WO2024230742A1 - 一种抗体或其抗原结合片段、抗体-药物偶联物及其应用 - Google Patents

一种抗体或其抗原结合片段、抗体-药物偶联物及其应用 Download PDF

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WO2024230742A1
WO2024230742A1 PCT/CN2024/091754 CN2024091754W WO2024230742A1 WO 2024230742 A1 WO2024230742 A1 WO 2024230742A1 CN 2024091754 W CN2024091754 W CN 2024091754W WO 2024230742 A1 WO2024230742 A1 WO 2024230742A1
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seq
antibody
amino acid
heavy chain
acid sequence
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PCT/CN2024/091754
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English (en)
French (fr)
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陈思萌
王蕾蕾
张伟
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上海翰森生物医药科技有限公司
常州恒邦药业有限公司
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Publication of WO2024230742A1 publication Critical patent/WO2024230742A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention belongs to the field of antibodies, and specifically relates to an antibody specifically binding to mammals, especially human SEZ6, and a preparation method, a conjugate and an application thereof; the drug is prepared by connecting an anti-SEZ6 antibody and an anti-tumor drug 9106 via a linker.
  • Antibody-Drug conjugate is a drug that selectively binds to cancer cells by coupling cytotoxins to antibodies that selectively bind to the surface of cancer cells, and selectively delivers cytotoxins into tumor cells through antigen-antibody-mediated internalization. When the toxins accumulate to a certain level, the tumor cells are killed. As an ADC, it consists of three parts: antibodies, linkers, and toxins. ADC drugs can effectively improve the precise tumor targeting of toxins and increase the therapeutic window, so the target selection of ADC is crucial. It needs to be specifically highly expressed in tumors and not expressed in normal tissues.
  • Small cell lung cancer is a highly malignant tumor. About 250,000 people are diagnosed with it each year worldwide, accounting for about 15% of lung cancers. Among them, 70% of patients have metastases when diagnosed. The occurrence of tumors is closely related to smoking and is accompanied by mutations in multiple tumor suppressor genes. The median survival time of confirmed patients is only 8 to 12 months, and very few patients survive for more than 5 years.
  • the standard first-line treatment for small cell lung cancer is platinum plus etoposide. Patients have a high response rate to first-line chemotherapy, but after relapse and drug resistance, there is a lack of new effective treatment options. The response rate of second-line topotecan treatment is low, and there is no significant improvement in survival.
  • the treatment of small cell lung cancer urgently needs new breakthroughs and progress (Nature Reviews Clinical Oncology volume 14, pages 549–561 (2017)).
  • Human SEZ6 (epilepsy-related protein 6, hereinafter referred to as hSEZ6) is a single transmembrane protein, which is mainly expressed in brain tissue and almost not expressed in the periphery. In the direction of neurological diseases, knockout of mouse SEZ6 promotes the formation of short and multiple synapses (Neuron 56, 621–639, November 21, 2007); SEZ6 is a substrate of BACE1, and soluble SEZ6 in cerebrospinal fluid can be used as a marker for Alzheimer's disease (Molecular Neurodegeneration 2016 11: 67).
  • SEZ6 is a tumor-specific marker that is highly expressed in neuroendocrine tumor tissues. Immunohistochemical analysis of clinical specimens showed that SEZ6 is highly expressed in small cell lung cancer, and the expression level is much higher than that in normal tissues. In 174 tumor tissue sections of small cell lung cancer patients, about 70% of the patients' tumors were positive for SEZ6 (Public Patent: US2016287720A1).
  • AbbVie Pharmaceuticals Based on the high specific expression of SEZ6 in tumors, AbbVie Pharmaceuticals has developed an ADC targeting SEZ6, ABBV-011, for small cell lung cancer (public patent: WO2013126810A). Calicheomicin is used as a toxin with a DAR value of 2 and a non-degradable linker. The ADC drug entered Phase I clinical trials in 2019 (NCT03639194).
  • This patent obtains SEZ6 antibodies with high affinity and high endocytosis activity through mouse and camel immunization, and conjugates toxin compound 169106 (public patent: WO2020063676A) to obtain innovative ADC Drug.
  • 169106 is an inhibitor of topoisomerase I (also described herein as 9106), and previous studies have shown that this antibody-conjugated toxin has good drugability and in vivo antitumor activity and safety.
  • the present disclosure relates to anti-SEZ6 antibodies or antigen-binding fragments thereof, ADCs thereof, and medical uses thereof. More specifically, a monoclonal antibody or antigen-binding fragment thereof that binds to the amino acid sequence or three-dimensional structure of the ECD region of SEZ6 is provided, and an ADC drug thereof coupled to a cytotoxic substance exotecan analogue is provided.
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) or a heavy chain single domain (VHH), wherein the heavy chain variable region or the heavy chain single domain comprises: (1) heavy chain HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 37, 38 and 39 amino acid sequences; or (2) heavy chain HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 43, 44 and 45 amino acid sequences; or (3) heavy chain HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 49, 50 and 51 amino acid sequences; or (4) heavy chain HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 65, 66 and 67 amino acid sequences; or (5) heavy chain HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 68, 69 and 70 amino acid sequences.
  • VH heavy chain variable region
  • VHH heavy chain single domain
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region comprise: (1) heavy chain HCDR1, HCDR2, HCDR3 as shown in the amino acid sequences of SEQ ID NOs: 37, 38 and 39, and light chain LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NOs: 40, 41 and 42; or (2) light chain HCDR1, HCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NOs: or (3) heavy chain HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 43, 44 and 45, and light chain LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 46, 47 and 48. Or (4) heavy chain HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 49, 50 and 51
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, wherein the heavy chain single domain comprises an amino acid sequence as shown in SEQ ID NO: 63, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto; or as shown in SEQ ID NO: 64, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, wherein the heavy chain single domain comprises an amino acid sequence as shown in SEQ ID NO: 63 or as shown in SEQ ID NO: 64.
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, wherein the heavy chain single domain amino acid sequence is as shown in SEQ ID NO: 63, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto; or as shown in SEQ ID NO: 64, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.
  • the present disclosure provides an anti-DLL3 antibody or an antigen-binding fragment thereof, wherein the heavy chain single domain amino acid sequence is shown in SEQ ID NO: 63 or SEQ ID NO: 64.
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 35, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 35; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36.
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region amino acid sequence is selected from SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 35, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto; the light chain variable region amino acid sequence is selected from SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region amino acid sequence is selected from SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 35; the light chain variable region amino acid sequence is selected from SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36.
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (1) the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 29, or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 30, or a sequence having at least 85%, 90%, 95%, 99% homology thereto; or (2) the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 31, or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 32, or a sequence having at least 85%, 90%, 95%, 99% homology thereto.
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 35, or a sequence with at least 85%, 90%, 95%, 99% homology thereto
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 36, or a sequence with at least 85%, 90%, 95%, 99% homology thereto.
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (1) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 30; or (2) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 31, and the light chain variable region comprises the amino acid sequence or (3) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34; or (4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 36.
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise: (1) the heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 29, or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and the light chain variable region amino acid sequence is as shown in SEQ ID NO: 30, or a sequence having at least 85%, 90%, 95%, 99% homology thereto; or (2) the heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 31, or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and the light chain variable region amino acid sequence is as shown in SEQ ID NO: 32.
  • the heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 35, or a sequence with at least 85%, 90%, 95%, 99% homology thereto
  • the light chain variable region amino acid sequence is as shown in SEQ ID NO: 36, or a sequence with at least 85%, 90%, 95%, 99% homology thereto.
  • the present disclosure provides an anti-SEZ6 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise: (1) the heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 29, and the light chain variable region amino acid sequence is as shown in SEQ ID NO: 30; or (2) the heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 31, and the light chain variable region amino acid sequence is as shown in SEQ ID NO: 32; or (3) the heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 31, and the light chain variable region amino acid sequence is as shown in SEQ ID NO: 34; or (4) the heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 35, and the light chain variable region amino acid sequence is as shown in SEQ ID NO: 36.
  • the aforementioned anti-DLL3 antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), single-domain antibody (sdAb), dimerized V region (diabody), disulfide bond-stabilized V region (dsFv) and an antigen-binding fragment of a peptide comprising CDR.
  • the aforementioned anti-SEZ6 antibody or antigen-binding fragment thereof comprises an antibody constant region; wherein the heavy chain constant region is derived from human IgG1, IgG2, IgG3 or IgG4, or has at least 95% sequence identity thereto, or a variant thereof; and/or the light chain constant region is derived from human antibody ⁇ , ⁇ chain, or has at least 95% sequence identity thereto, or a variant thereof; preferably, the amino acid sequence of the heavy chain constant region is derived from human IgG1, or has at least 95% sequence identity thereto, or a variant thereof; and/or the light chain constant region is derived from human antibody ⁇ chain, or has at least 95% sequence identity thereto, or a variant thereof; more preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 33, and/or the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 59.
  • the aforementioned anti-SEZ6 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein: (1) the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:55, or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:56, or a sequence having at least 85%, 90%, 95%, 99% homology thereto; or (2) the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:57, or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:58, or a sequence having at least 85% , 90%, 95%, 99% homologous thereto; or (3) the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:57, or a sequence with at least 85%, 90%, 95%, 99% homology thereto, and the light chain comprises the amino acid sequence
  • the aforementioned anti-SEZ6 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein: (1) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:55, and the light chain comprises the amino acid sequence shown in SEQ ID NO:56; or (2) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:57, and the light chain comprises the amino acid sequence shown in SEQ ID NO:58; or (3) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:57, and the light chain comprises the amino acid sequence shown in SEQ ID NO:60; or (4) the heavy chain comprises the amino acid sequence shown in SEQ ID NO:61, and the light chain comprises the amino acid sequence shown in SEQ ID NO:62.
  • the aforementioned anti-SEZ6 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain and the light chain comprise: (1) a heavy chain sequence shown in SEQ ID NO:55 or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and a light chain sequence shown in SEQ ID NO:56 or a sequence having at least 85%, 90%, 95%, 99% homology thereto; or (2) a heavy chain sequence shown in SEQ ID NO:57 or a sequence having at least 85%, 90%, 95%, 99% homology thereto, and a light chain sequence shown in SEQ ID NO:58 or a sequence having at least 85%, 90%, 95%, 99% homology thereto.
  • the aforementioned anti-SEZ6 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain and the light chain comprise: (1) the heavy chain sequence shown by SEQ ID NO:55 and the light chain sequence shown by SEQ ID NO:56; or (2) the heavy chain sequence shown by SEQ ID NO:57 and the light chain sequence shown by SEQ ID NO:58; or (3) the heavy chain sequence shown by SEQ ID NO:57 and the light chain sequence shown by SEQ ID NO:60; or (4) the heavy chain sequence shown by SEQ ID NO:61 and the light chain sequence shown by SEQ ID NO:62. or
  • the aforementioned anti-DLL3 antibody or antigen-binding fragment thereof which consists of a heavy chain
  • the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:71, or a sequence having at least 85%, 90%, 95%, 99% homology thereto; or (2) the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:72, or a sequence having at least 85%, 90%, 95%, 99% homology thereto.
  • the aforementioned anti-SEZ6 antibody or its antigen-binding fragment is composed of a heavy chain, wherein: (1) the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:71; or (2) the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:72.
  • the aforementioned anti-SEZ6 antibody or its antigen-binding fragment which is composed of a heavy chain, comprises: (1) a heavy chain sequence shown in SEQ ID NO:71 or a sequence having at least 85%, 90%, 95%, or 99% homology thereto; or (2) a heavy chain sequence shown in SEQ ID NO:72 or a sequence having at least 85%, 90%, 95%, or 99% homology thereto.
  • the aforementioned anti-SEZ6 antibody or its antigen-binding fragment, which is composed of a heavy chain comprises: (1) the heavy chain sequence shown by SEQ ID NO:71; or (2) the heavy chain sequence shown by SEQ ID NO:72.
  • the present disclosure provides an isolated anti-DLL3 antibody or antigen-binding fragment thereof that competes with any of the aforementioned antibodies or antigen-binding fragments thereof for binding to DLL3.
  • the present invention discloses an isolated nucleic acid encoding any of the aforementioned anti-DLL3 antibodies or antigen-binding fragments thereof.
  • the present invention discloses a vector comprising the nucleic acid as described above.
  • the present disclosure provides a host cell comprising the vector as described above.
  • the present disclosure provides a method for preparing an anti-DLL3 antibody or an antigen-binding fragment thereof, comprising culturing the aforementioned host cell under conditions suitable for expressing the anti-DLL3 antibody or the antigen-binding fragment thereof.
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, comprising the anti-DLL3 antibody or antigen-binding fragment thereof as described in any of the preceding items.
  • the aforementioned antibody-drug conjugate or its pharmaceutically acceptable salt or solvate, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof wherein the drug is selected from cytotoxic agents, radioactive markers, fluorophores, chromophores, imaging agents, immunomodulators, angiogenesis inhibitors, cell proliferation inhibitors, pro-apoptotic agents, cell lytic enzymes, and any combination thereof; preferably, the drug is selected from: DNA damaging agents, topoisomerase inhibitors, microtubule inhibitors, protein degraders, STING agonists, and any combination thereof.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof has a structure represented by general formula (I):
  • Dr is selected from: camptothecin, auristatin, maytansinoid, pyrrolobenzodiazepine (PBD), calicheamicin, duokamycin, daunorubicin, doxorubicin, calicheamicin, anthramycin, neomycin, amatoxin, hemiesterin, eribulin, Tubulysin, and analogs or derivatives thereof.
  • PBD pyrrolobenzodiazepine
  • n is 1 to 10, preferably n is 2 to 8, more preferably n is 3 to 5 or 4 to 8, n is a decimal or an integer, further preferably n is 4 to 6, n is a decimal or an integer, further preferably n is 5 to 6, n is a decimal or an integer, further preferably, n is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8;
  • L and Y are linker units; Pc is the aforementioned anti-SEZ6 antibody or an antigen-binding fragment thereof.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof has a structure represented by the general formula (Pc-LY-Dr), general formula (II), general formula (III), or general formula (IV):
  • Y is selected from -O-( CRaRb ) m - CR1R2 - C(O)-, -O-CR1R2- ( CRaRb)m-, -O - CR1R2- , -NH- ( CRaRb )m- CR1R2 - C (O)- or -S-(CRaRb) m -CR1R2 - C ( O)-;
  • Ra and Rb are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group; or, Ra and Rb together with the carbon atom to which they are attached form
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof wherein n is 2 to 8, preferably 3 to 5 or 4 to 8, n is a decimal or an integer, more preferably 4 to 6, n is a decimal or an integer, further preferably 5 to 6, n is a decimal or an integer, and further preferably, n is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8.
  • L1 is s 1 is an integer from 2 to 8; L 2 is a chemical bond; L 3 is a tetrapeptide residue; L 4 is -NR 5 (CR 6 R 7 )t-, R 5 , R 6 or R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, and t is 1 or 2.
  • L1 is s1 and s2 are independently selected from integers from 0 to 8; preferably s1 is 2 and s2 is an integer from 2 to 8; more preferably, s1 is 2 and s2 is 2, 3, 4, 5 or 6; L2 is a chemical bond; L3 is a tetrapeptide residue; L4 is -NR5 ( CR6R7 )t-, R5 , R6 or R7 are the same or different and are each independently a hydrogen atom or an alkyl group, and t is 1 or 2.
  • L2 is a chemical bond; L3 is a tetrapeptide residue of GGFG; R1 is a haloalkyl group or a C3-6 cycloalkyl group; R2 is selected from a hydrogen atom, a haloalkyl group or a C3-6 cycloalkyl group; or, R1 and R2 together with the carbon atom to which they are connected form a C3-6 cycloalkyl group; R5 , R6 or R7 are the same or different and are each independently a hydrogen atom or an alkyl group; s1 is an integer from 2 to 8; m is an integer from 0 to 4.
  • the antibody-drug conjugate or a pharmaceutically acceptable A salt or solvate, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof which is an antibody-drug conjugate represented by the general formula (Pc-L a -Y-Dr) or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof:
  • W is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl or a straight-chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • L 2 is selected from -NR 4 (CH 2 CH 2 O) p1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O) p1 CH 2 C(O)-, -S(CH 2 ) p1 C(O)- or a chemical bond, and p 1 is an integer from 1 to 20;
  • L R3 is a peptide residue consisting of 2 to 7 amino acids, which
  • the substituent When substituted, the substituent may be substituted at any available attachment point, and the substituent is one or more independently selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • R1 is selected from halogen, halogenated alkyl, deuterated alkyl, cycloalkyl, heterocyclic group, aryl or heteroaryl;
  • R2 is selected from hydrogen atom, halogen, halogenated alkyl, deuterated alkyl, cycloalkyl, heterocyclic group, aryl or heteroaryl; or, R1 and R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group;
  • R4 and R5 are the same or different and are each independently selected from hydrogen atom, alkyl, halogenated alkyl, deuterated alkyl and hydroxyalkyl;
  • the antibody-drug conjugate represented by the general formula (Pc-LY-Dr) as described above, or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof is an antibody-drug conjugate represented by the general formula (Pc-L b -Y-Dr) or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof:
  • s1 is an integer of 2 to 8;
  • R1 , R2 , R5 to R7 , m and n are as defined in the general formula (Pc-La-Y-Dr).
  • the antibody-drug conjugate as described above or its pharmaceutically acceptable salt or solvate, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, is selected from:
  • n is 1 to 10, preferably n is 2 to 8, more preferably n is 3 to 5 or 4 to 8, n is a decimal or an integer, further preferably n is 4 to 6, n is a decimal or an integer, further preferably n is 5 to 6, n is a decimal or an integer, further preferably, n is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8;
  • Pc is the aforementioned anti-SEZ6 antibody or an antigen-binding fragment thereof.
  • the antibody-drug conjugate as described above or its pharmaceutically acceptable salt or solvate, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, is selected from:
  • n is 1 to 10, preferably n is 2 to 8, more preferably n is 3 to 5 or 4 to 8, n is a decimal or an integer, further preferably n is 4 to 6, n is a decimal or an integer, further preferably n is 5 to 6, n is a decimal or an integer, and further preferably n is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8.
  • the present disclosure further provides a method for preparing an antibody-drug conjugate as represented by the general formula (Pc-L a -Y-Dr) or a pharmaceutically acceptable salt or solvate thereof, comprising the following steps:
  • Pc is reduced, it is coupled with the general formula (L a -Y-Dr) to obtain a compound represented by the general formula (Pc-L a -Y-Dr); wherein: Pc is the anti-SEZ6 antibody or antigen-binding fragment thereof as described in any of the preceding items.
  • W, L 2 , L 3 , R 1 , R 2 , R 5 to R 7 , m and n are as defined in the general formula (Pc-L a -Y-Dr).
  • R 1 , R 2 , R 5 to R 7 , s 1 and m are as defined in the general formula Pc-L b -Y-Dr.
  • the present disclosure provides a kit comprising the anti-DLL3 antibody or antigen-binding fragment thereof described in the present disclosure, or the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof described in the present disclosure, or a tautomer, mesoform, racemate, enantiomer, diastereomer, or a mixture thereof.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-DLL3 antibody or antigen-binding fragment thereof according to the present disclosure, or the antibody-drug conjugate according to the present disclosure or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesoform, racemate, enantiomer, diastereomer, or a mixture thereof, and one or more pharmaceutically acceptable carriers.
  • the present disclosure provides the use of the anti-SEZ6 antibody or antigen-binding fragment thereof according to the present disclosure, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutical composition comprising the same in the preparation of a medicament for treating a disease or condition mediated by SEZ6, wherein the disease or condition mediated by SEZ6 is a cancer with high SEZ6 expression.
  • the present disclosure provides use of the antibody-drug conjugate according to the present disclosure or a pharmaceutically acceptable salt or solvate thereof, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutical composition comprising the same in the preparation of a medicament for treating or preventing a tumor or cancer;
  • the cancer is preferably: lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer.
  • the active compound may be formulated for administration by any appropriate route, preferably in a unit dosage form, or in a form in which a patient can self-administer a single dose.
  • the unit dosage form of the disclosed compound or composition may be tablets, capsules, cachets, bottled liquids, powders, granules, lozenges, suppositories, reconstituted powders or liquid preparations.
  • the dosage of the compound or composition used in the disclosed treatment methods will generally vary with the severity of the disease, the patient's weight and the relative efficacy of the compound. However, as a general guide, a suitable unit dosage may be 0.1 to 1000 mg.
  • the pharmaceutical composition of the present disclosure may contain one or more excipients in addition to the active compound, and the excipients are selected from the following ingredients: fillers (diluents), binders, wetting agents, disintegrants or excipients, etc.
  • the composition may contain 0.1 to 99% by weight of the active compound.
  • the anti-SEZ6 antibody or antigen-binding fragment thereof disclosed herein has no non-specific binding, has a better level of antigen binding, and has a unique binding epitope.
  • the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate disclosed herein, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, has excellent in vitro cytotoxic and in vivo tumor inhibition effects, and has excellent in vivo pharmacokinetic parameters.
  • Applicants intend to include formulations of the trade name product, generic drugs, and the active drug portion of the trade name product.
  • cytotoxic drug refers to a cytotoxic drug, which is represented by Dr and is a chemical molecule that has a strong ability to destroy the normal growth of tumor cells.
  • cytotoxic drugs can kill tumor cells at sufficiently high concentrations, but due to the lack of specificity, while killing tumor cells, they can also cause apoptosis of normal cells, resulting in serious side effects.
  • toxins such as small molecule toxins or enzyme-active toxins from bacteria, fungi, plants or animals, radioactive isotopes (such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.
  • radioactive isotopes such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu
  • toxic drugs chemotherapeutic drugs
  • antibiotics and nucleolytic enzymes preferably toxic drugs.
  • linker unit refers to a chemical structure fragment or bond that is connected to an antibody at one end and to a drug at the other end, and may be connected to a drug after being connected to other linkers.
  • the preferred embodiment of the present disclosure is represented by L and L1 to L4 , wherein the L1 end is connected to an antibody, and the L4 end is connected to a structural unit Y and then connected to a drug (Dr).
  • Linkers including extenders, spacers, and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the linker can be a "cleavable linker" that facilitates release of the drug in the cell.
  • an acid-labile linker e.g., hydrazone
  • a protease-sensitive linker e.g., peptidase-sensitive
  • a photolabile linker e.g., peptidase-sensitive linker
  • a dimethyl linker e.g., a disulfide-containing linker
  • ligand-drug conjugate refers to a ligand connected to a biologically active drug via a stable linker.
  • ligand-drug conjugate is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment connected to a biologically active toxic drug via a stable linker.
  • ADC antibody-drug conjugate
  • antibody refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain respectively.
  • immunoglobulin isotypes namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain respectively.
  • the same type of Ig is based on its hinge region amino acid composition and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are divided into ⁇ chains or ⁇ chains according to the difference in the constant region.
  • Each of the five types of Ig can have ⁇ chains or ⁇ chains.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region.
  • the variable region includes three hypervariable regions (HVRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of the CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments described in the present disclosure conform to the known Kabat numbering convention (LCDR1-3, HCDR2-3), or conform to the numbering convention of Kabat and Chothia (HCDR1).
  • the term “fully human antibody” or “fully human antibody”, also known as “fully human monoclonal antibody”, is an antibody whose variable and constant regions are both human, eliminating immunogenicity and toxic side effects.
  • the development of monoclonal antibodies has gone through four stages, namely: mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology.
  • the "fully human antibody” in the present disclosure is obtained by phage display technology. Phage display technology is to separate B cells from human PBMC, spleen, and lymph node tissues, construct a natural single-chain phage human antibody library, or screen the obtained antibodies by immunizing transgenic mice that can express human antibody light and heavy chains.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody.
  • binding fragments included in "antigen-binding fragments" include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge on the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody; (v) single domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of VHH domains; and (vi) isolated complementarity determining regions (CDRs) or (vii) combinations
  • the two domains of the Fv fragment, VL and VH are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, thereby enabling them to be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • single-chain Fv single-chain antibodies
  • Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody.
  • Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.
  • the antibodies can be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, among fragments obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which about half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.
  • F(ab')2 is an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, obtained by digesting the portion below two disulfide bonds in the hinge region of IgG with the enzyme pepsin, and comprising two Fab regions linked at the hinge position.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F(ab')2.
  • the Fab' fragment of the antibody can be produced by inserting a DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic organism or a eukaryotic organism to express the Fab'.
  • single-chain antibody means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker.
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using variants of 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • single domain antibody refers to a new type of antibody that does not contain the CH1 region and light chain of the heavy chain, only contains a single heavy chain variable region, and retains the complete antigen binding activity characteristics, also known as a nanobody. Like a complete antibody, it can selectively bind to a specific antigen. Compared with the mass of 150-160kDa of a complete antibody, a single domain antibody is much smaller, about only 12-15kDa.
  • the first single domain antibody was artificially engineered from the heavy chain antibody of a camel, called the "VHH segment".
  • single domain antibody “heavy chain variable region domain of a heavy chain antibody”, “VHH segment”, and “nanobody” are used interchangeably, and a single domain antibody is a variable region of a heavy chain antibody. Typically, a single domain antibody contains three CDRs and four FRs.
  • Single domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits and cattle.
  • naturally occurring VHH molecules can be derived from antibodies provided by camelid species (e.g., camels, dromedaries, llamas and guanacos).
  • camelid species e.g., camels, dromedaries, llamas and guanacos.
  • Single domain antibodies are able to selectively bind to specific antigens.
  • Single domain antibodies can contain only the variable domains of immunoglobulin chains, which have CDR1, CDR2 and CDR3 and framework regions.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain.
  • antibody framework refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • epitope refers to a site on an antigen to which an immunoglobulin or antibody specifically binds.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
  • binding refers to the binding of an antibody to a predetermined epitope on an antigen.
  • the antibody binds with an affinity (KD) of about less than 10-7M, such as about less than 10-8M, 10-9M or 10-10M or less.
  • nucleic acid refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.
  • expression vector refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto.
  • the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA segments can be connected.
  • the vector is a viral vector, in which other DNA segments can be connected to the viral genome.
  • the vector disclosed herein can replicate autonomously in the host cell introduced therein (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome (e.g., non-additional mammalian vectors).
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR region.
  • Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulins, 2001 ISBN012441351.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacterial, microbial, plant or animal cells.
  • Bacteria that are susceptible to transformation include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.
  • the engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region.
  • Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies.
  • the culture fluid that secretes antibodies can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer.
  • Non-specifically bound components are washed away.
  • the bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected using SDS-PAGE and collected.
  • the antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.
  • the resulting product must be immediately frozen, such as at -70°C, or freeze-dried.
  • peptide refers to a compound fragment between amino acids and proteins. It is composed of two or more amino acid molecules connected to each other by peptide bonds. It is a structural and functional fragment of protein. Hormones, enzymes, etc. are essentially peptides.
  • sucrose refers to a biological macromolecule composed of three elements: C, H, and O, which can be divided into monosaccharides, disaccharides, and polysaccharides.
  • fluorescent probe refers to a class of fluorescent molecules that have characteristic fluorescence in the ultraviolet-visible-near infrared region, and whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime and polarization, etc.) can change sensitively with the properties of the environment, such as polarity, refractive index, viscosity, etc. They non-covalently interact with nucleic acids (DNA or RNA), proteins or other macromolecular structures to change one or several fluorescence properties, and can be used to study the properties and behaviors of macromolecules.
  • DNA or RNA nucleic acids
  • proteins or other macromolecular structures to change one or several fluorescence properties
  • toxic drug refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction. It includes toxins and other compounds that can be used for tumor treatment.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5 or 6 carbon atoms).
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl 2-Methylpentyl, 3-Methylpentyl, 4-Methylpentyl, 2,3-Dimethylbutyl, n-Heptyl, 2-Methylhexyl, 3-Meth
  • lower alkyl groups having 1 to 6 carbon atoms are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like.
  • the alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.
  • alkylene refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of a parent alkane by removing two hydrogen atoms, and is a straight or branched group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms (including 1, 2, 3, 4, 5 or 6 carbon atoms).
  • alkylene groups include, but are not limited to, methylene (—CH 2 —), 1,1-ethylene (—CH(CH 3 )—), 1,2-ethylene (—CH 2 CH 2 )—, 1,1-propylene (—CH(CH 2 CH 3 )—), 1,2-propylene (—CH 2 CH(CH 3 )—), 1,3-propylene (—CH 2 CH 2 CH 2 —), 1,4-butylene (—CH 2 CH 2 CH 2 CH 2 —), and 1,5-butylene (—CH 2 CH 2 CH 2 CH 2 CH 2 —), and the like.
  • the alkylene group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is preferably independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio and oxo.
  • alkoxy refers to-O-(alkyl) and-O-(non-substituted cycloalkyl), wherein the definition of alkyl or cycloalkyl is as described above.
  • alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy.
  • Alkoxy can be optionally substituted or non-substituted, and when substituted, substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms (including 3, 4, 5, 6, 7 or 8 carbon atoms).
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spiro, fused, and bridged cycloalkyl groups.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are heteroatoms selected from nitrogen, oxygen or S(O) m (wherein m is an integer of 0, 1 or 2), but excluding the ring part of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon.
  • it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms (1, 2, 3 or 4 heteroatoms); more preferably, the cycloalkyl ring contains 3 to 10 ring atoms (including 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms).
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc.
  • Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups.
  • spiro heterocyclic group refers to a polycyclic heterocyclic group in which one atom (called a spiral atom) is shared between 5 to 20 monocyclic rings, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated ⁇ electron system. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • the spiral heterocyclic group is divided into a single spiral heterocyclic group, a double spiral heterocyclic group or a multi-spiro heterocyclic group, preferably a single spiral heterocyclic group and a double spiral heterocyclic group. More preferably, it is a 4-yuan/4-yuan, 4-yuan/5-yuan, 4-yuan/6-yuan, 5-yuan/5-yuan or 5-yuan/6-yuan single spiral heterocyclic group.
  • spiral heterocyclic groups include:
  • fused heterocyclic group refers to a polycyclic heterocyclic group of 5 to 20 yuan, each ring in the system shares a pair of atoms adjacent to other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated ⁇ electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S (O) m (wherein m is an integer 0, 1 or 2) heteroatoms, and the remaining ring atoms are carbon.
  • O oxygen
  • m is an integer 0, 1 or 2
  • 6 to 14 yuan more preferably 7 to 10 yuan (7 yuan, 8 yuan, 9 yuan or 10 yuan ring).
  • the number of constituent rings it can be divided into a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic group, preferably a bicyclic or tricyclic, more preferably a 5 yuan/5 yuan or 5 yuan/6 yuan bicyclic fused heterocyclic group.
  • fused heterocyclic groups include:
  • bridged heterocyclic group refers to a polycyclic heterocyclic group of 5 to 14 members, any two rings sharing two atoms that are not directly connected, which may contain one or more double bonds, but no ring has a completely conjugated ⁇ electron system, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (wherein m is an integer of 0, 1 or 2), and the remaining ring atoms are carbon.
  • it is 6 to 14 members, more preferably 7 to 10 members (7-membered, 8-membered, 9-membered or 10-membered rings).
  • bridged heterocyclic groups include:
  • the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:
  • the heterocyclic group may be optionally substituted or unsubstituted.
  • the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.
  • aryl refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated ⁇ electron system, preferably 6- to 10-membered (6-, 7-, 8-, 9-, or 10-membered), such as phenyl and naphthyl, preferably phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, non-limiting examples of which include:
  • the aryl group may be substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.
  • heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms (1, 2, 3 or 4 heteroatoms), 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.
  • 1-membered (5-membered, 6-membered, 7-membered, 8-membered, 9-membered or 10-membered heteroaryl), more preferably 5-membered or 6-membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc.
  • the heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:
  • the heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.
  • amino protecting group is to protect the amino group with a group that is easily removed in order to keep the amino group unchanged when other parts of the molecule react.
  • Non-limiting examples include 9-fluorenylmethoxycarbonyl, tert-butyloxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl. These groups can be optionally substituted with 1-3 substituents (1, 2 or 3 substituents) selected from halogen, alkoxy or nitro.
  • the amino protecting group is preferably 9-fluorenylmethoxycarbonyl.
  • haloalkyl refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.
  • hydroxyalkyl refers to an alkyl group substituted with one or more hydroxy groups, wherein alkyl is as defined above.
  • hydroxy refers to an -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH2 .
  • nitro refers to -NO2 .
  • cyano refers to -CN.
  • amido refers to -C(O)N(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.
  • carboxylate refers to -C(O)O(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.
  • the present disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of compounds of formula (I) with reference to relevant literature. Commercially available deuterated starting materials can be used when preparing deuterated forms of compounds of formula (I), or they can be synthesized using deuterated reagents using conventional techniques, and deuterated reagents include but are not limited to deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide, etc.
  • Optional or “optionally” means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.
  • a heterocyclic group optionally substituted with an alkyl group Meaning that an alkyl group may but need not be present, the description includes both the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group.
  • Substituted means that one or more hydrogen atoms, preferably up to 5, more preferably 1, 2 or 3 hydrogen atoms in a group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (e.g. olefinic) bonds.
  • composition refers to a mixture containing one or more compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.
  • pharmaceutically acceptable salt refers to a salt of the antibody-drug conjugate of the present disclosure, or a salt of a compound described in the present disclosure. Such salts are safe and effective when used in mammals and have the desired biological activity.
  • the antibody-drug conjugate of the present disclosure contains at least one amino group and can therefore form salts with acids.
  • Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogensulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.
  • solvate refers to a pharmaceutically acceptable solvate formed by the antibody-drug conjugate of the present disclosure and one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate.
  • drug loading refers to the average number of cytotoxic drugs loaded on each antibody in the formula (I) molecule, and can also be expressed as the ratio of the amount of drug to the amount of antibody.
  • the range of drug loading can be 0-12, preferably 1-10 cytotoxic drugs (D) connected to each antibody (Pc).
  • the drug loading is expressed as n, which can also be called the DAR value, and is exemplified by the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the average number of drugs per ADC molecule after the coupling reaction can be identified by conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test and HPLC characteristics.
  • the cytotoxic drug is coupled to the N-terminal amino group and/or the ⁇ -amino group of the lysine residue of the antibody via a linker.
  • the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum value.
  • the loading capacity of the antibody cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:
  • carrier is used in the present disclosure to refer to a drug that can change the way the drug enters the human body and
  • the drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure, and preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, and can be used as excellent drug carriers.
  • excipient refers to the additives in pharmaceutical preparations other than the main drug, which can also be called auxiliary materials.
  • auxiliary materials for example, the binders, fillers, disintegrants, and lubricants in tablets; the matrix part in semi-solid preparations such as ointments and creams; preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid preparations can all be called excipients.
  • diluent is also called filler, and its main purpose is to increase the weight and volume of the tablet.
  • the addition of diluent not only ensures a certain volume, but also reduces the dosage deviation of the main ingredients and improves the compression molding of the drug.
  • an absorbent needs to be added to absorb the oily substance and keep it in a “dry” state to facilitate tableting.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous solution.
  • acceptable solvents and solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution.
  • the sterile injectable preparation may be a sterile injectable water-in-oil microemulsion in which the active ingredient is dissolved in the oil phase.
  • the active ingredient is dissolved in a mixture of soybean oil and lecithin.
  • the oil solution is then added to a mixture of water and glycerol and treated to form a microemulsion.
  • the injection or microemulsion may be injected into the patient's bloodstream by local mass injection.
  • a continuous intravenous drug delivery device may be used.
  • An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous injection pump.
  • the pharmaceutical composition can be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration.
  • the suspension can be prepared according to known techniques with suitable dispersants or wetting agents and suspending agents as described above.
  • Sterile injection preparations can also be sterile injection solutions or suspensions prepared in non-toxic parenterally acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol.
  • sterile fixed oils can be conveniently used as solvents or suspension media. For this purpose, any blended fixed oils including synthetic mono- or diglycerides can be used.
  • fatty acids such as oleic acid can also be used to prepare injections.
  • the present disclosure relates to a type of cleavable linker with a specific structure and an active substance with a specific structure, and an antibody-drug conjugate (ADC) composed of the linker, the active substance and the antibody.
  • ADC antibody-drug conjugate
  • This type of ADC is a complex formed by connecting a toxic substance to an antibody via a spacer.
  • the antibody-drug conjugate (ADC) is degraded in the body to release active molecules, thereby playing an anti-tumor role.
  • Figure 1 Activity test results of recombinant human SEZ6 protein.
  • FIG. 2A Detection results of stable cell lines expressing human SEZ6
  • FIG. 2B Detection results of stable cell lines expressing monkey SEZ6.
  • Figure 3A Mouse chimeric antibody binding activity test results for human SEZ6 on the cell membrane
  • Figure 3B Mouse The results of the binding activity test of the chimeric antibody to monkey SEZ6 on the cell membrane.
  • Figure 4 Results of mouse chimeric antibody internalization and immunotoxin killing activity assays.
  • Figure 5 Binding activity test results of humanized antibodies to human cell membrane proteins.
  • FIG. 6A The results of the internalization and immunotoxin killing activity detection of the humanized antibody on H69 cells
  • FIG. 6B The results of the internalization and immunotoxin killing activity detection of the humanized antibody on H82 cells.
  • FIG. 7A The results of the binding activity detection of chimeric nanobodies to human SEZ6 on the cell membrane
  • FIG. 7B The results of the binding activity detection of chimeric nanobodies to monkey SEZ6 on the cell membrane.
  • Figure 8 Results of internalization and immunotoxin killing activity assay of chimeric nanobodies on H69 cells.
  • Figure 9 Results of the binding activity test of humanized nanobodies to human SEZ6 protein on the cell membrane.
  • Figure 10 Results of internalization and immunotoxin killing activity assay of humanized nanobodies on H69 cells.
  • FIG. 11 Results of the binding activity test of PR3178 and PR0071 to SEZ6 homologous family proteins.
  • FIG. 12 Results of identification of affinity epitopes of PR3178 and PR0071 for SEZ6.
  • FIG. 13A In vitro inhibitory activity of PR3178-9106 and PR0071-9106 in H69 cells
  • FIG. 13B In vitro inhibitory activity of PR3178-9106 and PR0071-9106 in H82 cells.
  • Figure 14 Anti-tumor activity of PR3178-9106 and PR0071-9106 in the H69 xenograft model.
  • Figure 15 Single-dose pharmacokinetic evaluation of PR3178-9106 and PR0071-9106 in SCID mice.
  • Recombinant human SEZ6-Fc fusion protein is a human IgG1 Fc tag protein fused to the C-terminus of the human SEZ6 protein ECD region (see GenBank database, accession number: NP_849191, SEQ ID NO: 1).
  • Recombinant human SEZ6-His6 protein is a polyhistidine tag His6 fused to the C-terminus of the human SEZ6 extracellular domain (ECD) (see GenBank database, accession number: NP_849191).
  • Recombinant monkey SEZ6-His6 protein is a His tag fused to the C-terminus of the monkey SEZ6 ECD region (see GenBank database, accession number: XP_005583333.2, SEQ ID NO: 2).
  • Recombinant mouse SEZ6-His6 protein is a His tag fused to the C-terminus of the mouse SEZ6 ECD region (1-922) (see GenBank database, accession number: NP_067261, SEQ ID NO: 3). The extracellular domain sequence is underlined.
  • the ELISA (enzyme-linked immunosorbent assay) method was used for verification. Specifically, the recombinant human SEZ6-His6 protein was diluted to 1 ⁇ g/mL with PBS, added to the ELISA microplate, 100 ⁇ L per well, and incubated at 4°C overnight; ELISA blocking solution (PBS phosphate buffer containing 1% BSA (w/v), pH 7.4) was added, and after blocking at 37°C for 2 hours, gradient dilutions of anti-SEZ6 detection antibody hSC200 (hSC17.200, heavy chain SEQ ID NO: 6, light chain SEQ ID NO: 7) were added in sequence, incubated at 37°C for 1 hour, and the plate was washed 2-3 times with plate washing solution; horseradish peroxidase (HRP)-labeled secondary antibody was added, incubated at 37°C for 1 hour, and the plate was washed 2-3 times with plate washing solution.
  • HRP horseradish peroxidase
  • WO2019232241 (AbbVie Stemcentrx LLC) discloses the hSC200 sequence as follows:
  • an overexpression cell line was constructed for the screening process. Specifically, after CHO-K1 cells were transfected with a plasmid encoding a recombinant human SEZ6 protein or a plasmid encoding a cynomolgus monkey SEZ6 protein, a cell line that stably expressed recombinant human or cynomolgus monkey SEZ6 protein was obtained by screening. The nucleotide sequence encoding the human or cynomolgus monkey SEZ6 protein was cloned and connected to a lentiviral expression vector and a plasmid was prepared.
  • the CHO-K1 cell line was transfected using a lentiviral transfection method well known in the industry, selectively cultured for two weeks using F-12 medium containing Puromycin (purchased from Gibco), subcloned in a 96-well culture plate by a limiting dilution method, and cultured in a cell culture incubator. After two weeks, the selected monoclones were detected and screened by a flow cytometry method.
  • the results in Figure 2 show that the cell line can stably express human SEZ6 or monkey SEZ6 at a high level and can be used in subsequent antibody screening processes.
  • this experiment used the expression plasmid of full-length human SEZ6, recombinant human SEZ6-Fc and human SEZ6-His6 proteins as immunogens to immunize 6-8 week-old Balb/c and SJL/J mice, and recombinant human SEZ6-His6 was used as an immunogen to immunize Balb/c and SJL/J mice.
  • the initial immunization dose of the protein was 50 ⁇ g per mouse. Two weeks after the initial immunization, booster immunization was performed with an immunization dose of 25 ⁇ g per mouse, and each booster immunization was performed 2 weeks apart.
  • Serum samples were collected one week after each booster immunization, and the antibody titer in mouse serum was detected by ELISA.
  • 1 ⁇ g/mL recombinant human SEZ6-His6 was coated on the plate, overnight at 4°C, blocked with PBST buffer containing 1% BSA for 1 hour, and washed 3 times.
  • the mouse serum was diluted 10 times in the blocking buffer starting at 1:100, incubated at 37°C for 1 hour, washed 3 times, and incubated with the secondary antibody (HRP labeled) against mouse IgG-Fc for 1 hour.
  • PBST was washed 3 times, 100 ⁇ L TMB substrate was added to each well, and the reaction was terminated with 2M HCl after 15 minutes to measure the absorbance at 450nm.
  • the sera of immune mice have different degrees of binding to the immunogen, and the highest dilution of the serum is 1:10 5 , which can still show antigen-antibody reaction.
  • mice were killed and the spleen was taken out, and the spleen cells were collected by grinding.
  • NH4OH with a final concentration of 1% (w/w) was added to lyse the mixed red blood cells in the spleen cells, obtain the spleen cell suspension, and the cells were washed by centrifugation at 1000rpm for 3 times.
  • Mouse spleen cells were mixed with mouse myeloma cells SP2/0 at a ratio of 5:1 in the number of live cells, and cell fusion was performed using a high-efficiency electrofusion method.
  • the fused cells were diluted into a 96-well cell culture plate using DMEM medium containing 20% fetal bovine serum and 1 ⁇ HAT (w/w), with a total of 1 x 10 5 cells per well in 200 ⁇ L, and placed in a 37°C incubator with 5% (v/v) CO2. After 14 days, the fused cells were screened by ELISA and FACS, and the positive clones with OD450nm>1.0 and FACS positive cell ratio greater than 20% were expanded to 24-well cell plates and continued to be cultured at 37°C, 5% (v/v) CO2 using DMEM medium containing 10% (w/w) HT fetal bovine serum.
  • Recombinant chimeric antibodies were obtained by replacing the constant region of mouse monoclonal antibodies.
  • the nucleotide sequence encoding the variable region of mouse monoclonal antibodies was then cloned into the pTT5 vector containing the protein sequence encoding the human heavy and light chain constant regions (Human IgG1, kappa).
  • the gene sequence was synthesized and transfected into HEK293 cells. After 5 days, the cells were removed by centrifugation and the cell culture fluid was collected and filtered.
  • the harvested cell culture supernatant was loaded onto a protein A column (MabSelect SuRe, GE), the bound antibody was eluted with glycine, the eluate was neutralized with 1M Tris, and the buffer was replaced with PBS to obtain the chimeric antibody.
  • a protein A column MobSelect SuRe, GE
  • this experiment added immunized camels.
  • the specific method is: 2 healthy camels were immunized with recombinant human SEZ6-Fc. After 4-5 immunizations, 200 ml of peripheral blood was collected from each camel to separate PBMC. RNA was extracted with TriZol, reverse transcribed to prepare cDNA, and then PCR amplified the antibody gene. After enzyme digestion, ligation, and transformation, a VHH phage display library (library capacity) was constructed. 1.2 ⁇ 10 9 ). Random sequencing results showed that the positive rate of antibody gene insertion reached 96%.
  • the previously constructed immune antibody library was screened with biotinylated recombinant human SEZ6-His6 protein, and affinity panning was performed for 3-5 rounds. Single clones were randomly selected to prepare phages, and phage ELISA was performed. Positive clones were selected for sequencing and sequence analysis. The clones with unique sequences were used to construct VHH-hFc expression vectors, which were transiently expressed in 293 cells and tested by ELISA and FACS. The amino acid sequences of some VHH antibodies are shown in Table 3.
  • mouse and camel antibodies In order to reduce the immune response to the human body caused by non-human species, it is necessary to humanize mouse and camel antibodies.
  • the specific implementation method is as follows: for humanized antibodies, after predicting the structure of mouse monoclonal antibodies through homology modeling, the mouse antibody CDR is embedded in the appropriate human GermLine framework (Bioinformation. 2014; 10(4): 180–186; Methods Mol Biol. 2019; 1904: 213-230), and then reverse mutations are introduced at sites that may affect antibody-antigen binding.
  • nucleotide sequence encoding the variable region of the humanized monoclonal antibody is cloned into the pTT5 vector containing the protein sequence encoding the human heavy and light chain constant regions (Human IgG1, kappa), and HEK293 cells are transfected using a similar method as mentioned above to produce humanized antibodies.
  • the sequence of the Human IgG1 constant region is as follows: (SEQ ID NO: 33)
  • the Kappa constant region sequence is as follows: (SEQ ID NO: 59)
  • variable region framework selection used for humanized antibodies from mouse antibodies is shown in Table 4, the variable region sequences of humanized antibodies are shown in Table 5, the heavy chain CDR (HCDR1/HCDR2/HCDR3) and light chain CDR (LCDR1/LCDR2/LCDR3) are shown in Table 6, and the full length of the amino acid sequence of the humanized antibody is shown in Table 7.
  • PR3178 is the humanized antibody of cAb3009
  • PR3121 and PR3123 are the humanized antibodies of cAb3014
  • PR3132 is the humanized antibody of cAb3056.
  • variable region framework used by Nanobodies for humanized antibodies is shown in Table 8, the humanized antibody variable region sequence is shown in Table 9, the heavy chain CDR (HCDR1/HCDR2/HCDR3) is shown in Table 10, and the humanized antibody amino acid sequence fused to the human IgG1-Fc fragment is shown in Table 11.
  • PR0069 is a humanized antibody of cAb025, and PR0071 is a humanized antibody of cAb314.
  • the fluorescence signal of the antibody bound to the cell surface was detected, and the binding strength of the antibody was evaluated according to the strength of the fluorescence signal.
  • the gradient diluted antibody molecules and control molecules were incubated with 1x10 5 cells at 4°C for 1 hour, the excess antibody was washed off, and the anti-human Fc antibody labeled with mouse Alexa Flour 647 was added, incubated at 4°C for 30 minutes, and the excess antibody was washed off and resuspended in 200 ⁇ L 1% BSA/PBS buffer, and the fluorescence signal on the cell surface was read by Thermo Attune NxT flow cytometer.
  • Table 12 shows the binding characteristics of mouse chimeric anti-antibodies (Figure 3), Table 13 shows the binding characteristics of mouse humanized antibodies (Figure 5), Table 14 shows the binding characteristics of nano chimeric antibodies ( Figure 7), and Table 15 shows the affinity characteristics of nano humanized antibodies ( Figure 9).
  • the results show that the candidate antibodies have SEZ6 membrane protein binding activity close to hSC200.
  • DT3C is a recombinantly expressed fusion protein with a molecular weight of 70KD. It is a fusion of Fragment A of diphtheria toxin (toxin part only) and 3C fragment of group G streptococcus (IgG binding part). This protein has a high affinity with the IgG part of the antibody and enters the cell together when the antibody is internalized. Under the action of intracellular furin, toxic DT is released. DT can inhibit the activity of EF2-ADP ribosylation, block the protein translation process, and ultimately lead to cell death. By using this system, the cell killing effect caused by antibody internalization and immunotoxin can be observed simultaneously (Yamaguchi, M., Hama, H., et al., Biochemical and Biophysical Research Communications 454 (2014) 600-603).
  • DT3C internalization and immunotoxin activity
  • sterile filtered DT3C and the chimeric antibody to be tested are mixed in a volume ratio of 1:1, incubated at 37°C for 30 minutes, and then diluted with complete medium, added to cells (H69 or H82) (3000 cells/well), and incubated at 37°C in a 5% carbon dioxide incubator for 6 days.
  • cells H69 or H82
  • Add CellTiter-Glo incubate at room temperature in the dark for 10 minutes, and read chemiluminescence on PerkinElmer.
  • Mouse chimeric antibodies cAb3009, cAb3014 and cAb3056 have stronger internalization and immunotoxin killing activity than the control molecule hSC200.
  • the endocytic activity of a series of chimeric antibodies with similar sequences, cAb3003, cAb3005, cAb3006, and cAb3009 was significantly better than that of the control molecule hSC200: the general formulas of CDRH1, CHDRH2, and CDRH3 of this series of sequences were XYEMH, GIDPEXXNTVYNQKFKX, and GDWYFDX; CDRL1, The general formula of CDRL2 and CDRL3 is KSSQSLLNSRTRENYLA, WASTRXX, XQSYNLFT.
  • humanized anti-SEZ6 mouse antibodies PR3178, PR3121, PR3123 and PR3132 have internalization and immunotoxin killing activities comparable to or slightly stronger than the control molecule hSC200.
  • camel-derived chimeric antibodies cAb025 and cAb314 have internalization and immunotoxin killing activities stronger than the control molecule hSC200.
  • humanized anti-SEZ6 camel-derived antibodies PR0069 and PR0071 have internalization and immunotoxin killing activities stronger than the control molecule hSC200.
  • ELISA In order to screen out antibodies with specific affinity to SEZ6, the binding of antibodies to SEZ6 homologous family proteins was detected by ELISA. Specifically: recombinant human SEZ6-Fc fusion protein, SEZ6L-Fc protein and SEZ6L2-Fc were diluted to 1 ⁇ g/mL with PBS, added to ELISA microplates, 100 ⁇ L per well, and incubated overnight at 4°C; ELISA blocking solution (PBS phosphate buffer containing 1% BSA (w/v), pH 7.4) was added, and after blocking at 37°C for 2 hours, 10 nM detection antibodies were added, incubated at 37°C for 1 hour, and the plates were washed 2-3 times with washing solution; horseradish peroxidase (HRP)-labeled secondary antibodies were added, incubated at 37°C for 1 hour, and the plates were washed 2-3 times with washing solution.
  • HRP horseradish peroxidase
  • the antibody endocytosis activity is correlated with the antigen affinity epitope.
  • This experiment identified the antigen affinity epitope of the antibody by ELISA.
  • the recombinant human SEZ6-SUSHI1 protein, SEZ6L-SUSHI2 protein and SEZ6-N1 protein were diluted to 1 ⁇ g/mL with PBS, added to the ELISA microplate, 100 ⁇ L per well, and incubated at 4°C overnight; ELISA blocking solution (PBS phosphate buffer containing 1% BSA (w/v), pH7.4) was added, and after blocking at 37°C for 2 hours, 10nM detection antibody was added, incubated at 37°C for 1 hour, and the plate was washed 2-3 times with plate washing solution; horseradish peroxidase (HRP)-labeled secondary antibody was added, incubated at 37°C for 1 hour, and the plate was washed 2-3 times with plate washing solution.
  • HRP horseradish peroxidase
  • TMB substrate 100 ⁇ L was added to each well, and after incubation at room temperature for 15 minutes, 50 ⁇ L of stop solution (2M HCl) was added to each well.
  • stop solution 2M HCl
  • the OD450nm value was read using an ELISA plate reader (SpectraMax M5e).
  • PR3178 binds to the SUSHI4 domain of SEZ6 protein
  • PR0071 binds to a separate domain of SEZ6 other than SUSHI1, SUSHI4, and N1.
  • the screened antibody molecules all have unique affinity epitopes and exhibit better endocytic activity.
  • the affinity activity of PR3178 and PR0071 antibodies was detected by SPR using the BIAcore 8K (Cityva) system.
  • the sensor chip protein A and related reagents used for detection were purchased from Cytiva.
  • Antibodies PR3178, PR0071 and control antibodies were diluted to 1 ⁇ g/ml with HBS-EP+ buffer, the flow rate was set to 10 ⁇ l/min, and the antibody was captured to 200RU level.
  • the his-tagged SEZ6 antigen was diluted with HBS-EP+ buffer in a certain proportion to a concentration gradient of 0nM, 3.125nM, 6.25nM, 12.5nM (two replicates), 25nM, 50nM, 100nM, and 200nM.
  • the flow rate was set to 30 ⁇ l/min during sample analysis.
  • the binding time was 120s and the dissociation time was 900s. Then regeneration was performed, using pH 1.5 Gly-HCl buffer as the regeneration buffer, the regeneration flow rate was set to 30 ⁇ l/min, and regeneration was performed for 30s.
  • the response signal was based on the analysis time as the horizontal axis and the response value as the vertical axis.
  • the obtained data were fitted by BIAcore 8K analysis software, and the 1:1 Langmuir binding model was used to determine the kinetic constants such as the binding rate constant (Ka), dissociation rate constant (Kd) and dissociation equilibrium constant (KD).
  • Ka binding rate constant
  • Kd dissociation rate constant
  • KD dissociation equilibrium constant
  • this experiment requires the coupling of antibodies PR3178 and PR0071 with small molecule toxin 9106.
  • TCEP-HCl tris(2-carboxyethyl)phosphine hydrochloride
  • the structures of the antibody drug conjugates PR3178-9106 and PR0071-9106 were obtained as follows.
  • the drug loading was calculated by RP-HPLC.
  • Example 12 Determination of the activity of ADC in inhibiting the proliferation of tumor cell lines in vitro
  • the in vitro activity of the ADC was reflected by the inhibition of tumor cell line growth.
  • the test cells H69 and H82 were grown overnight in 96-well culture plates, with a density of 3000 per well. The next day, equal volumes of gradient dilutions of toxin-coupled drugs were added. After 5 days, the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) was used as described in the manufacturer's protocol to determine Cell viability. Cell viability was evaluated as the percentage of control untreated cells. As shown in Figure 13, the ADC drug had good killing activity on H69 and H82 tumor cell lines.
  • the H69 xenograft tumor model was used to detect the changes in tumor growth size to reflect the anti-tumor activity of ADC.
  • SCID mice were subcutaneously inoculated with H69 cells 5x10 ⁇ 6/0.2ml (PBS+gel) at the right back.
  • the drug was administered on the day of grouping, and intraperitoneal injection was given twice on Day 0 and Day 7.
  • the tumor volume and body weight were detected twice a week, and the data were recorded.
  • Tumor volume V 1/2 ⁇ a ⁇ b 2 , where a and b represent length and width, respectively.
  • Relative tumor growth rate T/C (%) (T-T0)/(C-C0) ⁇ 100, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.
  • Tumor inhibition rate TGI (%) 1-T/C (%).
  • human IgG control antibody was used as a negative control.
  • TGI represents the maximum tumor growth inhibition during the experiment.
  • ADC has significant antitumor activity on the growth of H69 xenograft tumors at equimolar dose levels.
  • vs control group hIgG1 *p ⁇ 0.05, **p ⁇ 0.001, ***p ⁇ 0.0001
  • ELISA was used to detect the ADC concentration in mouse serum after a single dose. Specifically, SCID mice were used, and equimolar doses of ADC drugs were injected into the tail vein. After a single dose, blood was collected at the following time points: 15min, 6h, 24h, 72h, 144h, 240h, and 336h after the first dose. The blood drug concentration was detected by ELISA. The plate was coated with anti-hFc and incubated overnight at 4°C. After washing once with 300 ⁇ L PBST buffer, the diluted test serum was added and incubated at room temperature for 1 hour. Each tube of serum was tested on two plates.
  • the use and welfare of experimental animals in this disclosure are carried out in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
  • AALAC Association for Assessment and Accreditation of Laboratory Animal Care
  • the health status and mortality of animals are monitored daily, and routine inspections include observing the effects of test substances and drugs on the daily behavior of animals, such as behavioral activities, weight changes, physical signs, etc.

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Abstract

本发明涉及一种抗体或其抗原结合片段、其抗体-药物偶联物及其应用。具体而言，本发明提供一种SEZ6抗体、其抗原结合片段，以及一种SEZ6抗体-药物偶联物，编码所述抗体、其抗原结合片段的核酸分子，包含所述核酸分子的载体和宿主细胞，以及制备抗体偶联毒素的方法。还提供了包含所述抗体、其抗原结合片段或抗体偶联毒素的药物组合物及相关制药用途。

Description

一种抗体或其抗原结合片段、抗体-药物偶联物及其应用 技术领域
本发明属于抗体领域，具体涉及特异性结合哺乳动物、特别是人的SEZ6的抗体及制备方法、其偶联物和应用；该药物是将抗SEZ6抗体与抗肿瘤性药物9106通过连接子连接而成的。
背景技术
抗体-药物偶联物(Antibody-Drug conjugate；ADC)是通过利用将细胞毒素偶联至选择性结合癌细胞表面的抗体上，并通过抗原抗体介导的内化，将细胞毒素选择性地递送入肿瘤细胞内部，在毒素蓄积到一定程度，达到杀伤肿瘤细胞的目的。作为ADC，由三部分组成，分别为抗体，连接子和毒素。ADC药物能够有效提高毒素的肿瘤精准靶向，提高治疗窗，所以ADC的靶点选择至关重要，需要在肿瘤中特异性地高表达并且在正常组织中不表达。
小细胞肺癌是恶性程度很高的肿瘤，全球每年约有25万人确诊，约占肺癌的15％，其中70％的病人确诊时已发生转移；肿瘤发生跟吸烟密切相关，并伴随多种抑癌基因突变；确诊病人中位生存时间只有8～12个月，极少数患者生存超过5年。针对小细胞肺癌标准一线疗法是铂类加依托泊苷，患者对一线化疗响应率高，但复发耐药后，缺乏新的有效治疗方案。二线拓扑替康治疗响应率低，对生存期没有明显改善。小细胞肺癌的治疗急需新的突破和进展(Nature Reviews Clinical Oncology volume 14,pages549–561(2017))。
人SEZ6(癫痫相关蛋白6，以下表示为hSEZ6)是单次跨膜蛋白，主要在脑组织中表达，外周几乎不表达。在神经疾病方向，小鼠SEZ6的敲除促进短而多突触形成(Neuron 56,621–639,November 21,2007)；SEZ6是BACE1的底物，脑脊液中可溶性的SEZ6可以作为阿尔兹海默的标记物(Molecular Neurodegeneration 2016 11:67)。
SEZ6作为肿瘤特异标志物在神经内分泌肿瘤组织中特异性地高表达，对临床标本进行免疫组织化学的分析表明，SEZ6在小细胞肺癌上高表达，并且表达量远远高于正常组织。在174个小细胞肺癌病人肿瘤组织切片，约70％的病人肿瘤呈SEZ6阳性表达(公开专利：US2016287720A1)。
基于上述SEZ6在肿瘤中特异性高表达，艾伯维制药开发了针对SEZ6的ADC，ABBV-011，适应症为小细胞肺癌(公开专利：WO2013126810A)。利用卡奇霉素(Calicheamicin)作为毒素，DAR值为2，采用不可降解的连接子。该ADC药物于2019年进入I期临床(NCT03639194)。
本专利通过小鼠免疫和骆驼免疫获得具有高亲和活性和高内吞活性的SEZ6抗体，偶联毒素化合物169106(公开专利：WO2020063676A)来获得创新型ADC 药物。169106是拓扑异构酶Ⅰ的抑制剂(本文也描述为9106)，之前的研究表明，该抗体偶联毒素具有良好成药性以及体内抗肿瘤活性和安全性。
发明内容
本公开涉及抗SEZ6抗体或其抗原结合片段，其ADC，以及其医药用途。更具体的，提供与SEZ6的ECD区的氨基酸序列或三维结构结合的单克隆抗体或抗原结合片段，其与细胞毒性物质依喜替康类似物偶联的ADC药物。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，包含重链可变区(VH)或重链单结构域(VHH)，其中重链可变区或重链单结构域包含：(1)如SEQ ID NO：37、38和39氨基酸序列所示的重链HCDR1、HCDR2、HCDR3；或(2)如SEQ ID NO：43、44和45氨基酸序列所示的重链HCDR1、HCDR2、HCDR3；或(3)如SEQ ID NO：49、50和51氨基酸序列所示的重链HCDR1、HCDR2、HCDR3；或(4)如SEQ ID NO：65、66和67氨基酸序列所示的重链HCDR1、HCDR2、HCDR3；或(5)如SEQ ID NO：68、69和70氨基酸序列所示的重链HCDR1、HCDR2、HCDR3。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，包含重链可变区(VH)和轻链可变区(VL)，其中重链可变区和轻链可变区包含：(1)如SEQ ID NO：37、38和39氨基酸序列所示的重链HCDR1、HCDR2、HCDR3，和如SEQ ID NO：40、41和42氨基酸序列所示的轻链LCDR1、LCDR2和LCDR3；或(2)如SEQ ID NO：43、44和45氨基酸序列所示的重链HCDR1、HCDR2、HCDR3，和如SEQ ID NO：46、47和48氨基酸序列所示的轻链LCDR1、LCDR2和LCDR3；或(3)如SEQ ID NO：49、50和51氨基酸序列所示的重链HCDR1、HCDR2、HCDR3，和如SEQ ID NO：52、53和54氨基酸序列所示的轻链LCDR1、LCDR2和LCDR3。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，其中重链单结构域包含氨基酸序列如SEQ ID NO：63所示，或与其具有至少85％，90％，95％，99％同源性的序列；或如SEQ ID NO：64所示，或与其具有至少85％，90％，95％，99％同源性的序列。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，其中重链单结构域包含氨基酸序列如SEQ ID NO：63所示或如SEQ ID NO：64所示。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，其中重链单结构域氨基酸序列如SEQ ID NO：63所示，或与其具有至少85％，90％，95％，99％同源性的序列；或如SEQ ID NO：64所示，或与其具有至少85％，90％，95％，99％同源性的序列。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，其中重链单结构域氨基酸序列如SEQ ID NO：63或SEQ ID NO：64所示。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中：重链可变区包含氨基酸序列如SEQ ID NO：29，SEQ ID NO：31，SEQ ID NO：35所示，或与其具有至少85％，90％，95％，99％同源性的序列；轻链可变区包含氨基酸序列如SEQ ID NO：30，SEQ ID NO：32，SEQ ID NO：34，SEQ ID NO：36所示，或与其具有至少85％，90％，95％，99％同源性的序列。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中：重链可变区包含氨基酸序列如SEQ ID NO：29，SEQ ID NO：31，SEQ ID NO：35所示；轻链可变区包含氨基酸序列如SEQ ID NO：30，SEQ ID NO：32，SEQ ID NO：34，SEQ ID NO：36所示。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中：重链可变区氨基酸序列选自SEQ ID NO：29，SEQ ID NO：31，SEQ ID NO：35，或与其具有至少85％，90％，95％，99％同源性的序列；轻链可变区氨基酸序列选自SEQ ID NO：30，SEQ ID NO：32，SEQ ID NO：34，SEQ ID NO：36，或与其具有至少85％，90％，95％，99％同源性的序列。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中：重链可变区氨基酸序列选自SEQ ID NO：29，SEQ ID NO：31，SEQ ID NO：35；轻链可变区氨基酸序列选自SEQ ID NO：30，SEQ ID NO：32，SEQ ID NO：34，SEQ ID NO：36。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中：(1)所述重链可变区包含氨基酸序列如SEQ ID NO：29所示，或与其具有至少85％，90％，95％，99％同源性的序列，所述轻链可变区包含氨基酸序列如SEQ ID NO：30所示，或与其具有至少85％，90％，95％，99％同源性的序列；或(2)所述重链可变区包含氨基酸序列如SEQ ID NO：31所示，或与其具有至少85％，90％，95％，99％同源性的序列，所述轻链可变区包含氨基酸序列如SEQ ID NO：32所示，或与其具有至少85％，90％，95％，99％同源性的序列；或(3)所述重链可变区包含氨基酸序列如SEQ ID NO：31所示，或与其具有至少85％，90％，95％，99％同源性的序列，所述轻链可变区包含氨基酸序列如SEQ ID NO：34所示，或与其具有至少85％，90％，95％，99％同源性的序列；或(4)所述重链可变区包含氨基酸序列如SEQ ID NO：35所示，或与其具有至少85％，90％，95％，99％同源性的序列，所述轻链可变区包含氨基酸序列如SEQ ID NO：36所示，或与其具有至少85％，90％，95％，99％同源性的序列。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中：(1)所述重链可变区包含氨基酸序列如SEQ ID NO：29所示，所述轻链可变区包含氨基酸序列如SEQ ID NO：30所示；或(2)所述重链可变区包含氨基酸序列如SEQ ID NO：31所示，所述轻链可变区包含氨基酸序 列如SEQ ID NO：32所示；或(3)所述重链可变区包含氨基酸序列如SEQ ID NO：31所示，所述轻链可变区包含氨基酸序列如SEQ ID NO：34所示；或(4)所述重链可变区包含氨基酸序列如SEQ ID NO：35所示，所述轻链可变区包含氨基酸序列如SEQ ID NO：36所示。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中重链可变区和轻链可变区包含：(1)所述重链可变区氨基酸序列如SEQ ID NO：29所示，或与其具有至少85％，90％，95％，99％同源性的序列，所述轻链可变区氨基酸序列如SEQ ID NO：30所示，或与其具有至少85％，90％，95％，99％同源性的序列；或(2)所述重链可变区氨基酸序列如SEQ ID NO：31所示，或与其具有至少85％，90％，95％，99％同源性的序列，所述轻链可变区氨基酸序列如SEQ ID NO：32所示，或与其具有至少85％，90％，95％，99％同源性的序列；或(3)所述重链可变区氨基酸序列如SEQ ID NO：31所示，或与其具有至少85％，90％，95％，99％同源性的序列，所述轻链可变区氨基酸序列如SEQ ID NO：34所示，或与其具有至少85％，90％，95％，99％同源性的序列；或(4)所述重链可变区氨基酸序列如SEQ ID NO：35所示，或与其具有至少85％，90％，95％，99％同源性的序列，所述轻链可变区氨基酸序列如SEQ ID NO：36所示，或与其具有至少85％，90％，95％，99％同源性的序列。
在一些实施方案中，本公开提供一种抗SEZ6抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中重链可变区和轻链可变区包含：(1)所述重链可变区氨基酸序列如SEQ ID NO：29所示，所述轻链可变区氨基酸序列如SEQ ID NO：30所示；或(2)所述重链可变区氨基酸序列如SEQ ID NO：31所示，所述轻链可变区氨基酸序列如SEQ ID NO：32所示；或(3)所述重链可变区氨基酸序列如SEQ ID NO：31所示，所述轻链可变区氨基酸序列如SEQ ID NO：34所示；或(4)所述重链可变区氨基酸序列如SEQ ID NO：35所示，所述轻链可变区氨基酸序列如SEQ ID NO：36所示。
在一些实施方案中，前述的抗SEZ6抗体或其抗原结合片段，选自Fab、Fab'、F(ab')2、单链抗体(scFv)、单域抗体(sdAb)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
在一些实施方案中，前述的抗SEZ6抗体或其抗原结合片段，其包含抗体恒定区；其中重链恒定区来源于人IgG1、IgG2、IgG3或IgG4，或与其具有至少95％序列同一性，或其变体；和/或轻链恒定区来源于人抗体κ、λ链，或与其具有至少95％序列同一性，或其变体；优选地，重链恒定区的氨基酸序列源于人IgG1，或与其具有至少95％序列同一性，或其变体；和/或轻链恒定区来源于人抗体κ链，或与其具有至少95％序列同一性，或其变体；更优选地，所述重链恒定区的氨基酸序列如SEQ ID NO：33所示，和/或轻链恒定区的氨基酸序列如SEQ ID NO：59所示。
在一些实施方案中，前述的抗SEZ6抗体或其抗原结合片段，包含重链和轻链，其中：(1)重链包含氨基酸序列如SEQ ID NO:55所示，或与其具有至少85％，90％，95％，99％同源性的序列，和轻链包含氨基酸序列如SEQ ID NO:56所示，或与其具有至少85％，90％，95％，99％同源性的序列；或(2)重链包含氨基酸序列如SEQ ID NO:57所示，或与其具有至少85％，90％，95％，99％同源性的序列，和轻链包含氨基酸序列如SEQ ID NO:58所示，或与其具有至少85％，90％，95％，99％同源性的序列；或(3)重链包含氨基酸序列如SEQ ID NO:57所示，或与其具有至少85％，90％，95％，99％同源性的序列，和轻链包含氨基酸序列如SEQ ID NO:60所示，或与其具有至少85％，90％，95％，99％同源性的序列；或(4)重链包含氨基酸序列如SEQ ID NO:61所示，或与其具有至少85％，90％，95％，99％同源性的序列，和轻链包含氨基酸序列如SEQ ID NO:62所示，或与其具有至少85％，90％，95％，99％同源性的序列。
在一些实施方案中，前述的抗SEZ6抗体或其抗原结合片段，包含重链和轻链，其中：(1)重链包含氨基酸序列如SEQ ID NO:55所示，和轻链包含氨基酸序列如SEQ ID NO:56所示；或(2)重链包含氨基酸序列如SEQ ID NO:57所示，和轻链包含氨基酸序列如SEQ ID NO:58所示；或(3)重链包含氨基酸序列如SEQ ID NO:57所示，和轻链包含氨基酸序列如SEQ ID NO:60所示；或(4)重链包含氨基酸序列如SEQ ID NO:61所示，和轻链包含氨基酸序列如SEQ ID NO:62所示。
在一些实施方案中，前述的抗SEZ6抗体或其抗原结合片段，包含重链和轻链，其中重链和轻链包含：(1)由SEQ ID NO:55所示的重链序列或与其具有至少85％，90％，95％，99％同源性的序列，和SEQ ID NO:56所示的轻链序列或与其具有至少85％，90％，95％，99％同源性的序列；或(2)由SEQ ID NO:57所示的重链序列或与其具有至少85％，90％，95％，99％同源性的序列，和SEQ ID NO:58所示的轻链序列或与其具有至少85％，90％，95％，99％同源性的序列；或(3)由SEQ ID NO:57所示的重链序列或与其具有至少85％，90％，95％，99％同源性的序列，和SEQ ID NO:60所示的轻链序列或与其具有至少85％，90％，95％，99％同源性的序列；或(4)由SEQ ID NO:61所示的重链序列或与其具有至少85％，90％，95％，99％同源性的序列，和SEQ ID NO:62所示的轻链序列，或与其具有至少85％，90％，95％，99％同源性的序列。
在一些实施方案中，前述的抗SEZ6抗体或其抗原结合片段，包含重链和轻链，其中重链和轻链包含：(1)由SEQ ID NO:55所示的重链序列和SEQ ID NO:56所示的轻链序列；或(2)由SEQ ID NO:57所示的重链序列和SEQ ID NO:58所示的轻链序列；或(3)由SEQ ID NO:57所示的重链序列和SEQ ID NO:60所示的轻链序列；或(4)由SEQ ID NO:61所示的重链序列和SEQ ID NO:62所示的轻链序列。或
在一些实施方案中，前述的抗SEZ6抗体或其抗原结合片段，其由重链组成， 其中：(1)重链包含氨基酸序列如SEQ ID NO:71所示，或与其具有至少85％，90％，95％，99％同源性的序列；或(2)重链包含氨基酸序列如SEQ ID NO:72所示，或与其具有至少85％，90％，95％，99％同源性的序列。
在一些实施方案中，前述的抗SEZ6抗体或其抗原结合片段，其由重链组成，其中：(1)重链包含氨基酸序列如SEQ ID NO:71所示；或(2)重链包含氨基酸序列如SEQ ID NO:72所示。
在一些实施方案中，前述的抗SEZ6抗体或其抗原结合片段，其由重链组成，包含：(1)由SEQ ID NO:71所示的重链序列或与其具有至少85％，90％，95％，99％同源性的序列；或(2)由SEQ ID NO:72所示的重链序列或与其具有至少85％，90％，95％，99％同源性的序列。
在一些实施方案中，前述的抗SEZ6抗体或其抗原结合片段，其由重链组成，包含：(1)由SEQ ID NO:71所示的重链序列；或(2)由SEQ ID NO:72所示的重链序列。
在另一方面，本公开提供一种分离的抗SEZ6抗体或其抗原结合片段，其与前述任一项抗体或其抗原结合片段竞争结合SEZ6。
在另一方面，本公开一种分离的核酸，其编码前述任一项抗SEZ6抗体或其抗原结合片段。
在另一方面，本公开一种载体，其包含如前所述的核酸。
在又一方面，本公开提供一种宿主细胞，其包含如前所述的载体。
在一个方面，本公开提供一种制备抗SEZ6抗体或其抗原结合片段的方法，其包括在适于表达所述抗SEZ6抗体或其抗原结合片段的条件下培养前述的宿主细胞。
在又一个方面，本公开提供一种抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其包含如前任一项所述的抗SEZ6抗体或其抗原结合片段。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中所述的药物选自细胞毒性剂、放射性标记物、荧光团、发色团、显像剂、免疫调节剂、血管生成抑制剂、细胞增殖抑制剂、促细胞凋亡剂、细胞裂解酶，以及其任何组合；优选地，所述的药物选自：DNA损伤剂、拓扑异构酶抑制剂、微管抑制剂、蛋白质降解剂、STING激动剂，以及其任何组合。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其具有通式(I)所示的结构：
其中，Dr选自：喜树碱、澳瑞他汀、美登木素生物碱、吡咯并苯二氮卓类(PBD)、加利车霉素、多卡霉素、柔红霉素、多柔比星、卡奇霉素、安曲霉素、新霉素、鹅膏素毒素、哈米特林、艾日布林、Tubulysin，以及其类似物或衍生物。优选地，Dr选自：Exatecan、MMAE、MMAF、MMAD、SN-38、DM1、DM4、吡咯苯并二氮卓(PBD)二聚体；
n为1至10，优选的n为2至8，更优选的n为3至5或4至8，n是小数或整数，进一步优选的n为4至6，n是小数或整数，进一步优选的n为5至6，n是小数或整数，更进一步优选的，n为2，2.5，3，3.5，4，4.5，5，5.5，6，6.5，7，7.5或8；
L、Y为接头单元；Pc为前述抗SEZ6抗体或其抗原结合片段。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其具有通式(Pc-L-Y-Dr)、通式(II)、通式(III)、通式(IV)所示的结构：
其中：Y选自-O-(CRaRb)m-CR1R2-C(O)-、-O-CR1R2-(CRaRb)m-、-O-CR1R2-、-NH-(CRaRb)m-CR1R2-C(O)-或-S-(CRaRb)m-CR1R2-C(O)-；Ra和Rb相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；或者，Ra和Rb与其相连接的碳原子一起形成环烷基或杂环基；R1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基或杂芳基；R2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基或杂芳基；或者，R1和R2与其相连的碳原子一起形成环烷基或杂环基；或者，Ra和R2与其相连的碳原子一起形成环烷基或杂环基；m为0至4的整数；n为1至10，优选 的n为2至8，更优选的n为3至5或4至8，n是小数或整数，进一步优选的n为4至6，n是小数或整数，进一步优选的n为5至6，n是小数或整数，更进一步优选的，n为2，2.5，3，3.5，4，4.5，5，5.5，6，6.5，7，7.5或8；L为接头单元；Pc为如前任一项所述的抗SEZ6抗体或其抗原结合片段。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中n为2至8，优选为3至5或4至8，n是小数或整数，更优选为4至6，n是小数或整数，进一步优选为5至6，n是小数或整数，更进一步优选的，n为2，2.5，3，3.5，4，4.5，5，5.5，6，6.5，7，7.5或8。
在一些实施方案中，如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中：Y为-O-(CRaRb)m-CR1R2-C(O)-；Ra和Rb相同或不同，且各自独立地选自氢原子、氘原子、卤素或烷基；R1为卤代烷基或C3-6环烷基；R2选自氢原子、卤代烷基或C3-6环烷基；或者，R1和R2与其相连接的碳原子一起形成C3-6环烷基；m为0或1。
在一些实施方案中，如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中Y选自：
在一些实施方案中，如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中Y的O端与接头单元L相连。
在一些实施方案中，如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中接头单元-L-为-L1-L2-L3-L4-，
L1为s1为2至8的整数；L2为化学键；L3为四肽残基；L4为-NR5(CR6R7)t-，R5、R6或R7相同或不同，且各自独立地为氢原子或烷基，t为1或2。
在一些实施方案中，如前任一项所述的抗体-药物偶联物或其药学上可接受的 盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中接头单元-L-为-L1-L2-L3-L4-，
L1为s1和s2独立的选自0至8的整数；优选的s1为2，s2为2至8的整数；更优选的，s1为2，s2为2，3，4，5或6；L2为化学键；L3为四肽残基；L4为-NR5(CR6R7)t-，R5、R6或R7相同或不同，且各自独立地为氢原子或烷基，t为1或2。
在一些实施方案中，如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中所述的接头单元-L-，其L1端与Pc相连，L4端与Y相连。
在一些实施方案中，如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中所述的L3的四肽残基为由两个或多个选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸中的氨基酸形成的氨基酸残基；优选为GGFG的四肽残基。
在一些实施方案中，如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中所述-L-Y-为以下结构：
L2为化学键；L3为GGFG的四肽残基；R1为卤代烷基或C3-6环烷基；R2选自氢原子、卤代烷基或C3-6环烷基；或者，R1和R2与其相连接的碳原子一起形成C3-6环烷基；R5、R6或R7相同或不同，且各自独立地为氢原子或烷基；s1为2至8的整数；m为0至4的整数。
在一些实施方案中，如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中-L-Y-选自：

在一些实施方案中，如前任一项所述的抗体-药物偶联物或其药学上可接受的 盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其为通式(Pc-La-Y-Dr)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式：
其中：W选自C1-8烷基、C1-8烷基-环烷基或1至8个原子的直链杂烷基，所述杂烷基包含1至3个选自N、O或S的杂原子，其中所述的C1-8烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；L2选自-NR4(CH2CH2O)p1CH2CH2C(O)-、-NR4(CH2CH2O)p1CH2C(O)-、-S(CH2)p1C(O)-或化学键，p1为1至20的整数；L3为由2至7个氨基酸构成的肽残基，氨基酸可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基为一个或多个独立地选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基；R1选自卤素、卤代烷基、氘代烷基、环烷基、杂环基、芳基或杂芳基；R2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、杂环基、芳基或杂芳基；或者，R1和R2与其相连接的碳原子一起形成环烷基或杂环基；R4和R5相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；R6和R7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基；m为0至4的整数；n为1至10，优选的n为2至8，更优选的n为3至5或4至8，n是小数或整数，进一步优选的n为4至6，n是小数或整数，进一步优选的n为5至6，n是小数或整数，更进一步优选的，n为2，2.5，3，3.5，4，4.5，5，5.5，6，6.5，7，7.5或8；Pc为如前任一项所述的抗SEZ6抗体或其抗原结合片段。
在一些实施方案中，如前所述的通式(Pc-L-Y-Dr)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其为通式(Pc-Lb-Y-Dr)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式：
其中：s1为2至8的整数；R1、R2、R5～R7、m和n如通式(Pc-La-Y-Dr)中所定义。
在一些实施方案中，如前所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，所述抗体-药物偶联物选自：






n为1至10，优选的n为2至8，更优选的n为3至5或4至8，n是小数或整数，进一步优选的n为4至6，n是小数或整数，进一步优选的n为5至6，n是小数或整数，更进一步优选的，n为2，2.5，3，3.5，4，4.5，5，5.5，6，6.5，7，7.5或8；
Pc为前述抗SEZ6抗体或其抗原结合片段。
在一些实施方案中，如前所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，所述抗体-药物偶联物选自：


其中n为1至10，优选的n为2至8，更优选的n为3至5或4至8，n是小数或整数，进一步优选的n为4至6，n是小数或整数，进一步优选的n为5至6，n是小数或整数，更进一步优选的，n为2，2.5，3，3.5，4，4.5，5，5.5，6，6.5，7，7.5或8。
本公开进一步提供一种制备如通式(Pc-La-Y-Dr)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化物的方法，其包括以下步骤：
Pc还原后，与通式(La-Y-Dr)偶联反应，得到通式(Pc-La-Y-Dr)所示的化合物；其中：Pc为如前任一项所述的抗SEZ6抗体或其抗原结合片段。W、L2、L3、R1、R2、R5～R7、m和n如通式(Pc-La-Y-Dr)中所定义。
在另一个实施方式中，提供另一种方法，其中所述的通式La-Y-Dr为通式Lb-Y-Dr所示的化合物：
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中R1、R2、R5～R7、s1和m如通式Pc-Lb-Y-Dr中所定义。
在本公开的一个优选的实施方案中，根据本公开所述的通式(Pc-La-Y-Dr)或通式(Pc-Lb-Y-Dr)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化物的方法，其中通式(La-Y-Dr)所示的化合物或通式(Lb-Y-Dr)所示的化合物选自：
另一方面，本公开提供一种试剂盒，其包含本公开所述的抗SEZ6抗体或其抗原结合片段，或本公开所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式。
另一方面，本公开提供一种药物组合物，其包含根据本公开所述的抗SEZ6抗体或其抗原结合片段，或根据本公开所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，以及一种或多种药学上可接受的载体。
另一方面，本公开提供根据本公开所述的抗SEZ6抗体或其抗原结合片段，本公开所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或包含其的药物组合物在制备用于治疗SEZ6介导的疾病或病症的药物中的用途。其中所述SEZ6介导的疾病或病症为SEZ6高表达癌症。
另一方面，本公开提供根据本公开所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或包含其的药物组合物在制备用于治疗或预防肿瘤或癌症的药物中的用途；其中所述癌症优选：肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌。
可将活性化合物制成适合于通过任何适当途径给药的形式，活性化合物优选是以单位剂量的方式，或者是以患者可以以单剂自我给药的方式。本公开化合物或组合物的单位剂量的表达方式可以是片剂、胶囊、扁囊剂、瓶装药水、药粉、颗粒剂、锭剂、栓剂、再生药粉或液体制剂。
本公开治疗方法中所用化合物或组合物的剂量通常将随疾病的严重性、患者的体重和化合物的相对功效而改变。不过，作为一般性指导，合适的单位剂量可以是0.1～1000mg。
本公开的药物组合物除活性化合物外，可含有一种或多种辅料，所述辅料选自以下成分：填充剂(稀释剂)、粘合剂、润湿剂、崩解剂或赋形剂等。根据给药方法的不同，组合物可含有0.1至99重量％的活性化合物。
本公开的抗SEZ6抗体或其抗原结合片段无非特异性结合，与抗原结合水平更优异，且具有独特的结合表位。本公开的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，具有优异的体外细胞毒和体内肿瘤抑制效果，且具有优异的体内药代动力学参数。
发明的详细说明
一、术语
除非另有限定，本文所用的所有技术和科学术语均与本公开所属领域普通技术人员的通常理解一致。虽然也可采用与本文所述相似或等同的任何方法和材料实施或测试本公开，但本文描述了优选的方法和材料。描述和要求保护本公开时，依据以下定义使用下列术语。
当本公开中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“药物”是指细胞毒性药物，药物表示为Dr，能在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。该术语包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32和Lu的放射性同位素)，毒性药物，化疗药物，抗生素和核溶酶，优选为毒性药物。
术语“接头单元(或连接片段)”是指一端与抗体连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。本公开的优选方案表示为L和L1至L4，其中L1端与抗体相连，L4端与结构单元Y相连后与药物(Dr)相连。
接头，包括延伸物、间隔物和氨基酸单元，可以通过本领域已知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Research 52:127-131(1992)；美国专利No.5,208,020)。
术语“配体-药物偶联物”指配体通过稳定的连接单元与具有生物活性的药物相连。在本公开中“配体-药物偶联物”优选为抗体-药物偶联物(antibody drug conjugate，ADC)，指将单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的毒性药物相连。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位 置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR2-3)，或者符合kabat和chothia的编号规则(HCDR1)。
术语“完全人源抗体”或“全人抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。全人源抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。本公开中的“完全人抗体”采用噬菌体展示技术获得。噬菌体展示技术，从人PBMC、脾脏、淋巴结组织分离B细胞，构建天然单链噬菌体人抗体库，或者通过免疫可表达人抗体轻重链的转基因小鼠，筛选获得的抗体。
术语“抗原结合片段”是指抗体的保持特异性结合抗原的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。“抗原结合片段”中包含的结合片段的实例包括：(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab')2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VHH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如， IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
术语“单域抗体”、“单结构域抗体”或“sdAb”意指一种不包含重链的CH1区及轻链、仅包含单一重链可变区，同时保留了完整的抗原结合活性特征的新型抗体，也称为纳米抗体(Nanobody)。和完整的抗体一样，它可以选择性的和特定抗原结合。与完整抗体的150－160kDa的质量相比，单域抗体则显得小得多，大约只有12－15kDa。第一个单域抗体是从骆驼的重链抗体中人造工程制作出来的，称为“VHH区段”。
术语“单域抗体”、“重链抗体的重链可变区结构域”、“VHH区段”、“纳米抗体”可互换使用，单域抗体是重链抗体的可变区。通常，单域抗体含有三个CDR和四个FR。
单域抗体可以衍生自任何物种，包括小鼠、人、骆驼、美洲驼、山羊、兔和牛。例如，天然存在的VHH分子可以衍生自骆驼科物种(例如骆驼、单峰骆驼、美洲驼和原驼)提供的抗体。像完整的抗体一样，单域抗体能够选择性地结合特定抗原。单域抗体可以仅含有免疫球蛋白链的可变结构域，该结构域具有CDR1、CDR2和CDR3以及框架区。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的CDR2和CDR3(CDR H2、CDR H3或H2、H3)。
术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10-7M，例如大约小于10-8M、10-9M或10-10M或更小的亲和力(KD)结合。
术语“核酸”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“表达载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)； 链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
术语“肽”是指介于氨基酸和蛋白质之间的化合物片段，由2个或2个以上氨基酸分子通过肽键相互连接而成，是蛋白质的结构与功能片段，如激素、酶类等本质上都是肽。
术语“糖”是指由C、H、O三种元素组成的生物大分子，可分为单糖、二糖和多糖等。
术语“荧光探针”是指在紫外-可见-近红外区有特征荧光，并且其荧光性质(激发和发射波长、强度、寿命和偏振等)可随所处环境的性质，如极性、折射率、粘度等改变而灵敏地改变的一类荧光性分子，其与核酸(DNA或RNA)、蛋白质或其他大分子结构非共价相互作用而使一种或几种荧光性质发生改变，可用于研究大分子物质的性质和行为。
术语“毒性药物”是指抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质。包括毒素和其他能用于肿瘤治疗的化合物。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子(包含1个、2个、3个、4个、5个或6个碳原子)的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己 基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷基如上所定义。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子，更优选含有1至6个碳原子(包含1个、2个、3个、4个、5个或6个碳原子)的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH2-)、1,1-亚乙基(-CH(CH3)-)、1,2-亚乙基(-CH2CH2)-、1,1-亚丙基(-CH(CH2CH3)-)、1,2-亚丙基(-CH2CH(CH3)-)、1,3-亚丙基(-CH2CH2CH2-)、1,4-亚丁基(-CH2CH2CH2CH2-)和1,5-亚丁基(-CH2CH2CH2CH2CH2-)等。亚烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个取代基所取代。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子(包含3个、4个、5个、6个、7个或8个碳原子)。 单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0、1或2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子(1个、2个、3个或4个杂原子)；更优选环烷基环包含3至10个环原子(包含3个、4个、5个、6个、7个、8个、9个或10个环原子)。单环杂环基的非限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
术语“螺杂环基”指5至20元的单环之间共用一个原子(称螺原子)的多环杂环基团，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺杂环基分为单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺杂环基和双螺杂环基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。螺杂环基的非限制性实例包括：
术语“稠杂环基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0、1或2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元(7元、8元、9元或10元环)。根据组成环的数目可以分为双环、三环、四环或多环稠杂环基，优选为双环或三环，更优选为5元/5元或5元/6元双环稠杂环基。稠杂环基的非限制性实例包括：
术语“桥杂环基”指5至14元，任意两个环共用两个不直接连接的原子的多环杂环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0、1或2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元(7元、8元、9元或10元环)。根据组成环的数目可以分为双环、三环、四环或多环桥杂环基，优选为双环、三环或四环，更优选为双环或三环。桥杂环基的非限制性实例包括：
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，其非限制性实例包括：
等。
杂环基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元(6元、7元、8元、9元或10元)，例如苯基和萘基，优选苯基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“杂芳基”指包含1至4个杂原子(1个、2个、3个或4个杂原子)、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10 元(5元、6元、7元、8元、9元或10元杂芳基)，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“氨基保护基”是为了使分子其它部位进行反应时氨基保持不变，用易于脱去的基团对氨基进行保护。非限制性实施例包含9-芴甲氧羰基、叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基等。这些基团可任选地被选自卤素、烷氧基或硝基中的1-3个取代基(1个、2个或3个取代基)所取代。所述氨基保护基优选为9-芴甲氧羰基。
术语“卤代烷基”指烷基被一个或多个卤素取代，其中烷基如上所定义。
术语“氘代烷基”指烷基被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟烷基”指烷基被一个或多个羟基取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH2。
术语“硝基”指-NO2。
术语“氰基”指-CN。
术语“酰氨基”指-C(O)N(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
术语“羧酸酯基”指-C(O)O(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
本公开还包括各种氘化形式的式(I)化合物。与碳原子连接的各个可用的氢原子可独立地被氘原子替换。本领域技术人员能够参考相关文献合成氘化形式的式(I)化合物。在制备氘代形式的式(I)化合物时可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂包括但不限于氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团” 意味着烷基可以但不必须存在，该说明包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1个、2个或3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的盐”或“可药用盐”是指本公开抗体-药物偶联物的盐，或本公开中所述的化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性，本公开抗体药物偶联物至少含有一个氨基，因此可以与酸形成盐，可药用盐的非限制性实例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
术语“溶剂合物”指本公开的抗体-药物偶联物与一种或多种溶剂分子形成可药用的溶剂化物，溶剂分子的非限制性实例包括水、乙醇、乙腈、异丙醇、DMSO、乙酸乙酯。
术语“载药量”是指式(I)分子中每个抗体上加载的细胞毒性药物平均数量，也可以表示为药物量和抗体量的比值，药物载量的范围可以是每个抗体(Pc)连接0-12个，优选1-10个细胞毒性药物(D)。在本公开的实施方案中，载药量表示为n，也可称为DAR值，示例性的为1、2、3、4、5、6、7、8、9、10的均值。可用常规方法如UV/可见光光谱法、质谱、ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的药物品均数量。
本公开的一个实施方式中，细胞毒性药物通过连接单元偶联在抗体的N端氨基和/或赖氨酸残基的ε-氨基上，一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。
可以用以下非限制性方法控制抗体细胞毒性药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
常规的药物组合物的制备见中国药典。
术语“载体”用于本公开的药物，是指能改变药物进入人体的方式和在体内 的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
术语“赋形剂”是在药物制剂中除主药以外的附加物，也可称为辅料。如片剂中的粘合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。如淀粉、乳糖、钙的无机盐、微晶纤维素等。
药物组合物可以是无菌注射水溶液形式。可在使用的可接受的溶媒和溶剂中有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。例如将活性成分溶于大豆油和卵磷脂的混合物中。然后将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入患者的血流中。或者，最好按可保持本公开化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的实例是Deltec CADD-PLUS.TM.5400型静脉注射泵。
药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液，例如1,3-丁二醇中制备的溶液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。为此目的，可使用包括合成甘油单或二酯在内的任何调和固定油。此外，脂肪酸例如油酸也可以制备注射剂。
本公开涉及一类可裂解的特定结构的连接臂和特定结构的活性物，及由连接臂、活性物与抗体组成的抗体药物偶联物(ADC)。此类ADC是经由间隔物将一种毒性物质连于抗体而形成的复合物。该抗体偶联药物(ADC)在体内经降解而释放出活性分子，从而起到抗肿瘤的作用。
附图说明
图1：重组人SEZ6蛋白的活性检测结果。
图2A：稳转细胞株的表达人SEZ6检测结果；图2B：稳转细胞株的表达猴SEZ6检测结果。
图3A：鼠源嵌合抗体对细胞膜上人SEZ6的结合活性检测结果；图3B：鼠 源嵌合抗体对细胞膜上猴SEZ6的结合活性检测结果。
图4：鼠源嵌合抗体内化和免疫毒素杀伤活性检测结果。
图5：人源化抗体对人细胞膜蛋白的结合活性检测结果。
图6A：人源化抗体在H69细胞上的内化和免疫毒素杀伤活性检测结果；图6B：人源化抗体在H82细胞上的内化和免疫毒素杀伤活性检测结果。
图7A：嵌合纳米抗体对细胞膜上人SEZ6的结合活性检测结果；图7B：嵌合纳米抗体对对细胞膜上猴SEZ6的结合活性检测结果。
图8：嵌合纳米抗体在H69细胞上的内化和免疫毒素杀伤活性检测结果。
图9：人源化纳米抗体对细胞膜上的人SEZ6蛋白的结合活性检测结果。
图10：人源化纳米抗体在H69细胞上的内化和免疫毒素杀伤活性检测结果。
图11：PR3178和PR0071对SEZ6同家族蛋白结合活性检测结果。
图12：PR3178和PR0071对SEZ6亲和表位鉴定结果。
图13A：PR3178-9106和PR0071-9106在H69细胞体外抑制活性；图13B：PR3178-9106和PR0071-9106在H82细胞体外抑制活性。
图14：PR3178-9106和PR0071-9106在H69异种移植模型中的抗肿瘤活性。
图15：PR3178-9106和PR0071-9106在SCID小鼠中的单剂量药代动力学评估。
具体实施方式
以下结合实施例用于进一步描述，但这些实施例并非限制的范围。
实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.重组SEZ6蛋白的活性检测
重组人SEZ6-Fc融合蛋白是将人IgG1 Fc标签蛋白融合在人SEZ6蛋白ECD区(参见GenBank数据库，登录号：NP_849191，SEQ ID NO：1)的C端。重组人SEZ6-His6蛋白是将多组氨酸标签His6融合在人SEZ6胞外结构域(ECD)(参见GenBank数据库，登录号：NP_849191)的C端。重组猴SEZ6-His6蛋白是将His标签融合在猴SEZ6 ECD区(参见GenBank数据库，登录号：XP_005583333.2，SEQ ID NO：2)的C端。重组小鼠SEZ6-His6蛋白是将His标签融合在鼠SEZ6 ECD区(1-922位)(参见GenBank数据库，登录号：NP_067261，SEQ ID NO：3)的C端。胞外结构域序列用下划线标注。
表1.用于免疫或筛选的蛋白氨基酸序列



为了检测重组蛋白良好的免疫活性，通过ELISA(酶联免疫吸附实验)方法进行验证。具体为：用PBS将重组人SEZ6-His6蛋白稀释至1μg/mL，加入ELISA微孔板，每孔各100μL，4℃孵育过夜；加入ELISA封闭液(含1％BSA(w/v),pH 7.4的PBS磷酸盐缓冲液)，37℃封闭2小时后，依次加入梯度稀释的抗SEZ6检测抗体hSC200(hSC17.200，重链SEQ ID NO：6，轻链SEQ ID NO：7)，37℃温育1小时，用洗板液洗板2-3次；加入辣根过氧化物酶标记(HRP)的二抗，37℃温育1小时，用洗板液洗板2-3次。加入TMB底物每孔100μL，室温孵育15分钟后，每孔加入50μL终止液(2M HCl)。用ELISA板机(SpectraMax M5e)读取OD450nm值，结果如图1所示：重组人SEZ6-His6蛋白能够与SEZ6抗体hSC200以较高亲和力结合，可用于后续体内蛋白免疫。
WO2019232241(AbbVie Stemcentrx LLC)公开hSC200序列如下：
实施例2.建立表达重组人SEZ6的稳定细胞株
为了能筛选出与膜蛋白亲和的候选抗体分子，采用构建过表达细胞系用于筛选过程。具体为，用编码重组人SEZ6蛋白的质粒或者编码食蟹猴SEZ6蛋白的质粒转染CHO-K1细胞后，通过筛选获得稳定表达重组人或者食蟹猴SEZ6白的细胞株。编码人或食蟹猴SEZ6蛋白的核苷酸序列被克隆连接到慢病毒表达载体并制备质粒。用行业内熟知的慢病毒转染方法对CHO-K1细胞系进行转染，使用含有Puromycin的F-12培养基(购自Gibco)选择性培养两周，有限稀释法在96孔培养板中进行亚克隆，置于细胞培养箱培养，两周后对挑选的单克隆通过流式分析方法进行检测筛选。图2结果显示细胞株能够稳定高水平表达人SEZ6或者猴SEZ6，可用于后续抗体筛选过程。
实施例3.重组SEZ6蛋白免疫小鼠获得鼠源抗SEZ6单克隆抗体
为筛选出能具有特异性结合人和猴SEZ6膜蛋白的抗体，本实验使用全长人SEZ6的表达质粒，重组人SEZ6-Fc以及人SEZ6-His6蛋白作为免疫原免疫6-8周龄的Balb/c和SJL/J小鼠，使用重组人SEZ6-His6作为免疫原免疫Balb/c和SJL/J小鼠。其中，蛋白初次免疫剂量为每只小鼠50μg。初次免疫2周后，加强免疫，免疫剂量为每只小鼠25μg，每次加强免疫间隔2周。
每次加强免疫一周后采集血清样品，ELISA检测小鼠血清中的抗体滴度。1μg/mL重组人SEZ6-His6包板，4℃过夜，用含1％ BSA的PBST缓冲液封闭1小时，洗板3次。在封闭缓冲液中以1：100开始，10倍梯度稀释小鼠血清，37℃孵育1小时，洗板3次，与抗小鼠IgG-Fc的二抗(HRP标记)温育1小时。PBST洗涤3次，每孔加入100μL TMB底物，15分钟后用2M HCl终止反应测定450nm的吸光度。免疫小鼠血清对免疫原有不同程度的结合，血清的最高稀释倍数为在1:105时，仍能呈现抗原抗体反应。
最后一次免疫腹腔注射25μg重组人SEZ6-His6蛋白，5天后处死小鼠后取脾脏，碾磨收集脾细胞。加入终浓度1％(w/w)的NH4OH裂解脾细胞中混杂的红细胞，获得脾细胞悬液，1000rpm离心清洗细胞3次。按活细胞数目比5:1将小鼠脾脏细胞与小鼠骨髓瘤细胞SP2/0混合，使用高效电融合方法进行细胞融合。使用20％胎牛血清、含1×HAT(w/w)的DMEM培养基稀释融合后的细胞至96孔细胞培养板中，200μL每孔共计1 x 105个细胞，置于5％(v/v)CO2的37℃培养箱中。14天后使用ELISA和FACS筛选融合后的细胞，将OD450nm>1.0、且FACS阳性细胞比例大于20％的阳性克隆扩增至24孔细胞板，在37℃,5％(v/v)CO2条件下，使用含10％(w/w)HT胎牛血清的DMEM培养基继续扩大培养。3天后取24孔细胞板培养液离心收集上清，用ELISA、FACS确定对SEZ6蛋白和SEZ6阳性细胞的结合活性。经过两轮单克隆化，结果取阳性克隆测序。鼠源抗体的可变区序列示于表2。
表2.鼠源抗SEZ6抗体序列

通过替换鼠源单克隆抗体的恒定区，获得重组嵌合型抗体，随后将编码鼠源单克隆抗体可变区的核苷酸序列克隆到含有编码人重、轻链恒定区(Human IgG1,kappa)蛋白序列的pTT5载体上，合成基因序列后转染HEK293细胞，5天后离心去除细胞收集并过滤细胞培养液。将收获的细胞培养液上清液上样至蛋白A柱(MabSelect SuRe，GE)，使用甘氨酸洗脱结合的抗体，1M Tris中和洗脱液，PBS置换缓冲液，即获得嵌合抗体。
实施例4.重组SEZ6蛋白免疫骆驼获得抗SEZ6纳米抗体
为筛选出更多特异性结合人和猴SEZ6膜蛋白抗体，本实验增加了免疫骆驼物种。具体方法为：用重组人SEZ6-Fc免疫2头健康骆驼，免疫4-5次后，每头骆驼采集200ml外周血，分离PBMC。TriZol提取RNA，进行反转录制备cDNA，然后PCR扩增抗体基因，经过酶切、连接、转化，构建VHH噬菌体展示文库(文库容量为 1.2×109)。随机测序结果显示抗体基因插入阳性率达到96％。
用生物素化的重组人SEZ6-His6蛋白筛选先前构建的免疫抗体库，进行亲和淘选3-5轮，随机挑选单克隆制备噬菌体，进行噬菌体ELISA检测，选择阳性克隆进行测序，分析序列。将独特序列的克隆构建VHH-hFc表达载体，293细胞瞬时表达，进行ELISA和FACS检测。部分VHH抗体的氨基酸序列示于表3。
表3.骆驼来源抗SEZ6抗体序列
实施例5.抗体的人源化
为了减少非人源物种引起对人体的免疫反应，需要对鼠源及驼源抗体进行人源化改造，具体实施方法为：对于人源化抗体，通过同源建模预测鼠源单抗的结构后，将鼠抗CDR嵌合到合适的人GermLine框架上(Bioinformation.2014；10(4):180–186；Methods Mol Biol.2019；1904:213-230)，随后在有可能影响抗体-抗原结合的位点引入回复突变，最后将人源化单克隆抗体编码可变区的核苷酸序列克隆到含有编码人重、轻链恒定区(Human IgG1,kappa)蛋白序列的pTT5载体上，如前述相似的方法转染HEK293细胞，生产人源化抗体。
Human IgG1恒定区序列如下：(SEQ ID NO:33)
Kappa恒定区序列如下：(SEQ ID NO:59)
鼠源抗体进行人源化抗体所使用的可变区框架选择见表4，人源化抗体可变区序列参见表5，重链CDR(HCDR1/HCDR2/HCDR3)和轻链CDR(LCDR1/LCDR2/LCDR3)见表6，人源化抗体氨基酸序列全长见表7。其中，PR3178是cAb3009的人源化抗体，PR3121和PR3123是cAb3014的人源化抗体，PR3132是cAb3056的人源 化抗体。
表4.鼠源抗体进行人源化抗体可变区的框架选择
表5.人源化抗SEZ6抗体的可变区序列
表6.人源化抗SEZ6抗体的CDR序列(kabat编号系统)
表7.人源化抗体的重轻链全长氨基酸序列

纳米抗体进行人源化抗体所使用的可变区框架见表8，人源化抗体可变区序列参见表9，重链CDR(HCDR1/HCDR2/HCDR3)见表10，人源化抗体氨基酸序列融合人IgG1-Fc片段见表11。其中，PR0069是cAb025的人源化的抗体，PR0071是cAb314的人源化抗体。
表8.人源化抗SEZ6纳米抗体可变区的框架选择
表9.人源化抗SEZ6纳米抗体的可变区序列
表10.人源化抗SEZ6纳米抗体的CDR序列(kabat编号系统)
表11.人源化抗SEZ6纳米抗体-Fc蛋白的氨基酸序列

实施例6抗SEZ6抗体在细胞上的SEZ6结合活性
为了筛选出能以较高亲和力结合人和猴SEZ6膜蛋白的嵌合抗体或人源化抗体，通过检测细胞表面结合抗体的荧光信号，根据荧光信号的强弱来评价抗体的结合强度。具体为：将梯度稀释的抗体分子和对照分子于1x105个细胞在4℃孵育1小时，洗掉多余的抗体，加入鼠源Alexa Flour 647标记的抗人Fc抗体，在4℃孵育30分钟，洗掉多余的抗体后200μL 1％BSA/PBS缓冲液重悬，通过Thermo Attune NxT流式细胞仪读取细胞表面的荧光信号。表12显示鼠源嵌合抗抗体的结合特性(图3)，表13显示鼠源人源化抗体的结合特征(图5)，表14显示纳米嵌合抗体的结合特征(图7)，表15显示纳米人源化抗体的亲和特征(图9)。结果表明：候选抗体具有和hSC200接近的SEZ6膜蛋白结合活性。
表12.鼠源SEZ6嵌合抗体的体外表征
表13.人源化抗SEZ6鼠源抗体的体外表征

表14.骆驼来源SEZ6嵌合抗体的体外表征
表15.人源化抗SEZ6骆驼源抗体的体外表征
实施例7抗SEZ6抗体体外细胞内化活性的测定
为了评价抗体的内吞活性，采用DT3C抗体内化活性评价体系。DT3C是重组表达的融合蛋白，分子量70KD，由白喉毒素的Fragment A(仅毒素部分)和G群链球菌的3C片段(IgG结合部分)融合而成，该蛋白能够与抗体的IgG部分高度亲和，在抗体发生内吞时一同进入细胞，在胞内弗林蛋白酶的作用下，释放出具有毒性的DT，DT能够抑制EF2-ADP核糖基化的活性，阻断蛋白翻译过程，最终导致细胞死亡。通过利用该系统，可同时观察因抗体的内化和免疫毒素而导致的细胞杀伤效果(Yamaguchi,M.，Hama，H.，等，Biochemical and Biophysical Research Communications 454(2014)600-603)。
依赖于DT3C的内化以及免疫毒素活性的评价，具体如下：将无菌过滤的DT3C和待检测嵌合抗体(DT3C摩尔浓度为抗体摩尔浓度的2倍)按照1:1的体积混匀，于37℃静置孵育30分钟后用完全培养基梯度稀释，加入细胞(H69或者H82)中(3000个细胞/孔)，5％二氧化碳培养箱中37℃孵育6天。加入CellTiter-Glo，室温下避光孵育10min，PerkinElmer上读取化学发光。如图4所示：鼠源嵌合抗体cAb3009、cAb3014和cAb3056有强于对照分子hSC200的内化和免疫毒素杀伤活性。另外cAb3003，cAb3005，cAb3006，cAb3009这一系列序列相似的嵌合抗体的内吞活性均显著优于对照分子hSC200：这一系列序列的CDRH1，CHDRH2，CDRH3通式为XYEMH，GIDPEXXNTVYNQKFKX，GDWYFDX；CDRL1， CDRL2，CDRL3通式为KSSQSLLNSRTRENYLA，WASTRXX，XQSYNLFT。
表16.人源化抗SEZ6鼠源抗体的体外表征
表17.骆驼来源SEZ6嵌合抗体的体外表征
表18.人源化抗SEZ6骆驼源抗体的体外表征
如表16和图6所示：人源化抗SEZ6鼠源抗体PR3178、PR3121、PR3123和PR3132有和对照分子hSC200相当或略强的内化和免疫毒素杀伤活性。如表17和图8所示：骆驼来源嵌合抗体cAb025和cAb314有强于对照分子hSC200的内化和免疫毒素杀伤活性。如表18和图10所示：人源化抗SEZ6骆驼源抗体PR0069和PR0071有强于对照分子hSC200的内化和免疫毒素杀伤活性。
实施例8抗SEZ6抗体对SEZ6同家族SEZ6L和SEZ6L2亲和检测
为了筛选出能与SEZ6特异性亲和的抗体，通过ELISA的方法检测抗体与SEZ6同家族蛋白的结合。具体为：用PBS将重组人SEZ6-Fc融合蛋白、SEZ6L-Fc蛋白和SEZ6L2-Fc分别稀释至1μg/mL，加入ELISA微孔板，每孔各100μL，4℃孵育过夜；加入ELISA封闭液(含1％BSA(w/v),pH7.4的PBS磷酸盐缓冲液)，37℃封闭2小时后，分别加入10nM的检测抗体，37℃温育1小时，用洗板液洗板2-3次；加入辣根过氧化物酶标记(HRP)的二抗，37℃温育1小时，用洗板液洗板2-3次。加入 TMB底物每孔100μL，室温孵育15分钟后，每孔加入50μL终止液(2M HCl)。用ELISA板机(SpectraMax M5e)读取OD450nm值。
结果如图11所示，PR3178和PR0071都和SEZ6蛋白结合，不结合SEZ6L蛋白和SEZ6L2蛋白，说明了PR3178和PR0071具有良好的亲和特异性。
实施例9抗SEZ6抗体对SEZ6亲和表位鉴定
抗体内吞活性与抗原亲和表位有相关性，该实验通过ELISA的方法鉴定抗体的抗原亲和表位。具体为：用PBS将重组人SEZ6-SUSHI1蛋白、SEZ6L-SUSHI2蛋白和SEZ6-N1蛋白分别稀释至1μg/mL，加入ELISA微孔板，每孔各100μL，4℃孵育过夜；加入ELISA封闭液(含1％BSA(w/v),pH7.4的PBS磷酸盐缓冲液)，37℃封闭2小时后，分别加入10nM的检测抗体，37℃温育1小时，用洗板液洗板2-3次；加入辣根过氧化物酶标记(HRP)的二抗，37℃温育1小时，用洗板液洗板2-3次。加入TMB底物每孔100μL，室温孵育15分钟后，每孔加入50μL终止液(2M HCl)。用ELISA板机(SpectraMax M5e)读取OD450nm值。
结果如图12显示，PR3178结合SEZ6蛋白的SUSHI4域，PR0071结合SEZ6非SUSHI1、SUSHI4、N1的单独域。筛选出的抗体分子都具有独特的亲和表位，并且表现出更优的内吞活性。
实施例10用表面等离子共振SPR技术评估抗体与SEZ6相互作用的动力学
用BIAcore 8K(Cityva)系统进行SPR检测PR3178和PR0071抗体的亲和活性。检测用的传感器芯片protein A及相关试剂购自Cytiva。分别将抗体PR3178，PR0071及对照抗体用HBS-EP+缓冲液稀释至1μg/ml,流速设置为10μl/min,捕获抗体至200RU水平。带his标签的SEZ6抗原用HBS-EP+缓冲液按一定比例进行稀释为浓度梯度分别为0nM，3.125nM，6.25nM，12.5nM(两个重复)，25nM，50nM，100nM，200nM。样品分析时设置流速为30μl/min。结合时间为120s，解离时间为900s。然后进行再生，用pH 1.5 Gly-HCl缓冲液作为再生缓冲液，再生流速设置为30μl/min，再生30s。其响应信号以分析时间为横坐标，响应值为纵坐标。所得数据通过BIAcore 8K分析软件进行拟合，采用1：1Langmuir结合模型，确定其结合速率常数(Ka)、解离速率常数(Kd)及解离平衡常数(KD)等动力学常数。结果显示PR3178和PR0071分子与人和食蟹猴抗原结合，且亲和力强于hSC200。
表19.抗SEZ6抗体的结合动力学参数
实施例11 PR3178和PR0071偶联小分子毒素
为将小分子毒素9106靶向肿瘤部位，本实验需要将抗体PR3178和PR0071与小分子毒素9106进行偶联。具体实验如下：在37℃条件下，向抗体的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，5.0mL，342.5nmol)加入配置好的的三(2-羧乙基)膦盐酸盐(TCEP-HCl)的水溶液(10mM，75.4μL，753.5nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将根据WO2020063676A1制备的化合物9106(3.7mg，3424.6nmol)溶解于250μL二甲亚砜中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用HiPrep 26/10进行脱盐(缓冲液：PBS pH7.2-7.4，流速10mL/min)，使用超滤管浓缩，得到PR3178-9106和PR0071-9106的PBS缓冲液(PR3178-9106浓度为1.0mg/mL，26mg；PR0071-9106浓度为2.0mg/mL，20mg)，于4℃冷藏储存。抗SEZ6检测抗体hSC200(hSC17.200，重链SEQ ID NO：6，轻链SEQ ID NO：7)与9106进行偶联方法同上。
得到抗体药物偶联物PR3178-9106和PR0071-9106结构如下，经RP-HPLC计算载药量，PR3178-9106和PR0071-9106的DAR值分别为：n＝4.1和n＝3.6。
实施例12 ADC体外抑制肿瘤细胞系增殖的活性测定
为了检测偶联后ADC分子的体外抗肿瘤活性，通过对肿瘤细胞系生长抑制反映ADC体外活性。具体如下，检测细胞H69和H82分别在96孔培养板中生长过夜，每孔密度3000。第二天加入等体积梯度稀释的毒素偶联药物。5天后使用CellTiter-Glo发光细胞活力测定试剂盒(Promega),如制造商的方案中所述，确定 细胞活力。将细胞活力评估为对照未处理细胞的百分比。如图13所示，ADC药物在H69和H82肿瘤细胞系上有良好的杀伤活性。
表20.ADC在不同表达水平细胞株的杀伤活性
实施例13 ADC体内抗肿瘤活性评估
为了检测ADC分子的体内抗肿瘤活性，使用H69异体移植瘤模型检测肿瘤生长大小变化来反映ADC的抗肿瘤活性。具体如下，为了产生异种移植物，SCID小鼠右后背位置皮下接种H69细胞5x10^6/0.2ml(PBS+gel)，肿瘤均值体积达128mm3时，按肿瘤体积及体重分成5组，每组6只，分组当天开始给药分别在Day0和Day7腹腔注射给药2次，每周2次检测瘤体积和体重，记录数据。
肿瘤体积V＝1/2×a×b2，其中a、b分别表示长、宽。相对肿瘤增殖率T/C(％)＝(T-T0)/(C-C0)×100其中T、C为实验结束时治疗组和对照组的肿瘤体积；T0、C0为实验开始时的肿瘤体积。抑瘤率TGI(％)＝1-T/C(％)。如上所述，将人IgG对照抗体用作阴性对照。TGI代表实验过程中最大的肿瘤生长抑制。
研究结果如图14和表21所示，ADC在等摩尔剂量水平下，对H69异种移植肿瘤的生长具有显著抗肿瘤活性。
表21.抗SEZ6 ADC体内抗肿瘤活性
备注：vs对照组hIgG1：*p<0.05，**p<0.001，***p＜0.0001
实施例14毒素偶联药物的血药浓度测定
为了检测ADC在小鼠体内的药代动力学特征，采取ELISA检测单次给药后小鼠血清中ADC浓度来计算。具体如下，采用SCID小鼠，尾静脉注射等摩尔剂量的ADC药物，单次给药，在以下时间点采集血液：第一次给药后的15min，6h，24h，72h，144h，240h，336h。对血药浓度通过ELISA方法进行检测。采用anti-hFc包被板子，4℃过夜孵育。300μLPBST缓冲液洗涤一次后，加入稀释好的待测血清，室温孵育1小时。每管血清分两个板子检测。一份血清板用300μLPBST缓冲液洗涤三次后，每孔加入100μLHRP标记的羊抗人IgG Fc溶液，室温孵育一小时。PBST 缓冲液洗涤五遍之后，加入显色液，显色完成加入终止液，酶标仪读取OD450。采用Phoenix软件的非房室模型进行PK参数的计算。结果参见图15和表22，结果说明PR3178-9106具有优于hSC200-9106的药代动力学特征。
表22.ADC药物在小鼠中的药代动力学参数
本公开中实验动物的使用及福利遵照“国际实验动物评估和认可委员会(AAALAC)”的规定执行。每天监测动物的健康状况及死亡情况，例行检查包括观察受试物和药物对动物日常行为表现的影响如行为活动，体重变化，外观体征等。
虽然以上描述了本公开的具体实施方案，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本公开的原理和实质的前提下，可以对这些实施方案做出多种变更或修改。因此，本公开的保护范围由所附权利要求书限定。

Claims (29)

  1. 一种抗SEZ6抗体或其抗原结合片段，包含重链可变区(VH)或重链单结构域(VHH)，其中重链可变区或重链单结构域包含：
    (1)如SEQ ID NO：37、38和39氨基酸序列所示的重链HCDR1、HCDR2、HCDR3；或
    (2)如SEQ ID NO：43、44和45氨基酸序列所示的重链HCDR1、HCDR2、HCDR3；或
    (3)如SEQ ID NO：49、50和51氨基酸序列所示的重链HCDR1、HCDR2、HCDR3；或
    (4)如SEQ ID NO：65、66和67氨基酸序列所示的重链HCDR1、HCDR2、HCDR3；或
    (5)如SEQ ID NO：68、69和70氨基酸序列所示的重链HCDR1、HCDR2、HCDR3。
  2. 根据权利要求1所述的抗SEZ6抗体或其抗原结合片段，包含重链可变区(VH)和轻链可变区(VL)，其中重链可变区和轻链可变区包含：
    (1)如SEQ ID NO：37、38和39氨基酸序列所示的重链HCDR1、HCDR2、HCDR3，和如SEQ ID NO：40、41和42氨基酸序列所示的轻链LCDR1、LCDR2和LCDR3；或
    (2)如SEQ ID NO：43、44和45氨基酸序列所示的重链HCDR1、HCDR2、HCDR3，和如SEQ ID NO：46、47和48氨基酸序列所示的轻链LCDR1、LCDR2和LCDR3；或
    (3)如SEQ ID NO：49、50和51氨基酸序列所示的重链HCDR1、HCDR2、HCDR3，和如SEQ ID NO：52、53和54氨基酸序列所示的轻链LCDR1、LCDR2和LCDR3。
  3. 根据权利要求1所述的抗SEZ6抗体或其抗原结合片段，其中重链单结构域包含氨基酸序列如SEQ ID NO：63所示，或与其具有至少85％，90％，95％，99％同源性的序列；或如SEQ ID NO：64所示，或与其具有至少85％，90％，95％，99％同源性的序列；
    优选地，重链单结构域包含氨基酸序列如SEQ ID NO：63所示，或如SEQ ID NO：64所示。
  4. 根据权利要求1或2中所述的抗SEZ6抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中：
    重链可变区包含氨基酸序列如SEQ ID NO：29，SEQ ID NO：31，SEQ ID NO：35所示，或与其具有至少85％，90％，95％，99％同源性的序列；
    轻链可变区包含氨基酸序列如SEQ ID NO：30，SEQ ID NO：32，SEQ ID NO：34，SEQ ID NO：36所示，或与其具有至少85％，90％，95％，99％同源性的序列。
  5. 根据权利要求4中所述的抗SEZ6抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中：
    (1)所述重链可变区包含氨基酸序列如SEQ ID NO：29所示，或与其具有至少85％，90％，95％，99％同源性的序列，所述轻链可变区包含氨基酸序列如SEQ ID NO：30所示，或与其具有至少85％，90％，95％，99％同源性的序列；或
    (2)所述重链可变区包含氨基酸序列如SEQ ID NO：31所示，或与其具有至少85％，90％，95％，99％同源性的序列，所述轻链可变区包含氨基酸序列如SEQ ID NO：32所示，或与其具有至少85％，90％，95％，99％同源性的序列；或
    (3)所述重链可变区包含氨基酸序列如SEQ ID NO：31所示，或与其具有至少85％，90％，95％，99％同源性的序列，所述轻链可变区包含氨基酸序列如SEQ ID NO：34所示，或与其具有至少85％，90％，95％，99％同源性的序列；或
    (4)所述重链可变区包含氨基酸序列如SEQ ID NO：35所示，或与其具有至少85％，90％，95％，99％同源性的序列，所述轻链可变区包含氨基酸序列如SEQ ID NO：36所示，或与其具有至少85％，90％，95％，99％同源性的序列；
    优选地，(1)所述重链可变区包含氨基酸序列如SEQ ID NO：29所示，所述轻链可变区包含氨基酸序列如SEQ ID NO：30所示；或
    (2)所述重链可变区包含氨基酸序列如SEQ ID NO：31所示，所述轻链可变区包含氨基酸序列如SEQ ID NO：32所示；或
    (3)所述重链可变区包含氨基酸序列如SEQ ID NO：31所示，所述轻链可变区包含氨基酸序列如SEQ ID NO：34所示；或
    (4)所述重链可变区包含氨基酸序列如SEQ ID NO：35所示，所述轻链可变区包含氨基酸序列如SEQ ID NO：36所示。
  6. 根据权利要求1至5中任一项所述的抗SEZ6抗体或其抗原结合片段，其中所述抗原结合片段选自Fab、Fab'、F(ab')2、单链抗体(scFv)、单域抗体(sdAb)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
  7. 根据权利要求1至6中任一项所述的抗SEZ6抗体或其抗原结合片段，其包含抗体恒定区；
    其中重链恒定区来源于人IgG1、IgG2、IgG3、IgG4或其变体；和/或轻链恒 定区来源于人抗体κ、λ链或其变体；
    优选地，所述重链恒定区的氨基酸序列源于人IgG1或其变体；和/或轻链恒定区来源于人抗体κ链或其变体；
    更优选地，所述重链恒定区包含氨基酸序列如SEQ ID NO：33所示，和/或轻链恒定区包含氨基酸序列如SEQ ID NO：59所示。
  8. 根据权利要求7所述的抗SEZ6抗体或其抗原结合片段，包含重链和轻链，其中：
    (1)重链包含氨基酸序列如SEQ ID NO:55所示，或与其具有至少85％，90％，95％，99％同源性的序列，和轻链包含氨基酸序列如SEQ ID NO:56所示，或与其具有至少85％，90％，95％，99％同源性的序列；或
    (2)重链包含氨基酸序列如SEQ ID NO:57所示，或与其具有至少85％，90％，95％，99％同源性的序列，和轻链包含氨基酸序列如SEQ ID NO:58所示，或与其具有至少85％，90％，95％，99％同源性的序列；或
    (3)重链包含氨基酸序列如SEQ ID NO:57所示，或与其具有至少85％，90％，95％，99％同源性的序列，和轻链包含氨基酸序列如SEQ ID NO:60所示，或与其具有至少85％，90％，95％，99％同源性的序列；或
    (4)重链包含氨基酸序列如SEQ ID NO:61所示，或与其具有至少85％，90％，95％，99％同源性的序列，和轻链包含氨基酸序列如SEQ ID NO:62所示，或与其具有至少85％，90％，95％，99％同源性的序列；
    优选地，(1)重链包含氨基酸序列如SEQ ID NO:55所示，和轻链包含氨基酸序列如SEQ ID NO:56所示；或
    (2)重链包含氨基酸序列如SEQ ID NO:57所示，和轻链包含氨基酸序列如SEQ ID NO:58所示；或
    (3)重链包含氨基酸序列如SEQ ID NO:57所示，和轻链包含氨基酸序列如SEQ ID NO:60所示；或
    (4)重链包含氨基酸序列如SEQ ID NO:61所示，和轻链包含氨基酸序列如SEQ ID NO:62所示。
  9. 根据权利要求3所述的抗SEZ6抗体或其抗原结合片段，其由重链组成，其中：
    (1)重链包含氨基酸序列如SEQ ID NO:71所示，或与其具有至少85％，90％，95％，99％同源性的序列；或
    (2)重链包含氨基酸序列如SEQ ID NO:72所示，或与其具有至少85％，90％，95％，99％同源性的序列；
    优选地，(1)重链包含氨基酸序列如SEQ ID NO:71所示；或
    (2)重链包含氨基酸序列如SEQ ID NO:72所示。
  10. 一种分离的核酸，其编码权利要求1至9中任一项所述的抗SEZ6抗体或其抗原结合片段。
  11. 一种载体，其包含权利要求10所述的分离的核酸。
  12. 一种宿主细胞，其包含权利要求11所述的载体。
  13. 一种制备抗SEZ6抗体或其抗原结合片段的方法，其包括在适于表达所述抗SEZ6抗体或其抗原结合片段的条件下培养权利要求12所述的宿主细胞。
  14. 一种抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其包含权利要求1至9中任一项所述的抗SEZ6抗体或其抗原结合片段和药物；其中所述的药物选自：细胞毒性剂、放射性标记物、荧光团、发色团、显像剂、免疫调节剂、血管生成抑制剂、细胞增殖抑制剂、促细胞凋亡剂、细胞裂解酶，以及其任何组合；
    优选地，所述的药物选自：DNA损伤剂、拓扑异构酶抑制剂、微管抑制剂、蛋白质降解剂、STING激动剂，以及其任何组合。
  15. 根据权利求14所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其具有通式(I)所示的结构：
    Pc-(L-Y-Dr)n
    (I)
    其中，Dr为权利要求14所述的药物；
    优选地，Dr选自：喜树碱、澳瑞他汀、美登木素生物碱、长春花生物碱、吡咯并苯二氮卓类(PBD)、加利车霉素、多卡霉素、柔红霉素、多柔比星、卡奇霉素、安曲霉素、新霉素、鹅膏素毒素、哈米特林、艾日布林、Tubulysin，以及其类似物或衍生物；
    更优选地，Dr选自：Exatecan、MMAE、MMAF、MMAD、SN-38、DM1、DM4、吡咯苯并二氮卓(PBD)二聚体，以及其类似物或衍生物；
    n为1至10，n是小数或整数，优选的n为2至8，更优选的n为3至5或4至8，进一步优选的n为4至6，进一步优选的n为5至6，更进一步优选的，n为2，2.5，3，3.5，4，4.5，5，5.5，6，6.5，7，7.5或8；
    L、Y为接头单元；
    Pc为权利要求1至9中任一项所述的抗SEZ6抗体或其抗原结合片段。
  16. 根据权利求14或15所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其具有通式(Pc-L-Y-Dr)、通式(II)、通式(III)、通式(IV)所示的结构：
    其中：
    Y选自-O-(CRaRb)m-CR1R2-C(O)-、-O-CR1R2-(CRaRb)m-、-O-CR1R2-、-NH-(CRaRb)m-CR1R2-C(O)-或-S-(CRaRb)m-CR1R2-C(O)-；
    Ra和Rb相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
    或者，Ra和Rb与其相连接的碳原子一起形成环烷基或杂环基；
    R1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基或杂芳基；
    R2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基或杂芳基；
    或者，R1和R2与其相连的碳原子一起形成环烷基或杂环基；
    或者，Ra和R2与其相连的碳原子一起形成环烷基或杂环基；
    m为0至4的整数；
    n为1至10，n是小数或整数，优选的n为2至8，更优选的n为3至5或4至8，进一步优选的n为4至6，进一步优选的n为5至6，更进一步优选的，n 为2，2.5，3，3.5，4，4.5，5，5.5，6，6.5，7，7.5或8；
    L为接头单元；
    Pc为权利要求1至9中任一项所述的抗SEZ6抗体或其抗原结合片段。
  17. 根据权利要求16所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，
    其中：
    Y为-O-(CRaRb)m-CR1R2-C(O)-；
    Ra和Rb相同或不同，且各自独立地选自氢原子、氘原子、卤素或烷基；
    R1为卤代烷基或C3-6环烷基；
    R2选自氢原子、卤代烷基或C3-6环烷基；
    或者，R1和R2与其相连接的碳原子一起形成C3-6环烷基；
    m为0或1。
  18. 根据权利要求16或17中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中Y选自：
  19. 根据权利要求16至18中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中Y的O端与接头单元L相连。
  20. 根据权利要求16至19中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中接头单元-L-为-L1-L2-L3-L4-，
    L1为s1为2至8的整数；
    L2为化学键；
    L3为四肽残基；
    L4为-NR5(CR6R7)t-，R5、R6或R7相同或不同，且各自独立地为氢原子或烷基，t为1或2。
  21. 根据权利要求16至19中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中接头单元-L-为-L1-L2-L3-L4-，
    L1为s1和s2独立的选自0至8的整数；优选的s1为2，s2为2至8的整数；更优选的，s1为2，s2为2，3，4，5或6；
    L2为化学键；
    L3为四肽残基；
    L4为-NR5(CR6R7)t-，R5、R6或R7相同或不同，且各自独立地为氢原子或烷基，t为1或2。
  22. 根据权利要求16至21中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中所述的接头单元-L-，其L1端与Pc相连，L4端与Y相连。
  23. 根据权利要求16至22中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，其中所述的L3的四肽残基为由两个或多个选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸中的氨基酸形成的氨基酸残基；优选为GGFG的四肽残基。
  24. 根据权利要求16至23中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，所述抗体-药物偶联物选自：





    其中Pc和n如权利要求16中所定义。
  25. 根据权利要求16至24中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，所述抗体-药物偶联物选自：


    其中n如权利要求16中所定义。
  26. 一种试剂盒，其包含权利要求1至9中任一项所述的抗SEZ6抗体或其抗原结合片段，或权利要求14至25中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非 对映异构体、或其混合物形式。
  27. 一种药物组合物，其包含权利要求1至9中任一项所述的抗SEZ6抗体或其抗原结合片段，或权利要求14至25中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，以及一种或多种药学上可接受的载体。
  28. 根据权利要求1至9中任一项所述的抗SEZ6抗体或其抗原结合片段，或权利要求14至25中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或根据权利要求27所述的药物组合物在制备用于治疗SEZ6介导的疾病或病症的药物中的用途；
    优选地，所述SEZ6介导的疾病或病症为SEZ6高表达癌症。
  29. 根据权利要求1至9中任一项所述的抗SEZ6抗体或其抗原结合片段，或权利要求14至25中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或根据权利要求27所述的药物组合物在制备用于治疗或预防肿瘤或癌症的药物中的用途；
    优选地，其中所述肿瘤或癌症选自：肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌。
PCT/CN2024/091754 2023-05-08 2024-05-08 一种抗体或其抗原结合片段、抗体-药物偶联物及其应用 WO2024230742A1 (zh)

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Publication number Priority date Publication date Assignee Title
CN104334580A (zh) * 2012-02-24 2015-02-04 施特姆森特Rx股份有限公司 抗sez6抗体及使用方法
CN112135843A (zh) * 2018-05-30 2020-12-25 艾伯维施特姆森特克斯有限责任公司 抗sez6抗体药物缀合物和使用方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104334580A (zh) * 2012-02-24 2015-02-04 施特姆森特Rx股份有限公司 抗sez6抗体及使用方法
CN112135843A (zh) * 2018-05-30 2020-12-25 艾伯维施特姆森特克斯有限责任公司 抗sez6抗体药物缀合物和使用方法

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