ light chain-HRP (Abcam, Cat#: ab202549) diluted in 1% PBS was added to each well at 100 μL / well and incubated at 37°C for 1 hour. After washing the plate again, tetramethylbenzidine (TMB) solution was added to each well of the 96-well plate as a substrate at a rate of 100 μL / well. After incubation at room temperature and in the dark for 5–10 minutes, 100 μL of stop solution (Beyotime, Cat#: P0215) was added to each well. The luminescence signal of the plate was measured at 450 nm and 630 nm to calculate the concentration. A standard curve was established using the absorbance values ​​of calibration samples prepared with each test product and their corresponding concentrations, as shown in the figure. Figures 5A to 5D and Figures 6A to 6D As shown.

[0240] The results showed that 1D12 can be used effectively in ELISA analysis to detect T-CPT2, T-VA-AZ0132, or T-GGFG-Dxd, which have strong responses and high sensitivity. Figure 5B , Figure 5C , Figure 5D However, no response was observed when used to test trastuzumab analogs. Figure 5A Conversely, anti-Dxd mIgG antibodies can be used for the efficient detection of T-GGFG-Dxd, which exhibits strong response and high sensitivity. Figure 6D However, it showed no response or extremely low sensitivity to T-CPT2, T-VA-AZ0132, and trastuzumab analogues. Figure 6A , Figure 6B , Figure 6C This indicates that 1D12 can specifically bind to T-CPT2, T-VA-AZ0132, and T-GGFG-Dxd, but not to trastuzumab analogs, suggesting its specificity for camptothecin derivatives. Conversely, the anti-Dxd mIgG antibody specifically binds only to T-GGFG-Dxd, making it suitable only for detecting components containing Dxd compounds.

[0241] In another experiment, the binding activity of 1D12 with T-CPT2, T-VA-AZ0132, and T-GGFG-Dxd was assessed using ELISA. T-CPT2, T-VA-AZ0132, and T-GGFG-Dxd were diluted to 1 μg / mL, introduced into ELISA plates, and incubated overnight at 2°C–8°C. After washing with PBS-T buffer, unbound areas were blocked with 2% casein blocking buffer at 37°C for 2 hours. Then, serially diluted 1D12 (maximum concentration: 1000 ng / mL, 3-fold dilution, 7 gradients) or anti-Dxd mIgG antibody was added to each corresponding well and incubated at 37°C for 1 hour. Next, anti-mouse IgG-HRP marker diluted in 0.5% casein was added to each well and incubated at 37°C for 1 hour. After washing, TMB solution was added and incubated at room temperature and in the dark for 5–10 minutes. 100 μL of stop solution was added to each corresponding well. The luminescence signal was measured at 450 nm and 570 nm to determine the antibody concentration. A fitted curve was generated based on the photometric values ​​and the corresponding antibody concentrations. Figure 7 As shown, 1D12 exhibits strong response and high sensitivity to T-CPT2, T-VA-AZ0132 and T-GGFG-Dxd.

[0242] In another experiment, the binding activity of 1D12 with Dxd, eczema, AZ0132, CPT2, SN38, and MMAE was assessed using ELISA. Human IgG1 was conjugated to compound Dxd via a GGFG linker to produce an ADC with a drug-antibody ratio (DAR) of approximately 8, termed ISO-GGFG-Dxd. Specifically, ISO-GGFG-Dxd was diluted to a concentration of 1 μg / mL and introduced into ELISA plates, incubated overnight at 2°C–8°C. HRP-labeled antibody 1D12 was diluted to a concentration of 5 ng / mL as a diluent (termed HRP-1D12 diluent). MMAE or camptothecin derivative Dxd, eczema, AZ0132, CPT2, or SN38 were serially diluted with HRP-1D12 diluent (maximum concentration 10000 nM, 3-fold dilution, 10 gradients) and then added to ELISA plates for incubation. After treatment with TMB solution and termination solution, the emission signal was measured at 450 nm and 570 nm. For example... Figure 8 As shown, the binding of 1D12 to ISO-GGFG-Dxd was inhibited by Dxd, eczema, AZ0132, CPT2, and SN38. However, it was not inhibited by MMAE, further demonstrating the specific inhibitory activity of 1D12 against camptothecin derivatives.

[0243] Example 4. Detection of the concentration of anti-EGFR / TROP2 bispecific antibody ADC in monkey serum.

[0244] The concentration of T-6F7-E-6C4-CPT2 in monkey serum was detected by ELISA.

[0245] Dilute 1D12 to 2 μg / mL with PBS and add 100 μL / well to a 96-well plate (ELISA plate), then incubate overnight at 2℃–8℃. Afterward, wash the plate three times with PBS-T buffer (PBS replenished with Tween™ 20). Unbound areas are cleaned with Blocker in PBS. TM Casein was blocked at 25°C for 2 hours. Then, the plate was washed three times with PBS-T buffer. All cynomolgus monkey serum samples containing T-6F7-E-6C4-CPT2 were blocked using a blocker. TM Casein was diluted 200-fold in PBS and added to each well (100 μL / well), then incubated at 25°C for 1.5 h. After washing the plate three times with PBS-T buffer, anti-human κ light chain goat IgG biotin (monkey absorbed) (IBL, Cat#: 17249) deionized water was added using a blocker. TM Casein was diluted with PBS and added to each well of the plate at 100 μL / well, and incubated at 25 °C for 1 h. After washing the plate again, streptavidin-HRP bridging solution was added to the wells at 100 μL / well, and incubated at 25 °C for 20 min. Then, 100 μL of tetramethylbenzidine (TMB) was added to the wells as a substrate. After incubation at 25 °C for 4-5 min, 100 μL of sulfuric acid stop solution was added to each well. The luminescence signal was measured at 450 nm and 630 nm to calculate the concentration.

[0246] The results showed that when 1D12 was used for coating, it exhibited good selectivity and stability within the quantitative range of 6.4 μg / mL to 0.2 μg / mL, indicating that 1D12 can be used to effectively detect the concentration of T-6F7-E-6C4-CPT2 in serum.

[0247] Example 5. Immunohistochemical (IHC) Analysis

[0248] The in vivo distribution of trastuzumab was evaluated using 1D12 in tumor-bearing mice with human gastric cancer cells NUGC-4. Trastuzumab is a HER2-targeting ADC composed of a humanized monoclonal antibody trastuzumab and Dxd covalently linked via a GGFG linker.

[0249] Specifically, approximately 5×10 6 NuGC-4 cells were subcutaneously injected into B-NDG mice (Biocytogen, Cat#: B-CM-002). When the tumor volume reached approximately 300 mm², the cells were incubated. 3Mice were randomly divided into two groups (n=2 per group) based on tumor volume. Mice were then administered either phosphate-buffered saline (PBS) (G1) or trastuzumab (G2) intravenously (iv). Tumors were collected 24 hours after administration. The tumors were fixed in 10% formalin solution and then subjected to FFPE (formalin-fixed paraffin embedding). Five-micrometer sections were then cut using a microtome for further analysis.

[0250] Immunohistochemical staining of FFPE slides was performed using the fully automated Bond-RX (Leica Biosystems) system. FFPE slides were dewaxed and then subjected to antigen retrieval with ER2 buffer for 20 minutes. After blocking, slides were incubated with biotinylated 1D12 (Biotin-1D12) at 37°C for 60 minutes. Slides were then incubated in ABC solution at 37°C for 30 minutes. DAB was used as the chromogen, and hematoxylin was used as the background staining agent. Images of the slides were captured using a Leica slide scanner, with each slide in each treatment group magnified at 20x and 200x.

[0251] like Figure 9 As shown, a distinct brown signal was observed in the G2 treatment group, indicating that 1D12 can effectively detect camptothecin and its derivatives.

[0252] Other implementation plans

[0253] It should be understood that although the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to a camptothecin derivative, said antibody or antigen-binding fragment comprising: The heavy chain variable region (VH) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein VH CDR1 contains an amino acid sequence having at least 80% identity with a selected VH CDR1 amino acid sequence, VH CDR2 contains an amino acid sequence having at least 80% identity with a selected VH CDR2 amino acid sequence, and VH CDR3 contains an amino acid sequence having at least 80% identity with a selected VH CDR3 amino acid sequence; and The system comprises light chain variable regions (VLs) including CDR1, 2, and 3, wherein VL CDR1 contains an amino acid sequence having at least 80% identity with a selected VL CDR1 amino acid sequence, VL CDR2 contains an amino acid sequence having at least 80% identity with a selected VL CDR2 amino acid sequence, and VL CDR3 contains an amino acid sequence having at least 80% identity with a selected VL CDR3 amino acid sequence. The selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1, 2, and 3, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 31, 32, and 33, respectively; (2) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4, 5, and 6, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 34, 35, and 36, respectively. (3) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7, 8, and 9, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 37, 38, and 39, respectively; (4) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10, 11, and 12, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 37, 38, and 39, respectively; (5) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13, 14, and 15, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 40, 41, and 42, respectively. (6) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16, 17, and 18, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 31, 32, and 33, respectively. (7) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 19, 20, and 21, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 34, 35, and 36, respectively. (8) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 22, 23, and 24, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 37, 38, and 39, respectively; (9) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 25, 26, and 27, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 37, 38, and 39, respectively; and (10) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 28, 29, and 30, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 40, 41, and 42, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 1, 2, and 3, respectively, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 31, 32, and 33, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 4, 5, and 6, respectively, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 34, 35, and 36, respectively.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 7, 8, and 9, respectively, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 37, 38, and 39, respectively.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 10, 11, and 12, respectively, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 37, 38, and 39, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 13, 14, and 15, respectively, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 40, 41, and 42, respectively.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the definition of Chothia, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 16, 17, and 18, respectively, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 31, 32, and 33, respectively.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the definition of Chothia, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 19, 20, and 21, respectively, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 34, 35, and 36, respectively.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the definition of Chothia, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 22, 23, and 24, respectively, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 37, 38, and 39, respectively.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the definition of Chothia, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 25, 26, and 27, respectively, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 37, 38, and 39, respectively.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the definition of Chothia, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 28, 29, and 30, respectively, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 40, 41, and 42, respectively.

12. An antibody or antigen-binding fragment thereof conjugated to a camptothecin derivative, said antibody or antigen-binding fragment comprising: Heavy chain variable region (VH), wherein the VH contains an amino acid sequence that is at least 90% identical to a selected VH sequence; and a light chain variable region (VL), said VL comprising an amino acid sequence having at least 90% identity with a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 44; (2) The selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 46; (3) The selected VH sequence is SEQ ID NO: 47, and the selected VL sequence is SEQ ID NO: 48; (4) The selected VH sequence is SEQ ID NO: 49, and the selected VL sequence is SEQ ID NO: 50; and (5) The selected VH sequence is SEQ ID NO: 51, and the selected VL sequence is SEQ ID NO:

52.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the VH comprises the sequence of SEQ ID NO: 43, and the VL comprises the sequence of SEQ ID NO:

44.

14. The antibody or antigen-binding fragment thereof according to claim 12, wherein the VH comprises the sequence of SEQ ID NO: 45, and the VL comprises the sequence of SEQ ID NO:

46.

15. The antibody or antigen-binding fragment thereof according to claim 12, wherein the VH comprises the sequence of SEQ ID NO: 47, and the VL comprises the sequence of SEQ ID NO:

48.

16. The antibody or antigen-binding fragment thereof according to claim 12, wherein the VH comprises the sequence of SEQ ID NO: 49, and the VL comprises the sequence of SEQ ID NO:

50.

17. The antibody or antigen-binding fragment thereof according to claim 12, wherein the VH comprises the sequence of SEQ ID NO: 51, and the VL comprises the sequence of SEQ ID NO:

52.

18. An antibody or antigen-binding fragment thereof conjugated to a camptothecin derivative, said antibody or antigen-binding fragment comprising: The heavy chain variable region (VH) includes VHCDR1, VH CDR2, and VH CDR3 identical to those of the selected VH sequence; and the light chain variable region (VL) includes VL CDR1, VL CDR2, and VL CDR3 identical to those of the selected VL sequence. The selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 43, and the selected VL sequence is SEQ ID NO: 44; (2) The selected VH sequence is SEQ ID NO: 45, and the selected VL sequence is SEQ ID NO: 46; (3) The selected VH sequence is SEQ ID NO: 47, and the selected VL sequence is SEQ ID NO: 48; (4) The selected VH sequence is SEQ ID NO: 49, and the selected VL sequence is SEQ ID NO: 50; and (5) The selected VH sequence is SEQ ID NO: 51, and the selected VL sequence is SEQ ID NO:

52.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the antibody or antigen-binding fragment thereof specifically binds to a camptothecin derivative. The camptothecin derivatives mentioned therein are compounds of the following general formula (I) or their salts, solvates, stereoisomers or isotopic variants: LG-LD (I), Where LG represents no ligand or no linker; L represents no linker or no linker; and D is a compound of the following structural formula (II) or a salt, solvate, stereoisomer or isotopic variant thereof: (II), R1 and R2 independently represent hydrogen atoms and hydroxyl groups, and may contain halogen atoms, nitro groups, or cyano groups. 1-6 alkyl group, C 2-6 alkenyl group, C 1-6 alkoxy group, C 1-6 Aminoalkoxy groups, halogen atoms, nitro groups, cyano groups, mercapto groups, alkylthio groups, amino groups that may contain protecting groups, or C groups that may contain protecting groups or have C atoms at the amino position. 1-6 C of alkyl group 1-6 The aminoalkyl group may contain a protecting group or a C at the amino position. 1-6 C of alkyl group 1-6 Aminoalkylamino groups, which may contain C 1-6 Alkyl, C 1-6 C of heterocycles with alkoxy, amino, halogen, nitro, or cyano groups 1-6 Alkyl groups, which may contain C 1-6 Alkyl, C 1-6 The carbonyl group of a heterocyclic compound containing an alkoxy, amino, halogen, nitro, or cyano group, which may contain a C 1-6 Alkyl, C 1-6 C of a heterocyclic ring consisting of an alkoxy group, an amino group (which may contain a protecting group), a halogen group, a nitro group, a cyano group, or a protecting group. 1-6 The alkylamino group may contain a protecting group or a C-shaped group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The amino-heterocyclic group of the alkyl group may contain a protecting group or a C-shaped protecting group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The alkyl group may contain a heterocyclic amino group, or may contain a protecting group or a C-shaped group. 1-6 The carbamoyl group of the alkyl group; R3 represents C 1-6 alkyl groups; R4 represents C 1-6 Alkyl groups, which may contain protecting amino groups, quaternary trialkylammonium groups such as -N.sup.+(CH3)3, and C groups that may contain protecting C groups. 1-6 Alkylamino groups, which may contain protecting groups, are C14 groups. 1-6 An aminoalkyl group, which may contain a protecting group, is a C-shaped group. 1-6 alkylaminoalkyl group, sulfonic acid group or carboxyl group; Z represents oxygen atom, sulfur atom, CR5 R6, where R5 and R6 each represent hydrogen atom or carbon atom. 1-6 Alkyl, or N-R7, where R7 represents a hydrogen atom, C 1-6 Alkyl groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 An alkylaminoalkyl group, or a protecting group of the amino group; and m and n represent 0, 1, or 2 respectively.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the antibody or antigen-binding fragment thereof specifically binds to a camptothecin derivative, wherein the camptothecin derivative is a compound of general formula (I) or a salt, solvate, stereoisomer, or isotopic variant thereof: LG-LD (I), Where LG represents no ligand or no linker; L represents no linker or no linker; and D is a compound of the following structural formula (VI) or a salt, solvate, stereoisomer, or isotopic variant thereof: (VI) R1 and R2 independently represent hydrogen atoms and hydroxyl groups, and may contain halogen atoms, nitro groups, or cyano groups. 1-6 alkyl group, C 2-6 alkenyl group, C 1-6 alkoxy group, C 1-6 Aminoalkoxy groups, halogen atoms, nitro groups, cyano groups, mercapto groups, alkylthio groups, amino groups that may contain protecting groups, or C groups that may contain protecting groups or have C atoms at the amino position. 1-6 C of alkyl group 1-6 The aminoalkyl group may contain a protecting group or a C at the amino position. 1-6 C of alkyl group 1-6 Aminoalkylamino groups, which may contain C 1-6 Alkyl, C 1-6 C of heterocycles with alkoxy, amino, halogen, nitro, or cyano groups 1-6 Alkyl groups, which may contain C 1-6 Alkyl, C 1-6 The carbonyl group of a heterocyclic compound containing an alkoxy, amino, halogen, nitro, or cyano group, which may contain a C 1-6 Alkyl, C 1-6 C of a heterocyclic ring consisting of an alkoxy group, an amino group (which may contain a protecting group), a halogen group, a nitro group, a cyano group, or a protecting group. 1-6 The alkylamino group may contain a protecting group or a C-shaped group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The amino-heterocyclic group of the alkyl group may contain a protecting group or a C-shaped protecting group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The alkyl group may contain a heterocyclic amino group, or may contain a protecting group or a C-shaped group. 1-6 The carbamoyl group of the alkyl group; Alternatively, R1 and R2, together with the atoms they are attached to, form a 3-, 4-, 5-, or 6-membered heterocyclic alkyl group or a 3-, 4-, 5-, or 6-membered cycloalkyl group, wherein the 3-, 6-membered heterocyclic alkyl group or 3-, 6-membered cycloalkyl group is optionally substituted by one, two, three, or four substituents selected from hydrogen atoms, hydroxyl groups, and C atoms that may contain halogen atoms, nitro groups, or cyano groups. 1-6 alkyl group, C 2-6 alkenyl group, C 1-6 alkoxy group, C 1-6 Aminoalkoxy groups, halogen atoms, nitro groups, cyano groups, mercapto groups, alkylthio groups, amino groups that may contain protecting groups, or C groups that may contain protecting groups or have C atoms at the amino position. 1-6 C of alkyl group 1-6 The aminoalkyl group may contain a protecting group or a C at the amino position. 1-6 C of alkyl group 1-6 Aminoalkylamino groups, which may contain C 1-6 Alkyl, C 1-6 C of heterocycles with alkoxy, amino, halogen, nitro, or cyano groups 1-6 Alkyl groups, which may contain C 1-6 Alkyl, C 1-6 The carbonyl group of a heterocyclic compound containing an alkoxy, amino, halogen, nitro, or cyano group, which may contain a C 1-6 Alkyl, C 1-6 C of a heterocyclic ring consisting of an alkoxy group, an amino group (which may contain a protecting group), a halogen group, a nitro group, a cyano group, or a protecting group. 1-6 The alkylamino group may contain a protecting group or a C-shaped group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The amino-heterocyclic group of the alkyl group may contain a protecting group or a C-shaped protecting group at the nitrogen atom of the heterocyclic moiety or at the amino position. 1-6 The alkyl group may contain a heterocyclic amino group, or may contain a protecting group or a C-shaped group. 1-6 The carbamoyl group of the alkyl group; R3 represents C 1-6 alkyl groups; Z' represents hydrogen atom, deuterium atom, oxygen atom, sulfur atom, halogen atom, nitro group, cyano group, hydroxyl group, and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 6-10 Aryl, C 3-14 Cycloalkyl, 5-14-membered heteroaryl, 4-14-membered heterocycloalkyl, C 6-10 Aryl-C 1-4 Alkyl-, C 3-14 cycloalkyl-C 1-4 Alkyl-, (5-14-membered heteroaryl)-C 1-4 Alkyl-, (4-14 membered heterocyclic alkyl)-C 1-4 Alkyl-, OR8, C(O)R8, C(O)NR8R8, C(O)OR8, S(O)2R8 and S(O)2NR8R8, wherein Z' of C 1-6 Alkyl, C 1-6 Haloalkyl, C 6-10 Aryl, C 3-14 Cycloalkyl, 5-14-membered heteroaryl, 4-14-membered heterocycloalkyl, C 6-10 Aryl-C 1-4 Alkyl-, C 3-14 cycloalkyl-C 1-4 Alkyl-, (5-14-membered heteroaryl)-C 1-4 Alkyl- and (4-14 membered heterocyclic alkyl)-C 1-4 Each alkyl group is optionally substituted by one, two, three, four, or five independently selected substituents selected from hydrogen atoms, deuterium atoms, halogen atoms, nitro groups, cyano groups, hydroxyl groups, C6 groups, and C7 groups. 1-6 Alkyl groups, amino groups that may contain protecting groups, quaternary trialkylammonium groups such as -N.sup.+(CH3)3, and C groups that may contain protecting groups 1-6 Alkylamino groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 alkylaminoalkyl groups, sulfonic acid groups, or carboxyl groups; and R8 is selected from hydrogen atom, deuterium atom, halogen atom, nitro group, cyano group, hydroxyl group, and C. 1-6 Alkyl groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 aminoalkyl groups, C groups that may contain protecting groups 1-6 Alkylaminoalkyl group, or a protecting group of the amino group.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, wherein the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or antigen-binding fragment thereof further comprises a detectable marker.

23. A nucleic acid comprising a polynucleotide encoding a polypeptide, said polypeptide comprising: (1) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 1, 2 and 3, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO:

44. (2) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 31, 32 and 33, respectively, and wherein the VL binds a camptothecin derivative when paired with a VH comprising an amino acid sequence as shown in SEQ ID NO:

43. (3) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 4, 5 and 6, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO:

46. (4) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 34, 35 and 36, respectively, and wherein the VL binds a camptothecin derivative when paired with a VH comprising an amino acid sequence as shown in SEQ ID NO:

45. (5) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 7, 8 and 9, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO:

48. (6) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 37, 38 and 39, respectively, and wherein the VL binds a camptothecin derivative when paired with a VH comprising an amino acid sequence as shown in SEQ ID NO:

47. (7) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 10, 11 and 12, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 50; (8) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 37, 38 and 39, respectively, and wherein the VL binds a camptothecin derivative when paired with a VH comprising an amino acid sequence as shown in SEQ ID NO:

49. (9) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 13, 14 and 15, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 52; (10) An immunoglobulin heavy chain or a fragment thereof comprising a light chain variable region (VL), said VL comprising complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 40, 41 and 42, respectively, and wherein said VL binds a camptothecin derivative when paired with a heavy chain variable region (VH) comprising an amino acid sequence as shown in SEQ ID NO: 51; (11) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 16, 17 and 18, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 44; (12) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 19, 20 and 21, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 46; (13) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 22, 23 and 24, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 48; (14) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), said VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 having amino acid sequences as shown in SEQ ID NO: 25, 26, and 27, respectively, and said VH binding a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 50; or (15) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 28, 29 and 30, respectively, and wherein the VH binds a camptothecin derivative when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO:

52.

24. The nucleic acid of claim 23, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprising an immunoglobulin heavy chain or a fragment thereof containing VH, the VH comprising CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 1, 2 and 3, respectively.

25. The nucleic acid of claim 23, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprising an immunoglobulin heavy chain or a fragment thereof containing VH, the VH comprising CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 16, 17 and 18, respectively.

26. The nucleic acid of claim 23, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprising an immunoglobulin light chain or fragment thereof containing a VL, the VL comprising CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 31, 32, and 33, respectively.

27. The nucleic acid according to any one of claims 23 to 26, wherein the VH specifically binds to a camptothecin derivative when paired with VL, or wherein the VL specifically binds to a camptothecin derivative when paired with VH.

28. The nucleic acid according to any one of claims 23 to 27, wherein the immunoglobulin heavy chain or a fragment thereof is a mouse immunoglobulin heavy chain or a fragment thereof (e.g., a mouse IgG1 heavy chain or a fragment thereof), and the immunoglobulin light chain or a fragment thereof is a mouse immunoglobulin light chain or a fragment thereof.

29. The nucleic acid according to any one of claims 23 to 28, wherein the nucleic acid encodes a single-stranded variable fragment (scFv).

30. The nucleic acid according to any one of claims 23 to 29, wherein the nucleic acid is cDNA.

31. A vector comprising one or more nucleic acids as claimed in any one of claims 23 to 30.

32. A vector comprising two nucleic acids as claimed in any one of claims 23 to 30, wherein the vector encodes a VL region and a VH region, the VL region and the VH region together binding a camptothecin derivative.

33. A pair of vectors, wherein each vector comprises a nucleic acid as claimed in any one of claims 23 to 30, wherein the vector pair together encodes a VL region and a VH region, the VL region and the VH region together binding a camptothecin derivative.

34. A cell comprising the carrier as described in claim 31 or 32, or the carrier pair as described in claim 33.

35. The cell of claim 34, wherein the cell is a CHO cell.

36. A cell comprising one or more nucleic acids as claimed in any one of claims 23 to 30.

37. A cell comprising two nucleic acids as claimed in any one of claims 23 to 30.

38. The cell of claim 37, wherein the two nucleic acids together encode a VL region and a VH region, wherein the VL region and the VH region together bind a camptothecin derivative.

39. A method for generating an antibody or an antigen-binding fragment thereof, the method comprising: (a) The cells are cultured under conditions sufficient to induce the cells, as described in any one of claims 34 to 38, to produce the antibody or the antigen-binding fragment; and (b) Collect the antibody or antigen-binding fragment produced by the cells.

40. A composition comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 22.

41. A kit comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 22.

42. A method for detecting a sample containing a camptothecin derivative, the method comprising contacting the sample with an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 22 or a composition as claimed in claim 40.

43. A method for detecting a sample containing a camptothecin derivative, the method comprising using a kit as described in claim 41.

44. The method according to claim 42 or 43, wherein the sample is a tissue sample, a blood sample, a serum sample, or a plasma sample.

45. The method according to any one of claims 42 to 44, wherein the sample is obtained from a subject who has been administered a therapeutic agent containing a camptothecin derivative.

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