WO2021213434A1 - 一种抗Nectin-4的抗体及其应用 - Google Patents

一种抗Nectin-4的抗体及其应用 Download PDF

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WO2021213434A1
WO2021213434A1 PCT/CN2021/088661 CN2021088661W WO2021213434A1 WO 2021213434 A1 WO2021213434 A1 WO 2021213434A1 CN 2021088661 W CN2021088661 W CN 2021088661W WO 2021213434 A1 WO2021213434 A1 WO 2021213434A1
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cdr
amino acid
acid sequence
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PCT/CN2021/088661
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French (fr)
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任红媛
朱戬
林鉴
王骊淳
徐晓红
邓小芳
毕建军
王晋
吴建
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迈威(上海)生物科技股份有限公司
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Priority to BR112022021322A priority Critical patent/BR112022021322A2/pt
Priority to AU2021260639A priority patent/AU2021260639A1/en
Priority to EP21791797.0A priority patent/EP4141029A1/en
Priority to JP2022563917A priority patent/JP2023522229A/ja
Priority to CN202180030277.7A priority patent/CN115427452A/zh
Priority to MX2022013163A priority patent/MX2022013163A/es
Priority to US17/996,403 priority patent/US20230265183A1/en
Priority to KR1020227040582A priority patent/KR20230007406A/ko
Priority to CA3176385A priority patent/CA3176385A1/en
Publication of WO2021213434A1 publication Critical patent/WO2021213434A1/zh

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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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Definitions

  • the present invention belongs to the field of antibody medicines. Specifically, the present invention relates to antibodies against human Nectin-4 and their use for preparing medicines.
  • Nectin-4 (also known as PVRL4, poliovirus receptor-like molecule 4) is a 66KD transmembrane glycoprotein, which belongs to the IG superfamily protein molecule of the Nectin family. Its extracellular domain consists of three Ig It is composed of VCC-like domain (VCC type) and participates in the formation and maintenance of adhesion junctions together with cadherin.
  • VCC VCC-like domain
  • Nectin-4 is closely related to the occurrence and development of a variety of tumor cells. It has been found that Nectin-4 is expressed in a variety of solid tumors, especially bladder cancer; in breast cancer, ovarian cancer and lung cancer, as a tumor-associated antigen, it accounts for 50% of breast cancer, 49% of ovarian cancer and 86% of lung cancer. The tissue expression detection rate of these epithelial malignancies, and plays a key role in the occurrence, invasion and metastasis of these epithelial malignant tumors. Therefore, Nectin-4 has become an important target for the diagnosis and treatment of many solid tumors.
  • the main drug for Nectin-4 is Enfortumab vedotin, which is an antibody-conjugated drug, which is a combination of a monoclonal antibody against Nectin-4 and a cell-killing drug monomethyl auristatin E (MMAE). It is used to treat bladder cancer, especially It is urothelial cancer, which has been approved by the FDA as a breakthrough therapy in March 2018.
  • adhesion factor Nnectin-4 can be used not only as an effective prognostic factor for breast cancer, but also as an effective therapeutic target for patients with triple-negative breast cancer (TNBC); in vivo and in vitro studies have confirmed that antibodies against Nectin-4 Conjugated drugs (ADC) have a good effect on local and metastatic TNBC.
  • ADC Nectin-4 Conjugated drugs
  • the technical problem to be solved by the present invention is to obtain a high-affinity antibody that specifically binds to Nectin-4 through hybridoma screening and humanization technology, wherein a fully human antibody sequence is obtained through humanization modification.
  • the purpose of the present invention is to provide an antibody molecule or fragment thereof that specifically binds to Nectin-4, especially human Nectin-4, and to provide its use.
  • the fragments of the antibody molecule of the present invention cover various functional fragments of antibodies, for example, antigen-binding parts thereof, such as Fab, F(ab') 2 or scFv fragments.
  • the present invention provides an antibody molecule or a fragment thereof, the antibody molecule or a fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region and the light chain
  • VH heavy chain variable region
  • VL light chain variable region
  • the variable regions respectively comprise a combination of heavy chain CDRs and light chain CDRs selected from:
  • CDR-H1 GYTFTTY
  • CDR-H2 YPGNVN
  • CDR-H3 GLYYFDY
  • SEQ ID NO: 35, 39, 43 in sequence and, shown in SEQ ID NO: 45, 47, 49 of CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), CDR-L3 (QQDYSSPYT);
  • CDR-H1 GYTFTTYYIH
  • CDR-H2 WIYPGNVNTK
  • CDR-H3 GLYYFDY
  • SEQ ID NO: 36, 40, 43 in sequence and, shown in SEQ ID NO: 45, 47, 49 of CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), CDR-L3 (QQDYSSPYT);
  • CDR-H1 (TYYIH), CDR-H2 (WIYPGNVNTKYNEKFKG), CDR-H3 (GLYYFDY) shown in SEQ ID NO: 37, 41, 43 in sequence; and, shown in SEQ ID NO: 45, 47, 49 of CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), CDR-L3 (QQDYSSPYT);
  • CDR-H1 TTYYIH
  • CDR-H2 WIGWIYPGNVNTK
  • CDR-H3 ARGLYYFD
  • SEQ ID NO: 38, 42, 44 SEQ ID NO: 44
  • SEQ ID NO: 46, 48, 50 CDR-L1 (SNDVAWY), CDR-L2 (LLIYYASNRY), CDR-L3 (QQDYSSPY);
  • CDR-H1 (GFSLIDY), CDR-H2 (WGDGK), CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 51, 55, 59 in sequence; and, shown in SEQ ID NO: 61, 63, 65 CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES), CDR-L3 (QQHYNTPFT);
  • CDR-H1 (GFSLIDYGVS), CDR-H2 (VIWGDGKIY), CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 52, 56, 59; and, shown in SEQ ID NO: 61, 63, 65 CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES), CDR-L3 (QQHYNTPFT);
  • CDR-H1 (DYGVS), CDR-H2 (VIWGDGKIYYNSVLKS), CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 53, 57, 59 in sequence; and, shown in SEQ ID NO: 61, 63, 65 CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES), CDR-L3 (QQHYNTPFT);
  • CDR-H1 IDYGVS
  • CDR-H2 word GVIWGDGKIY
  • CDR-H3 AKQGGLLFYAMD
  • SEQ ID NO: 54, 58, 60 shown in SEQ ID NO: 54, 58, 60; and, shown in SEQ ID NO: 62, 64, 66 CDR-L1 (LNSYSQKNYLAWY), CDR-L2 (LLIYFASTRE), CDR-L3 (QQHYNTPF);
  • CDR-H1 (GFSLIDY), CDR-H2 (WGDGK), CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 51, 55, 59; and, shown in SEQ ID NO: 67, 63, 65 CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES), CDR-L3 (QQHYNTPFT);
  • CDR-H1 (GFSLIDY), CDR-H2 (WGDAK), CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 51, 68, 59; and, shown in SEQ ID NO: 67, 63, 65 CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES), CDR-L3 (QQHYNTPFT);
  • CDR-H1 (GFSLIDY), CDR-H2 (WGGGK), CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 51, 69, 59; and, shown in SEQ ID NO: 67, 63, 65 CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES), CDR-L3 (QQHYNTPFT);
  • CDR-H1 GYTFTSY
  • CDR-H2 YPGNAN
  • CDR-H3 SVYYFDY
  • SEQ ID NO: 70, 74, 78 shown in SEQ ID NO: 45, 80, 49 of CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRNT), CDR-L3 (QQDYSSPYT);
  • CDR-H1 GYTFTSYYIH
  • CDR-H2 WIYPGNANNK
  • CDR-H3 SVYYFDY
  • SEQ ID NO: 71, 75, 78 in sequence and, shown in SEQ ID NO: 45, 80, 49 of CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRNT), CDR-L3 (QQDYSSPYT);
  • CDR-H1 SYYIH
  • CDR-H2 WIYPGNANNKYNENFKG
  • CDR-H3 SVYYFDY
  • SEQ ID NO: 72, 76, 78 in sequence; and, shown in SEQ ID NO: 45, 80, 49 of CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRNT), CDR-L3 (QQDYSSPYT);
  • CDR-H1 (TSYYIH), CDR-H2 (WIGWIYPGNANNK), CDR-H3 (ARSVYYFD) shown in SEQ ID NO: 73, 77, 79 in sequence; and, shown in SEQ ID NO: 46, 81, 50 CDR-L1 (SNDVAWY), CDR-L2 (LLIYYASNRN), CDR-L3 (QQDYSSPY);
  • CDR-H1 GYSFTDY
  • CDR-H2 NPNNGN
  • CDR-H3 EDRYAFAY
  • SEQ ID NO: 82, 86, 90 shown in SEQ ID NO: 82, 86, 90; and, shown in SEQ ID NO: 92, 94, 96 CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), CDR-L3 (QHTWEIPYT);
  • CDR-H1 GYSFTDYYMH
  • CDR-H2 RVNPNNGNTL
  • CDR-H3 EDRYAFAY
  • SEQ ID NO: 83, 87, 90 SEQ ID NO: 83, 87, 90
  • SEQ ID NO: 92, 94, 96 CDR-L1 RASQSVSTSSYTYMH
  • CDR-L2 YASNLES
  • CDR-L3 QHTWEIPYT
  • CDR-H1 (DYYMH), CDR-H2 (RVNPNNGNTLYNQKFRG), CDR-H3 (EDRYAFAY) shown in SEQ ID NO: 84, 88, 90; and, shown in SEQ ID NO: 92, 94, 96 CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), CDR-L3 (QHTWEIPYT);
  • CDR-H1 (TDYYMH), CDR-H2 (WIGRVNPNNGNTL), CDR-H3 (AREDRYAFA) shown in SEQ ID NO: 85, 89, 91 in sequence; and, shown in SEQ ID NO: 93, 95, 97 CDR-L1 (STSSYTYMHWY), CDR-L2 (LLIKYASNLE), CDR-L3 (QHTWEIPY).
  • the heavy chain variable region or the light chain variable region in the antibody molecule or fragment thereof of the present invention is divided into FR1-CDR1-FR2-
  • the sequence of CDR2-FR3-CDR3-FR4 includes the above-mentioned domain components, where FR is the framework region.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, 9 or 10 or has at least 75% identity with the amino acid sequence.
  • An amino acid sequence; and, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, 11 or 12 or an amino acid sequence having at least 75% identity with the amino acid sequence; or,
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13, 15, 16, 18, 19, 20, or 21 or an amino acid sequence having at least 75% identity with the amino acid sequence; and, the The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14, 22, 23 or 25 or an amino acid sequence having at least 75% identity with the amino acid sequence; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, 29 or 30 or an amino acid sequence having at least 75% identity with the amino acid sequence; and, the light chain variable region comprises the amino acid sequence shown in The amino acid sequence of SEQ ID NO: 28, 31 or 32 or an amino acid sequence having at least 75% identity with the amino acid sequence; or,
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity with the amino acid sequence; and, the light chain variable region includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence of 34 or an amino acid sequence having at least 75% identity with the amino acid sequence.
  • variable region of the heavy chain and the variable region of the light chain in the antibody molecule or a fragment thereof are selected from a combination of the following amino acid sequences:
  • amino acid sequence shown in SEQ ID NO: 7 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 7; and, the amino acid sequence shown in SEQ ID NO: 8 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 8;
  • amino acid sequence shown in SEQ ID NO: 9 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 9; and, the amino acid sequence shown in SEQ ID NO: 12 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 12;
  • amino acid sequence shown in SEQ ID NO: 10 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 10; and, the amino acid sequence shown in SEQ ID NO: 12 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 12;
  • amino acid sequence shown in SEQ ID NO: 33 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 33; and, the amino acid sequence shown in SEQ ID NO: 34 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 34.
  • the antibody molecule or fragment thereof provided by the present invention binds to poliovirus receptor-like molecule 4 (Nectin-4), preferably mammalian Nectin-4, more preferably primate Nectin-4, further preferably human or monkey Nectin -4, especially human Nectin-4.
  • Nectin-4 poliovirus receptor-like molecule 4
  • mammalian Nectin-4 more preferably primate Nectin-4, further preferably human or monkey Nectin -4, especially human Nectin-4.
  • the antibody molecule is a murine antibody, a chimeric antibody or a fully or partially humanized antibody; the fragment is any fragment of the antibody molecule that can specifically bind to Nectin-4, such as scFv, dsFv, ( dsFv) 2 , Fab, Fab', F(ab') 2 or Fv fragment.
  • Nectin-4 such as scFv, dsFv, ( dsFv) 2 , Fab, Fab', F(ab') 2 or Fv fragment.
  • the antibody molecule is a monoclonal antibody or a single chain antibody.
  • the antibody molecule or fragment thereof further comprises a human or murine constant region, preferably a murine or human heavy chain constant region (CH) and/or a light chain constant region (CL); preferably, the antibody molecule Or fragments thereof include a heavy chain and a light chain, for example, two heavy chains and a light chain. More preferably, the antibody molecule or a fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and/or a kappa or lambda light chain constant region.
  • the antibody molecule provided by the present invention is a monoclonal antibody, preferably a humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is of type IgG1, and the light chain constant region is ⁇ type.
  • the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity with the amino acid sequence;
  • the light chain constant region of the monoclonal antibody comprises The amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 75% identity with the amino acid sequence.
  • At least 75% identity is the identity of any percentage number between 75% and 100%, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
  • the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a light chain variable region, a heavy chain variable region, a heavy chain, or a light chain contained in the antibody molecule of the present invention or a fragment thereof .
  • the nucleic acid molecule of the present invention can be cloned into a vector, and then transformed or transfected into a host cell. Therefore, in yet another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention.
  • the vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like.
  • the vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell or enter the host cell in any manner for the purpose of preservation or expression of antibodies. Therefore, in another aspect, the present invention provides a host cell, which contains the nucleic acid molecule and/or vector of the present invention, or the host cell is transformed or transfected by the nucleic acid molecule and/or vector of the present invention.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
  • the antibody molecule provided by the present invention can be obtained by any method known in the art.
  • the heavy chain variable region and/or light chain variable region of the antibody can be obtained first from the nucleic acid molecule provided by the present invention, or the heavy chain and/or light chain of the antibody molecule can be obtained, and then combined with the antibody molecule
  • the optional other domains of the antibody are assembled into antibodies; or, in the host cell provided by the present invention, the heavy chain variable region and/or light chain variable region of the antibody molecule or the heavy chain and/or heavy chain of the antibody molecule are expressed in the host cell provided by the present invention.
  • the host cell is cultured.
  • the method further includes the step of recovering the antibody molecules produced.
  • the antibody molecules or fragments thereof, nucleic acid molecules, vectors, host cells, or fusion proteins provided by the present invention can be included in a composition, more particularly in a pharmaceutical preparation, so as to be used for various purposes according to actual needs. Therefore, in another aspect, the present invention also provides a composition comprising the antibody molecules or fragments thereof, nucleic acid molecules, vectors and/or host cells provided by the present invention.
  • the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.
  • the present invention also provides the use of the antibody molecule or fragments thereof, nucleic acid molecules, vectors, host cells and/or compositions in the preparation of reagents for detecting or diagnosing diseases or disorders. .
  • the present invention also provides a method for detecting or diagnosing a disease or disorder, which method comprises combining the antibody molecule or fragment, nucleic acid molecule, vector, host cell and/or composition of the present invention with a subject Contact with the sample of the person.
  • the subject is a mammal, preferably a primate, and more preferably a human.
  • the present invention also provides the use of the antibody molecule or fragments thereof in the preparation of antibody drug conjugates.
  • the present invention provides an antibody drug conjugate, which is formed by coupling the antibody molecule of the present invention or a fragment thereof with a cytotoxic moiety.
  • the cytotoxic part is a tubulin inhibitor, a topoisomerase inhibitor or a DNA binding agent.
  • the tubulin inhibitor is selected from maytansine derivatives, Monomethylauristatin E (MMAE), Monomethylauristatin F (MMAF), Monomethyl Dolastatin 10, Tubulysin derivatives, Cryptophycin derivatives and Taltobulin.
  • the topoisomerase inhibitor is selected from the PNU-159682 derivative of doxorubicin metabolite and the SN38 derivative of irinotecan (CPT-11) metabolite.
  • the DNA binding agent is selected from PBD derivatives and Duocarmycin derivatives.
  • the present invention also provides the use of the antibody molecule or fragments thereof, nucleic acid molecules, vectors, host cells, compositions and/or antibody drug conjugates in the preparation of drugs for preventing or treating diseases or disorders.
  • the present invention also provides a method for preventing or treating a disease or disorder, the method comprising administering the antibody molecule or fragment, nucleic acid molecule, vector, host cell, or combination of the present invention to a subject in need thereof And/or antibody-drug conjugates.
  • the subject is a mammal, more preferably a human.
  • the present invention provides a kit comprising the antibody molecule or fragments thereof, nucleic acid molecules, vectors, host cells, compositions and/or antibody drug conjugates of the present invention.
  • the kit can be used for treatment, detection or diagnosis purposes, such as treatment, detection or diagnosis of diseases or disorders.
  • the antibody molecule or its fragments, nucleic acid molecules, vectors, host cells, compositions and/or antibody drug conjugates can be used to treat diseases or disorders related to the high expression of Nectin-4.
  • the disease or disorder is a tumor or cancer with high expression of Nectin-4, especially a solid tumor.
  • the disease or disorder is bladder cancer, pancreatic cancer, breast cancer (including triple-negative subtypes and basal subtypes), non-small cell lung cancer, gastric cancer, esophageal cancer, ovarian cancer, etc.; especially bladder cancer, breast cancer, Ovarian cancer or lung cancer.
  • the present invention obtains a high-affinity antibody that specifically binds to Nectin-4 through hybridoma screening and humanization technology, wherein a fully human antibody sequence is obtained through humanization modification.
  • a fully human antibody sequence is obtained through humanization modification.
  • an effective clinical lead drug molecule sequence was obtained.
  • Figure 1 shows the results of flow cytometry identification of antigen-expressing cell lines, in which small figure 1A: HT-1376 bladder cancer cells, and small figure 1B: CHO-huNectin4S8 cells.
  • Figure 2 shows the results of the FACS binding activity assay of the culture supernatant of hybridoma cells, in which small figure 2A: the first round of screening, and small figure 2B: the second round of screening.
  • Figure 3 shows the results of the FACS binding experiment of the antibody and the cells expressing BT474.
  • Figure 4 shows the results of the endocytosis activity of the antibody in BT474 cells.
  • Figure 5 shows the results of FACS binding experiments between antibodies and different cells expressing huNectin4, muNectin4, and cynoNectin4.
  • Figure 6 shows the results of the binding experiment between antibodies and members of the Nectin protein family, in which panel 6A: Nectin-1, panel 6B: Nectin-2, panel 6C: Nectin-3, panel 6D: Nectin-4.
  • Figure 7 shows the results of the antibody's monkey serum stability test, in which small figure 7A: control antibody Enfortumab, small figure 7B: 42D20 hz10.
  • Figure 8 shows the metabolism of antibodies in mice, in which small figure 8A: control antibody Enfortumab, small figure 8B: 42D20 hz10.
  • the heavy chain amino acid sequence of the control antibody Enfortumab is shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown in SEQ ID NO: 2.
  • the antibody sequences of the present invention are shown in Table I to Table IV.
  • Antigen NECTIN4 recombinant protein (serial number: NP_002178.2, 32aa-349aa), shown in SEQ ID NO: 3.
  • Enfortumab antibody light chain variable region and heavy chain variable region genes were cloned into the eukaryotic expression vector pCDNA3.1 upstream of the human-kappa light chain constant region and human IgG1 heavy chain constant region coding genes, respectively, to obtain Enfortumab light and heavy chain expression plasmids were transformed into E. coli for amplification, and a large number of plasmids containing Enfortumab antibody light chain (SEQ ID NO: 2) and heavy chain (SEQ ID NO: 1) were isolated and obtained. Amine (PEI) was mixed and co-transfected into HEK293 cells. The cells were transfected for 5-6 days, the culture supernatant was taken, and the expression supernatant was purified by Mabselect affinity chromatography column to obtain Enfortumab antibody.
  • PKI Protein
  • Nectin-4 gene was cloned from the vector containing Nectin-4 cDNA (Beijing Yiqiao Shenzhou, Cat: HG19771-UT) by PCR, and the reading frame of Nectin-4 gene was digested by restriction enzyme method It was cloned into a stable expression vector containing glutamine synthetase (GS) screening gene, electrotransfected (Nucleofector IIb, Lonza) suspension cultured CHO-K1 cells, and the transfected cells were placed in a 50 ⁇ M MSX (Sigma , Cat: M5379) CD CHO AGTTM medium (Gibco, Cat: 12490-025), seeded in 96-well cell culture plate, 37°C, 5% CO 2 static culture for 2-3 weeks, screening by MSX pressure , Obtain 9 cell growth holes from pre-screening under the microscope, and expand them to 24-well cell culture plates. Finally, the high-expressing antigen clone S8 is selected by flow
  • the high-expressing clone S8 was named CHO-huNectin4 S8.
  • the comparison and identification results of this cell and the endogenously expressing Nectin-4 HT-1376 bladder cancer cells are shown in Figure 1.
  • mice Ten 8-week-old Balb/c mice were divided into two groups, using two methods of traditional immunization and rapid immunization respectively.
  • the CHO-huNectin4 S8 engineered cell line for traditional immunization was used as the immunizing agent, and the recombinant protein human Nectin4 (purchased from Nearshore protein, Cat: CJ19) is an immunizing agent.
  • mice Blood was collected from mice before immunization as a negative control.
  • the two immunizing agents were injected intraperitoneally, and the second and third immunizations were performed at 2 weeks intervals. Blood was collected one week after the third immunization to measure the titer.
  • the mice with high titer were selected for sprint immunization 3 days before the fusion.
  • mice The spleen and lymph nodes of the mice were aseptically taken, ground to prepare a cell suspension, filtered by a cell filter, and then red blood cells were lysed, combined and counted after lysis. Mix the B cells and SP20 myeloma cells at a ratio of 1:2. After centrifugation, wash the mixed cells twice with electrofusion solution, then resuspend the cells and adjust the density to about 1-2 ⁇ 10 7 /ml. Add the cell suspension to the electric shock cup for fusion.
  • the culture supernatant of hybridoma cells was taken for analysis by FACS, and the positive wells that could bind to CHO-huNECTIN4S8 cells stably expressing human Nectin4 antigen on the cell surface were screened and did not bind to blank CHOK1 cells.
  • the screened positive wells are single-celled by the limiting dilution method, and the subcloning is terminated when the positive results of the two consecutive subclones are 100%, and the obtained hybridoma cell line secretes only one antibody.
  • the obtained murine monoclonal antibody is named after the hybridoma cell line ID.
  • the total RNA of the cells is extracted according to the instructions of the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.); the hybridoma cells are extracted using 5 ⁇ PrimeScript RT Master Mix (Takara) Reverse transcription of total RNA into cDNA; use degenerate primers (Anke Krebber.
  • the heavy chain variable region sequence of the murine anti-human Nectin-4 monoclonal antibody and the publicly published heavy chain constant region sequence of the human monoclonal antibody IgG1 subclass (SEQ ID NO: 4) were spliced together to construct a mammal In the cell expression vector; the light chain variable region sequence of the murine anti-human Nectin-4 monoclonal antibody and the publicly published light chain constant region sequence of the human monoclonal antibody ⁇ subclass (SEQ ID NO: 5) are spliced together , Constructed into mammalian cell expression vectors.
  • the constructed anti-human Nectin-4 chimeric antibody heavy chain vector and light chain vector were paired and mixed, and HEK293 cells were transfected with PEI. The cell supernatant was collected about 7 days later, and the anti-human NECTIN4 chimeric antibody protein was purified by Mabselect.
  • murine antibody named xiIgG.
  • the humanized antibody is named "mouse antibody named hzmn", where m and n are the numbers of the humanized (hz) modified sequences (VH_hz and VL_hz) of the murine antibody VH and VL, respectively.
  • the ADC corresponding to the antibody is named as "antibody designation-E”.
  • the anti-human Nectin-4 control antibody Enfortumumab, the antibody of the present invention, or the ADC is diluted with a 2-fold gradient from the starting concentration of 100 nM to a total of 16 concentration points, and 10 ul of the antibody at each concentration point is added to a 384-well plate.
  • BT474 cells (breast cancer cells) expressing Nectin-4 on the cell surface by centrifugation at 100g room temperature for 5 minutes. Wash the cells once with PBS containing 0.5% BSA, centrifuge at 100g room temperature for 5 minutes, and resuspend the cells to a density of about 2x10 6 cells/ml , Take 10ul and add it to the wells of the 384-well plate that has been added with antibody. After incubating for 1 hour at 4°C, a fluorescently-labeled goat anti-human IgG secondary antibody was added. After continuing to incubate at 4°C for 1 hour, the average fluorescence reading of the cell population was analyzed by flow cytometry.
  • HEK293 cells expressing murine Nectin4 (NP_082169): HEK-muNectin4;
  • HEK293 cells expressing CynoNectin4 (SEQ ID NO: 6): HEK-cynoNectin4.
  • the His-tagged human NECTIN4 antigen is coupled to both the sensor chip CAP analysis channel and the control sample channel, and then the serially diluted antibody sample is passed through the analysis channel and the control sample channel (starting The concentration was 20 nM, and 8 concentration points were diluted 1:3, and the concentration point was set at 0.741 nm to repeat), and the light response value after the antibody antigen binding was measured. After instrument software fitting analysis (1:1 binding mode), the binding constant Kon, dissociation constant Koff, and affinity constant KD of the antibody are finally obtained.
  • Antibody (primary antibody): the antibody of the present invention.
  • CD111/Nectin-1/PVRL1 Antibody Rabbit Fab, 80244-RP01-100, Sinobiologics.
  • Anti-Nectin 2 antibody(ab233085) Rabbit antibody, Abcam.
  • Anti-Nectin 3 antibody (ab137961), Rabbit antibody, Abcam.
  • the antibody of the present invention and the control antibody have the same properties, can specifically recognize Nectin-4 antigen, have a dose-dependent binding effect, and do not have cross-binding activity with other proteins of the same family of Nectin-4. .
  • test antibody FBS
  • test antibody binding antigen anti-huIgG Fab monoclonal antibody
  • anti-huIgG Fab monoclonal antibody Sigma, I5260-1ML
  • HRP-labeled goat anti-human IgG secondary antibody Jackson, code: 109-035-098.
  • the result proves that after the antibody of the present invention is incubated at 37°C for 21 days, there is no change in the effective antibody content, that is, it can stably exist at 37°C for more than 21 days.
  • mice Female Balb/C mice, 3 mice/group, respectively in the tail vein or abdominal cavity, 200ug/mouse;
  • the antibody diluent dilutes the antibody standard, and the dilution of the standard is still adjusted according to the preliminary experiment, so that the standard can be fitted to a linear curve (if appropriate software is available, an S-shaped curve can also be fitted).
  • the lower limit of detection is 9.77ng/ml.

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Abstract

提供一种结合人Nectin-4的抗体分子或其片段，该抗体通过杂交瘤筛选和人源化技术获得，用于预防或治疗癌症，可作为临床先导药物分子。

Description

一种抗Nectin-4的抗体及其应用
相关申请的交叉引用
本专利申请要求于2020年4月21日提交的申请号为CN202010320420.3的中国发明专利申请的优先权权益，在此将其全部内容引入作为参考。
技术领域
本发明属于抗体药物领域，具体而言，本发明涉及针对人Nectin-4的抗体及其用于制备药物的用途。
背景技术
Nectin-4(又名PVRL4，脊髓灰质炎病毒受体样分子4)是一个大小为66KD的一型跨膜糖蛋白，属于Nectin家族的IG超家族蛋白分子，其细胞外结构域由三个Ig样结构域(VCC型)组成，与钙粘蛋白一起参与粘附连接的形成和维持。
Nectin-4与多种肿瘤细胞的发生，发展有着密切联系。已发现Nectin-4在多种实体瘤、尤其是膀胱癌中均有表达；在乳腺癌、卵巢癌和肺癌中，作为肿瘤相关抗原，分别占乳腺癌50％、卵巢癌49％和肺癌86％的组织表达检出率，并且在这些上皮恶性肿瘤的发生、侵袭和转移中起关键作用。因此，Nectin-4已成为许多实体瘤诊断和治疗的重要靶点。
目前针对Nectin-4的主要药物为Enfortumab vedotin，这是一种抗体偶联药物，由抗Nectin-4的单克隆抗体和细胞杀伤药物monomethyl auristatin E(MMAE)偶联而成，主治膀胱癌，尤其是尿路上皮癌，已在2018年3月获FDA突破性疗法认定。此外另有研究显示，粘附因子Nnectin-4不仅可作为乳腺癌的有效预后因子，同时可作为三阴性乳腺癌(TNBC)患者的有效治疗靶点；体内外研究证实，抗Nectin-4的抗体偶联药物(ADC)对于局部、转移性TNBC具有较好疗效。
发明内容
本发明要解决的技术问题是，通过杂交瘤筛选和人源化技术，获得特异性结合Nectin-4的高亲和力抗体，其中通过人源化改造，获得全人抗体序列。
针对上述技术问题，本发明的目的是提供一种特异性结合Nectin-4、特 别是人Nectin-4的抗体分子或其片段，并提供其用途。其中，本发明所述的抗体分子的片段涵盖抗体的各种功能性片段，例如其抗原结合部分，如Fab、F(ab’) 2或scFv片段。
本发明的技术方案如下：
一方面，本发明提供一种抗体分子或其片段，所述抗体分子或其片段包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区和轻链可变区分别包含选自以下的重链CDR和轻链CDR的组合：
(1)依次示于SEQ ID NO：35、39、43的CDR-H1(GYTFTTY)、CDR-H2(YPGNVN)、CDR-H3(GLYYFDY)；和，依次示于SEQ ID NO：45、47、49的CDR-L1(KASQSVSNDVA)、CDR-L2(YASNRYT)、CDR-L3(QQDYSSPYT)；
(2)依次示于SEQ ID NO：36、40、43的CDR-H1(GYTFTTYYIH)、CDR-H2(WIYPGNVNTK)、CDR-H3(GLYYFDY)；和，依次示于SEQ ID NO：45、47、49的CDR-L1(KASQSVSNDVA)、CDR-L2(YASNRYT)、CDR-L3(QQDYSSPYT)；
(3)依次示于SEQ ID NO：37、41、43的CDR-H1(TYYIH)、CDR-H2(WIYPGNVNTKYNEKFKG)、CDR-H3(GLYYFDY)；和，依次示于SEQ ID NO：45、47、49的CDR-L1(KASQSVSNDVA)、CDR-L2(YASNRYT)、CDR-L3(QQDYSSPYT)；
(4)依次示于SEQ ID NO：38、42、44的CDR-H1(TTYYIH)、CDR-H2(WIGWIYPGNVNTK)、CDR-H3(ARGLYYFD)；和，依次示于SEQ ID NO：46、48、50的CDR-L1(SNDVAWY)、CDR-L2(LLIYYASNRY)、CDR-L3(QQDYSSPY)；
(5)依次示于SEQ ID NO：51、55、59的CDR-H1(GFSLIDY)、CDR-H2(WGDGK)、CDR-H3(QGGLLFYAMDY)；和，依次示于SEQ ID NO：61、63、65的CDR-L1(KSSQSLLNSYSQKNYLA)、CDR-L2(FASTRES)、CDR-L3(QQHYNTPFT)；
(6)依次示于SEQ ID NO：52、56、59的CDR-H1(GFSLIDYGVS)、CDR-H2(VIWGDGKIY)、CDR-H3(QGGLLFYAMDY)；和，依次示于SEQ ID NO：61、63、65的CDR-L1(KSSQSLLNSYSQKNYLA)、CDR-L2(FASTRES)、CDR-L3(QQHYNTPFT)；
(7)依次示于SEQ ID NO：53、57、59的CDR-H1(DYGVS)、CDR-H2 (VIWGDGKIYYNSVLKS)、CDR-H3(QGGLLFYAMDY)；和，依次示于SEQ ID NO：61、63、65的CDR-L1(KSSQSLLNSYSQKNYLA)、CDR-L2(FASTRES)、CDR-L3(QQHYNTPFT)；
(8)依次示于SEQ ID NO：54、58、60的CDR-H1(IDYGVS)、CDR-H2(WLGVIWGDGKIY)、CDR-H3(AKQGGLLFYAMD)；和，依次示于SEQ ID NO：62、64、66的CDR-L1(LNSYSQKNYLAWY)、CDR-L2(LLIYFASTRE)、CDR-L3(QQHYNTPF)；
(9)依次示于SEQ ID NO：51、55、59的CDR-H1(GFSLIDY)、CDR-H2(WGDGK)、CDR-H3(QGGLLFYAMDY)；和，依次示于SEQ ID NO：67、63、65的CDR-L1(KSSQSLLNTYSQKNYLA)、CDR-L2(FASTRES)、CDR-L3(QQHYNTPFT)；
(10)依次示于SEQ ID NO：51、68、59的CDR-H1(GFSLIDY)、CDR-H2(WGDAK)、CDR-H3(QGGLLFYAMDY)；和，依次示于SEQ ID NO：67、63、65的CDR-L1(KSSQSLLNTYSQKNYLA)、CDR-L2(FASTRES)、CDR-L3(QQHYNTPFT)；
(11)依次示于SEQ ID NO：51、69、59的CDR-H1(GFSLIDY)、CDR-H2(WGGGK)、CDR-H3(QGGLLFYAMDY)；和，依次示于SEQ ID NO：67、63、65的CDR-L1(KSSQSLLNTYSQKNYLA)、CDR-L2(FASTRES)、CDR-L3(QQHYNTPFT)；
(12)依次示于SEQ ID NO：70、74、78的CDR-H1(GYTFTSY)、CDR-H2(YPGNAN)、CDR-H3(SVYYFDY)；和，依次示于SEQ ID NO：45、80、49的CDR-L1(KASQSVSNDVA)、CDR-L2(YASNRNT)、CDR-L3(QQDYSSPYT)；
(13)依次示于SEQ ID NO：71、75、78的CDR-H1(GYTFTSYYIH)、CDR-H2(WIYPGNANNK)、CDR-H3(SVYYFDY)；和，依次示于SEQ ID NO：45、80、49的CDR-L1(KASQSVSNDVA)、CDR-L2(YASNRNT)、CDR-L3(QQDYSSPYT)；
(14)依次示于SEQ ID NO：72、76、78的CDR-H1(SYYIH)、CDR-H2(WIYPGNANNKYNENFKG)、CDR-H3(SVYYFDY)；和，依次示于SEQ ID NO：45、80、49的CDR-L1(KASQSVSNDVA)、CDR-L2(YASNRNT)、CDR-L3(QQDYSSPYT)；
(15)依次示于SEQ ID NO：73、77、79的CDR-H1(TSYYIH)、CDR-H2 (WIGWIYPGNANNK)、CDR-H3(ARSVYYFD)；和，依次示于SEQ ID NO：46、81、50的CDR-L1(SNDVAWY)、CDR-L2(LLIYYASNRN)、CDR-L3(QQDYSSPY)；
(16)依次示于SEQ ID NO：82、86、90的CDR-H1(GYSFTDY)、CDR-H2(NPNNGN)、CDR-H3(EDRYAFAY)；和，依次示于SEQ ID NO：92、94、96的CDR-L1(RASQSVSTSSYTYMH)、CDR-L2(YASNLES)、CDR-L3(QHTWEIPYT)；
(17)依次示于SEQ ID NO：83、87、90的CDR-H1(GYSFTDYYMH)、CDR-H2(RVNPNNGNTL)、CDR-H3(EDRYAFAY)；和，依次示于SEQ ID NO：92、94、96的CDR-L1(RASQSVSTSSYTYMH)、CDR-L2(YASNLES)、CDR-L3(QHTWEIPYT)；
(18)依次示于SEQ ID NO：84、88、90的CDR-H1(DYYMH)、CDR-H2(RVNPNNGNTLYNQKFRG)、CDR-H3(EDRYAFAY)；和，依次示于SEQ ID NO：92、94、96的CDR-L1(RASQSVSTSSYTYMH)、CDR-L2(YASNLES)、CDR-L3(QHTWEIPYT)；
(19)依次示于SEQ ID NO：85、89、91的CDR-H1(TDYYMH)、CDR-H2(WIGRVNPNNGNTL)、CDR-H3(AREDRYAFA)；和，依次示于SEQ ID NO：93、95、97的CDR-L1(STSSYTYMHWY)、CDR-L2(LLIKYASNLE)、CDR-L3(QHTWEIPY)。
按照本领域公知的抗体中重链可变区、轻链可变区的结构域组成，本发明抗体分子或其片段中的重链可变区或轻链可变区以FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的顺序包含上述结构域组分，其中FR为框架区。
优选地，在本发明提供的抗体分子或其片段中，所述重链可变区包含示于SEQ ID NO：7、9或10的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：8、11或12的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；或者，
所述重链可变区包含示于SEQ ID NO：13、15、16、18、19、20或21的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：14、22、23或25的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；或者
所述重链可变区包含示于SEQ ID NO：27、29或30的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：28、31或32的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；或者，
所述重链可变区包含示于SEQ ID NO：33的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：34的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
根据本发明的具体实施方式，所述抗体分子或其片段中的重链可变区和轻链可变区选自以下氨基酸序列的组合：
(1)如SEQ ID NO：7所示的氨基酸序列或与如SEQ ID NO：7所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：8所示的氨基酸序列或与如SEQ ID NO：8所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(2)如SEQ ID NO：9所示的氨基酸序列或与如SEQ ID NO：9所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：11所示的氨基酸序列或与如SEQ ID NO：11所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(3)如SEQ ID NO：9所示的氨基酸序列或与如SEQ ID NO：9所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：12所示的氨基酸序列或与如SEQ ID NO：12所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(4)如SEQ ID NO：10所示的氨基酸序列或与如SEQ ID NO：10所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：11所示的氨基酸序列或与如SEQ ID NO：11所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(5)如SEQ ID NO：10所示的氨基酸序列或与如SEQ ID NO：10所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：12所示的氨基酸序列或与如SEQ ID NO：12所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(6)如SEQ ID NO：13所示的氨基酸序列或与如SEQ ID NO：13所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：14所示 的氨基酸序列或与如SEQ ID NO：14所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(7)如SEQ ID NO：16所示的氨基酸序列或与如SEQ ID NO：16所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：22所示的氨基酸序列或与如SEQ ID NO：22所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(8)如SEQ ID NO：16所示的氨基酸序列或与如SEQ ID NO：16所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：23所示的氨基酸序列或与如SEQ ID NO：23所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(9)如SEQ ID NO：16所示的氨基酸序列或与如SEQ ID NO：16所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：25所示的氨基酸序列或与如SEQ ID NO：25所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(10)如SEQ ID NO：19所示的氨基酸序列或与如SEQ ID NO：19所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：25所示的氨基酸序列或与如SEQ ID NO：25所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(11)如SEQ ID NO：21所示的氨基酸序列或与如SEQ ID NO：21所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：25所示的氨基酸序列或与如SEQ ID NO：25所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(12)如SEQ ID NO：27所示的氨基酸序列或与如SEQ ID NO：27所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：28所示的氨基酸序列或与如SEQ ID NO：28所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(13)如SEQ ID NO：29所示的氨基酸序列或与如SEQ ID NO：29所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：32所示的氨基酸序列或与如SEQ ID NO：32所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(14)如SEQ ID NO：30所示的氨基酸序列或与如SEQ ID NO：30所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：32所示 的氨基酸序列或与如SEQ ID NO：32所示的氨基酸序列具有至少75％同一性的氨基酸序列；或者
(15)如SEQ ID NO：33所示的氨基酸序列或与如SEQ ID NO：33所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：34所示的氨基酸序列或与如SEQ ID NO：34所示的氨基酸序列具有至少75％同一性的氨基酸序列。
基于本发明提供的上述重链可变区或轻链可变区的具体氨基酸序列，本领域技术人员可以常规地确定其中包含的重链CDR和轻链CDR的氨基酸序列，以本领域其他已知方法确定得到的重轻链CDR及其组合也被涵盖在本发明的范围内。
本发明提供的所述抗体分子或其片段结合脊髓灰质炎病毒受体样分子4(Nectin-4)，优选哺乳动物Nectin-4，更优选灵长类动物Nectin-4，进一步优选人或猴Nectin-4，特别是人Nectin-4。
优选地，所述抗体分子为鼠源抗体、嵌合抗体或者完全或部分人源化抗体；所述片段为所述抗体分子的能够特异性结合Nectin-4的任何片段，例如scFv、dsFv、(dsFv) 2、Fab、Fab′、F(ab′) 2或Fv片段。
优选地，所述抗体分子为单克隆抗体或单链抗体。
优选地，所述抗体分子或其片段还包含人或鼠的恒定区，优选包含鼠或人的重链恒定区(CH)和/或轻链恒定区(CL)；优选地，所述抗体分子或其片段包含重链和轻链，例如两条重链和轻链。更优选地，所述抗体分子或其片段包含IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区。
根据本发明的具体实施方式，本发明提供的抗体分子为单克隆抗体，优选为人源化的单克隆抗体；优选地，所述单克隆抗体的重链恒定区为IgG1型，轻链恒定区为κ型。例如，所述单克隆抗体的重链恒定区包含示于SEQ ID NO：4的氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列；所述单克隆抗体的轻链恒定区包含示于SEQ ID NO：5的氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列。
在本发明的上下文中，“至少75％同一性”为75％至100％之间的任何百分比数字的同一性，例如75％、80％、85％、90％，甚至91％、92％、93％、94％、95％、96％、97％、98％或99％同一性。
另一方面，本发明提供一种核酸分子，其包含编码本发明所述的抗体分子或其片段中包含的轻链可变区、重链可变区、重链或轻链的核苷酸序列。
本发明的核酸分子可以被克隆到载体中，进而转化或转染宿主细胞。因此，还一方面，本发明提供一种载体，其包含本发明的核酸分子。所述载体可以为真核表达载体、原核表达载体、人工染色体及噬菌体载体等。
本发明的载体或核酸分子可以用于转化或转染宿主细胞或者以任何方式进入宿主细胞内，用于保存或表达抗体等目的。因此，又一方面，本发明提供一种宿主细胞，所述宿主细胞包含本发明的核酸分子和/或载体，或者所述宿主细胞被本发明的核酸分子和/或载体转化或转染。宿主细胞可以是任何原核或真核细胞，例如细菌或昆虫、真菌、植物或动物细胞。
本发明提供的抗体分子可以利用本领域已知的任何方法获得。例如，可以先由本发明提供的核酸分子获得所述抗体的重链可变区和/或轻链可变区，或者获得所述抗体分子的重链和/或轻链，然后与所述抗体分子的任选其他结构域组装成抗体；或者，在允许本发明提供的宿主细胞表达所述抗体分子的重链可变区和/或轻链可变区或者所述抗体分子的重链和/或轻链以组装成所述抗体的情况下，培养所述宿主细胞。任选地，所述方法还包括回收产生的抗体分子的步骤。
本发明提供的抗体分子或其片段、核酸分子、载体、宿主细胞或融合蛋白可以被包含在组合物中，更特别地被包含在药物制剂中，从而根据实际需要用于各种目的。因此，在又一方面，本发明还提供一种组合物，所述组合物包含本发明提供的抗体分子或其片段、核酸分子、载体和/或宿主细胞。优选地，所述组合物为药物组合物，其可选地还包含药学上可接受的载体、辅料或赋形剂。
再一方面，本发明还提供所述抗体分子或其片段、核酸分子、载体、宿主细胞和/或组合物在制备用于检测或诊断疾病或障碍的试剂中的用途。。
由此相应地，本发明还提供一种检测或诊断疾病或障碍的方法，所述方法包括使本发明的抗体分子或其片段、核酸分子、载体、宿主细胞和/或组合物与来自受试者的样品相接触。其中，所述受试者为哺乳类动物，优选灵长类动物，更优选人。
再一方面，本发明还提供所述抗体分子或其片段在制备抗体药物偶联物中的用途。
由此相应地，本发明提供一种抗体药物偶联物，其由本发明的抗体分子或其片段与细胞毒性部分偶联形成。
优选地，所述细胞毒性部分为微管蛋白抑制剂、拓扑异构酶抑制剂或 DNA结合剂。优选地，所述微管蛋白抑制剂选自美登素衍生物、Monomethyl auristatin E(MMAE)、Monomethylauristatin F(MMAF)、Monomethyl Dolastatin 10、Tubulysin类衍生物、Cryptophycin类衍生物和Taltobulin。优选地，所述拓扑异构酶抑制剂选自阿霉素(Doxorubicin)代谢产物PNU-159682衍生物和伊立替康(irinotecan，CPT-11)代谢产物SN38衍生物。优选地，所述DNA结合剂选自PBD类衍生物和Duocarmycin类衍生物。
再一方面，本发明还提供所述抗体分子或其片段、核酸分子、载体、宿主细胞、组合物和/或抗体药物偶联物在制备预防或治疗疾病或障碍的药物中的用途。
另一方面，本发明还提供一种预防或治疗疾病或障碍的方法，所述方法包括给有此需要的受试者施用本发明的抗体分子或其片段、核酸分子、载体、宿主细胞、组合物和/或抗体药物偶联物。其中，所述受试者为哺乳类动物，更优选人。
由此相应地，又一方面，本发明提供一种试剂盒，所述试剂盒包括本发明的抗体分子或其片段、核酸分子、载体、宿主细胞、组合物和/或抗体药物偶联物。所述试剂盒可以用于治疗、检测或诊断用途，例如治疗、检测或诊断疾病或障碍。
根据本发明提供的各个技术方案，所述抗体分子或其片段、核酸分子、载体、宿主细胞、组合物和/或抗体药物偶联物可以用于与Nectin-4高表达相关的疾病或障碍的预防、治疗、检测或诊断中。优选地，所述疾病或障碍为Nectin-4高表达的肿瘤或癌症，特别是实体肿瘤。例如，所述疾病或障碍为膀胱癌、胰腺癌、乳腺癌(包括三阴性亚型和基底亚型)、非小细胞肺癌、胃癌、食管癌、卵巢癌等；特别是膀胱癌、乳腺癌、卵巢癌或肺癌。
相对于现有技术，本发明通过杂交瘤筛选和人源化技术，获得特异性结合Nectin-4的高亲和力抗体，其中通过人源化改造，获得全人抗体序列。并且，通过对该分子进行了理化性质和细胞学活性研究，确认得到了有效的临床先导药物分子序列。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示了抗原表达细胞株的流式细胞术鉴定结果，其中小图1A：HT-1376膀胱癌细胞，小图1B：CHO-huNectin4S8细胞。
图2显示了杂交瘤细胞培养上清的FACS结合活性测定结果，其中小图2A：第一轮筛选，小图2B：第二轮筛选。
图3显示了抗体与表达BT474细胞的FACS结合实验结果。
图4显示了抗体的BT474细胞内吞活性实验结果。
图5显示了抗体与表达huNectin4、muNectin4、cynoNectin4的不同细胞的FACS结合实验结果。
图6显示了抗体与Nectin蛋白质家族成员的结合实验结果，其中小图6A：Nectin-1，小图6B：Nectin-2，小图6C：Nectin-3，小图6D：Nectin-4。
图7显示了抗体的猴血清稳定性实验结果，其中小图7A：对照抗体Enfortumab，小图7B：42D20 hz10。
图8显示了抗体的小鼠体内代谢情况，其中小图8A：对照抗体Enfortumab，小图8B：42D20 hz10。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。其中：
对照抗体Enfortumab的重链氨基酸序列示于SEQ ID NO：1，轻链氨基酸序列示于SEQ ID NO：2。
本发明的抗体序列见附表I至附表IV。
抗原NECTIN4重组蛋白(序列号：NP_002178.2，32aa-349aa)，示于SEQ ID NO：3。
实施例1 对照抗体的合成与表达
将全合成的Enfortumab抗体轻链可变区和重链可变区基因分别克隆至装有人-kappa轻链恒定区和人IgG1重链恒定区编码基因上游的真核表达载体pCDNA3.1中，获得Enfortumab轻、重链表达质粒，转入大肠杆菌扩增，分离获得大量含Enfortumab抗体轻链(SEQ ID NO：2)和重链(SEQ ID NO：1)的质粒，将二者与聚乙烯亚胺(PEI)混合后共转染入HEK293细胞中。细胞转染5-6天，取培养上清，利用Mabselect亲和层析柱对表达上清进行纯化，获得Enfortumab抗体。
实施例2 Nectin-4抗原表达细胞株的制备
通过PCR的方法，从含有Nectin-4 cDNA的载体(北京义翘神州，Cat：HG19771-UT)中克隆出Nectin-4基因的阅读框，并将Nectin-4基因的阅读框通过酶切的方法克隆入含有谷氨酰氨合成酶(GS)筛选基因的稳定表达载体中，电转染(Nucleofector IIb，Lonza)悬浮培养的CHO-K1细胞，将转染后的细胞置于含有50μM MSX(Sigma，Cat：M5379)的CD CHO AGTTM培养基(Gibco，Cat：12490-025)中，接种于96孔细胞培养板，37℃，5％CO 2静置培养2-3周，通过MSX加压筛选，从镜下预筛获得9个细胞生长孔，并将其放大培养至24孔细胞培养板，最终通过流式细胞分析(FACS)挑选出抗原高表达克隆S8，进行放大培养和冻存。
将该高表达克隆S8命名为CHO-huNectin4 S8，该细胞与内源表达Nectin-4的HT-1376膀胱癌细胞的比较鉴定结果见图1。
实施例3 杂交瘤细胞的筛选和鉴定
1.小鼠免疫
10只8周龄Balb/c小鼠分成两组，分别采用传统免疫和快速免疫两个方法，其中传统免疫用CHO-huNectin4 S8工程细胞株为免疫剂，快速免疫用重组蛋白人Nectin4(购自近岸蛋白，Cat：CJ19)为免疫剂。
小鼠免疫前采血作为阴性对照。两种免疫剂分别腹腔注射，间隔2周进行第二、三次免疫，第三次免疫后一周采血测效价，选取效价高的小鼠在融合前3天进行冲刺免疫。
2.融合筛选
将SP20骨髓瘤细胞复苏、扩大培养至一定数量级。融合前一天给细胞换液，以保证融合时细胞生长状态良好。融合当天收集骨髓瘤细胞，离心后用基础培养基悬浮，计数待用。
分别无菌取小鼠的脾脏、淋巴结，研磨制备细胞悬液，细胞过滤器过滤，然后进行红细胞裂解，裂解后合并、计数。按B细胞与SP20骨髓瘤细胞的数量比为1：2进行混合，离心后用电融合液洗两遍混合的细胞，再重悬细胞并将密度调整至1-2×10 7/ml左右。将细胞悬液加入电击杯进行融合，融合后加入到完整培养基中，放入到37℃、8％CO 2培养箱中恢复30-240分钟，然后加入HAT培养基铺至384孔板中培养，第5天补液，第7天换成HT培养 基，第8-10天进行如下的阳性杂交瘤筛选。
取杂交瘤细胞培养上清用FACS进行分析，筛选得到能够结合细胞表面稳定表达人Nectin4抗原的CHO-huNECTIN4S8细胞，同时不结合空白CHOK1细胞的阳性孔。通过有限稀释法将筛选到的阳性孔单细胞化，待连续两次亚克隆的阳性都为100％时结束亚克隆，得到的杂交瘤细胞株只分泌一个抗体。
杂交瘤细胞培养上清的FACS结合活性测定结果见图2和表1至表3。
表1.第一轮筛选出的克隆
ID FACS FACS(*10 5)
5M21 1731598.512 173.1599
7I18 900458.7143 90.04587
26G13 493254.2857 49.32543
29B12 685057.4296 68.50574
30J3 440992.4 44.09924
表2.第二轮筛选出的克隆
ID FACS FACS(*10 5)
4A15 221093.1035 22.1
21N4 264347.4081 26.4
28J3 208131.2025 20.8
32A13 677419.4598 67.7
38D17 266536.8786 26.7
41024 1749182.695 174.9
42D20 533378.824 53.3
45F2 393898.7755 39.4
49C23 1510386.33 151.0
50I15 1299424.85 129.9
表3.第三轮筛选出的克隆
Figure PCTCN2021088661-appb-000001
Figure PCTCN2021088661-appb-000002
得到的鼠源单克隆抗体以杂交瘤细胞株ID命名。
实施例4 鼠源单克隆抗体的可变区序列鉴定
将分泌抗人Nectin-4抗体的杂交瘤细胞扩大培养后，按照RNAfast200试剂盒(上海飞捷生物技术有限公司)说明书步骤提取细胞总RNA；利用5×PrimeScript RT Master Mix(Takara)将杂交瘤细胞总RNA反转录成cDNA；使用简并引物(Anke Krebber.1997)和Extaq PCR试剂(Takara)扩增抗体轻链可变区IgVL(κ)和重链可变区VH序列；利用PCR clean-up Gel extraction试剂盒(Macherey-Nagel公司)纯化PCR扩增产物；按照pClone007 Simple Vector Kit试剂盒(擎科生物科技有限公司)说明书将扩增PCR产物连接至T载体并转化大肠杆菌感受态细胞，菌株扩增、抽提质粒后进行DNA测序获得单克隆抗体可变区序列。
实施例5 嵌合抗体的制备
将鼠源抗人Nectin-4单克隆抗体的重链可变区序列和公开发表的人单克隆抗体IgG1亚类的重链恒定区序列(SEQ ID NO：4)拼接在一起，构建到哺乳动物细胞表达载体中；将鼠源抗人Nectin-4单克隆抗体的轻链可变区序列和公开发表的人单克隆抗体κ亚类的轻链恒定区序列(SEQ ID NO：5)拼接在一起，构建到哺乳动物细胞表达载体中。构建好的抗人Nectin-4嵌合抗体的重链载体和轻链载体配对混合，使用PEI转染HEK293细胞，约7天后收集细胞上清，使用Mabselect纯化得到抗人NECTIN4嵌合抗体蛋白。
得到的嵌合抗体在本文以“鼠源抗体命名xiIgG”表示。
实施例6 鼠源抗体的人源化与人源化抗体制备
综合抗体编码方案，确定鼠源抗体的重链和轻链的6个抗原互补决定簇(CDR)的氨基酸序列区域及支撑抗体保守三维构象的框架区域(framework region)。随后通过分析搜索已知人源抗体序列，选择与鼠源抗体最为相似接近的人源抗体重链可变区序列，如IGHV1|IGHJ4*01，选择其抗体框架区 序列作为模板，将鼠源抗体重链CDR与人源抗体框架区结合，最终生成人源化抗体重链可变区序列。同样过程，生成人源化抗体轻链可变区序列。
鼠源抗体CDR直接移植至人框架区的抗体常出现结合活性急剧下降，因此需要将框架区个别氨基酸从人源的改回鼠源的。确定回复突变位点，一是对照设计好的人源化抗体序列和原始的鼠源抗体序列，检查有哪些氨基酸的不同；二是检查这些氨基酸是否对支持抗体结构起重要作用或者对与抗原的结合起重要作用。人源化设计后的序列的同时需要检查是否有一些潜在的翻译后修饰位点，如N(天冬酰胺)糖基化位点、N脱酰胺化位点、D(天冬氨酸)异构化位点等。
将人源化的重链可变区和轻链可变区两两组合，参考实施例5所述的嵌合抗体的制备，获得人源化抗体。将人源化抗体命名为“鼠源抗体命名hzmn”，其中m和n分别为鼠源抗体VH和VL的人源化(hz)改造序列(VH_hz和VL_hz)编号。
实施例7 抗体药物缀合物(ADC)的制备
在pH为7.4的PBS中，2.0-2.6当量TECP还原抗体2小时后，将vcMMAE的DMA溶液加入到还原后的抗体溶液中(vcMMAE和抗体的摩尔比为6：1)，2-8℃搅拌1小时后超滤换液除去DMA和小分子残留。紫外分光光度计测定偶联物在248nm～280nm处的吸光值，计算得到偶联物的浓度。样品分装到冻存管中-80℃保存。样品的DAR值(4.0±1)由HPLC-HIC测定。
将抗体对应的ADC以“抗体命名-E”来命名。
实施例8 体外细胞结合实验
将抗人Nectin-4对照抗体Enfortumumab、本发明的抗体或ADC从100nM的起始浓度开始做2倍的梯度倍比稀释，共16个浓度点，各个浓度点的抗体取10ul加入384孔板。
100g室温离心5分钟收集细胞表面表达Nectin-4的BT474细胞(乳腺癌细胞)，用含0.5％BSA的PBS洗涤细胞一次，100g室温离心5分钟，重悬细胞为密度约2x10 6个细胞/ml，取10ul加入到已加抗体的384孔板的孔中。4℃孵育1小时后，加入荧光标记的羊抗人IgG二抗。继续于4℃孵育1小时后，用流式细胞仪分析细胞群的平均荧光读值。
本发明的鼠抗分子与BT474细胞的FACS结合实验结果见图3中的图3A 和表4、表5。
表4.鼠源单克隆抗体与BT474细胞的结合
Figure PCTCN2021088661-appb-000003
表5.鼠源单克隆抗体与BT474细胞的结合
Figure PCTCN2021088661-appb-000004
本发明的人源化改造分子与BT474细胞的FACS结合实验结果见图3中的图3B、图3C和表6、表7。
表6.人源化抗体与BT474细胞的结合
Figure PCTCN2021088661-appb-000005
表7.人源化抗体与BT474细胞的结合
Figure PCTCN2021088661-appb-000006
Figure PCTCN2021088661-appb-000007
本发明的ADC与BT474细胞的FACS结合实验结果见图3中的图3D和表8。
表8.抗体药物缀合物与BT474细胞的结合
Figure PCTCN2021088661-appb-000008
实施例9 抗体的体外细胞学实验
9.1 BT474细胞内吞实验
1.收集BT474细胞，1200rpm离心8分钟，DPBS(Gibco Cat.：14190-136)洗两次。
2. 1E5每孔种细胞，抗体从10ug/ml浓度起，按照1∶2浓度梯度稀释做7个点，最后一点为空白孔，将混合物冰上孵育1h。
3.冰PBS洗两次，1200rpm离心8分钟，用补充L-glutamine和HEPES的RPMI 1640培养基重悬细胞，根据时间点等量分成相应的份数，置于37℃孵育不同的时间段，其中一份一直置于冰上，做0点无内吞对照，设未加抗体组做NC对照。
4.用pH 2.7的柠檬酸洗3.5分钟，用1M pH 9.5的Tris-HCl溶液中和，用PBS洗两次，用适量的1％BSA-PBS重悬，上IQplus仪器检测。
5.用GraphPad Prism软件分析处理数据。
本发明的鼠抗分子的BT474细胞内吞活性实验结果见图4中的图4A和表9。
表9.鼠源单克隆抗体的BT474细胞内吞活性
  内吞效率(％)
Enfortumab 63
mIgG 5M21 69
mIgG 26G13 37
mIgG 29B12 57
mIgG 30J3 56
mIgG 7I18 66
同型对照 16
本发明的人源化改造分子的BT474细胞内吞活性实验结果见图4中的图4B、4C和表10、表11。
表10.人源化抗体的BT474细胞内吞活性
  内吞效率(％)
Enfortumab 62.4
5M21 xiIgG 56.9
5M21 hz00 54.8
5M21 hz01 62.2
5M21 hz10 64.8
5M21 hz11 64.4
表11.人源化抗体的BT474细胞内吞活性
Figure PCTCN2021088661-appb-000009
Figure PCTCN2021088661-appb-000010
本发明的ADC的BT474细胞内吞活性实验结果见图4中的图4D和表12。
表12.抗体药物缀合物的BT474细胞内吞活性
  内吞效率(％)
Enfortumab-E 58.9
42D20 hz10-E 74
42D20 hz43-E 68.9
42D20 hz44-E 71.1
42D20 hz63-E 67.9
42D20 hz64-E 66.6
20M12 xiIgG-E 67
20M12 hz01-E 56.6
20M12 hz11-E 58.4
同型对照 --
9.2 BT474细胞增殖抑制试验
培养表达Nectin4的乳腺癌细胞BT474，胰蛋白酶消化收集细胞，400g离心5分钟，弃上清。按照4000细胞每孔的密度种板，37℃，5％CO2培养24小时。待测抗体溶于含1％BSA的培养基，起始浓度为200ug/ml，3倍稀释，9个稀释浓度，包含0浓度，加100ul每个浓度的抗体至96孔板。将100ul稀释好的抗体样品加100ul细胞混合至96孔板中，37℃，5％CO2孵育120 小时。使用含1％BSA的培养基配制浓度为5uM的CCK-8，加20ul CCK-8至96孔板中，37℃，5％CO2孵育4小时。最后取出细胞板室温静置15min后，将细胞板充分振荡混匀。以450nm为检测波长读板。以裸抗和ADC样品工作浓度(ng/ml)为X轴，测得吸光度值为Y轴，用SoftMax Pro进行四参数拟合，得到裸抗样品与ADC样品的EC50值。
结果见表13、表14和表15。
表13.鼠抗的BT474细胞增殖抑制活性
  EC50(ng/ml) 浓度最高点细胞杀伤效果百分比
Enfortumab 3.367 43.3％
mIgG 5M21 42.34 33.5％
mIgG 29B12 387.0 33.2％
mIgG 30J3 16.88 52.8％
同型对照 N/A N/A
表14.ADC的BT474细胞增殖抑制活性
Figure PCTCN2021088661-appb-000011
表15.ADC的BT474细胞增殖抑制活性
Figure PCTCN2021088661-appb-000012
Figure PCTCN2021088661-appb-000013
实施例10 抗体交叉抗原活性分析-Facs
细胞：
CHO-huNectin4 S8；
表达鼠Nectin4(NP_082169)的HEK293细胞：HEK-muNectin4；和
表达Cyno Nectin4(SEQ ID NO：6)的HEK293细胞：HEK-cynoNectin4。
参照实施例8所述进行实验，结果分别见图5的图5A、5B和5C，以及表16、表17和表18。
表16.人源化抗体与CHO-huNectin4 S8细胞的结合
Figure PCTCN2021088661-appb-000014
表17.人源化抗体与HEK-muNectin4细胞的结合
Figure PCTCN2021088661-appb-000015
Figure PCTCN2021088661-appb-000016
表18.人源化抗体与HEK-cynoNectin4细胞的结合
Figure PCTCN2021088661-appb-000017
实施例11 抗体的体外结合亲和力和动力学实验
采用GE公司BIAcore仪器S200测定抗体抗原相互作用力。参考GE公司Biotin CAPture Kit操作说明，首先在传感芯片CAP分析通道和对照样品通道都偶联His标签的人NECTIN4抗原，然后在分析通道和对照样品通道一起流过梯度稀释的抗体样品(起始浓度20nM，1∶3稀释8个浓度点，并且设定0.741nm浓度点重复)，测定抗体抗原结合后发生的光反应值。经仪器软件拟合分析(1∶1结合模式)，最终得到抗体的结合常数Kon和解离常数Koff，以及亲和力常数KD。
结果见表19。
表19.抗体的结合亲和力和动力学结果
  ka(1/Ms) kd(1/s) KD(M)
Enfortumab 1.12E+06 5.52E-03 4.94E-09
42D20 xiIgG 7.90E+05 7.98E-04 1.01E-09
42D20 hz10 8.31E+05 9.46E-04 1.14E-09
42D20 hz11 1.19E+06 1.15E-03 9.60E-10
42D20 hz13 1.19E+06 1.08E-03 9.04E-10
42D20 hz63 8.73E+05 1.57E-03 1.80E-09
实施例12 抗体与同家族抗原蛋白的交叉结合活性验证
抗原：
human Nectin-1(C-6His)Novoprotein Cat#C492
human Nectin-2(C-6His)Novoprotein Cat#C440
human Nectin-3(C-6His)Novoprotein Cat#C630
human Nectin-4(C-6His)Novoprotein Cat#CJ19
抗体(一抗)：本发明的抗体；
对照抗体；
CD111/Nectin-1/PVRL1 Antibody，Rabbit Fab，80244-RP01-100，Sinobiologics.
Anti-Nectin 2 antibody(ab233085)，Rabbit antibody，Abcam.
Anti-Nectin 3 antibody(ab137961)，Rabbit antibody，Abcam.
二抗：
Goat anti-Rabbit IgG-Fc Secondary antibody(HRP)Cat#SSA003，Jackson Immuno.
用1ug/mi抗原包板，4℃过夜孵育，然后依次加入梯度稀释的抗体，最后加入HRP标记的二抗来检测A450的吸收值。结果见图6的图6A、6B、6C和6D。
从结果可知，本发明的抗体和对照抗体表现的性质相一致，都能特异性识别Nectin-4抗原，有剂量依赖的结合效应，而且并不与Nectin-4同家族的其他蛋白有交叉结合活性。
实施例13 抗体体外猴血清稳定性研究
实验材料：待测抗体，FBS，待测抗体结合抗原，anti-huIgG Fab单克隆抗体(Sigma，I5260-1ML)，HRP标记山羊抗人IgG二抗(Jackson，code：109-035-098)。
实验仪器：37℃培养箱，酶标仪。
实验步骤：
样品制备：
1)待测抗体调浓度至20ug/ml，过滤除菌，分装为250ul/管备用；
2)猴血清等体积加入分装好的待测抗体，即终浓度为50％血清浓度，10ug/ml抗体浓度；
3)共制备7个样品，封口膜封口，放置于37℃，全程需保持无菌；
4)分别于第0天、3天、7天、10天、14天、21天取样放置于4℃保存待测，其中第21天取样应至少在4℃放置一天。
检测方法：
1)分别PBS包被结合抗原、anti-IgG Fab单克隆抗体于96孔酶联板，0.2ug/ml，100ul/孔，4℃过夜；
2)配置所需试剂：
封闭液5％BSA+PBS
抗体稀释液5％BSA+PBS+50％FBS
酶联板洗涤液0.1％Tween+PBS
3)包被好的酶联板PBS清洗三遍，300ul/孔，洗去游离的未包被抗原；
4)加入封闭液，200ul/孔，37℃封闭1h；
5)待测抗体使用抗体稀释液稀释至2ug/ml，并3倍稀释共8个梯度；
6)倒掉封闭液，两种包被方法的酶联板分别加入稀释好的抗体，100ul/孔，于37℃孵育1h；
7)PBST洗板3遍；
8)1∶5000稀释二抗，加入洗涤好的酶联板中，100ul/孔，37℃孵育40min；
9)PBST洗板3遍；
10)TMB显色，100ul/孔，避光10min；
11)加入50ul 2M HCL终止，450nm读数。
结果处理：
根据ELISA显色值制定结合曲线，观察放置不同时间结合曲线的变化，评估放置后抗体结合活性稳定性。结果见图7的图7A、7B。
结果证明本发明抗体在37℃孵育21天以后，有效抗体含量没有任何变化，即能够在37℃条件下稳定存在21天以上。
实施例14 抗体小鼠体内药物代谢分析
实验材料：待测抗体，小鼠不同时间点采集血清，待测抗体的结合抗原人Nectin-4，anti-huIgG Fab单克隆抗体(Sigma，I5260-1ML)，HRP标记山羊抗人IgG二抗(Jackson，code：109-035-098)。
实验方法：
血清收集：
1)雌性Balb/C小鼠，3只/组，分别于尾静脉或者腹腔给药，200ug/只；
2)按照实验设计时间点尾静脉采血，收集血样，室温放置30min以上，4000rpm，15min收集血清，-20℃保存，为防止血清蒸发，最后血清收集体积尽量大于20ul；
3)最后一次收集血清至少于-20℃冻存24h。
检测方法：
1)分别PBS包被结合抗原、anti-IgG Fab单克隆抗体于96孔酶联板，0.2ug/ml，100ul/孔，4℃过夜；
2)配制所需试剂：
封闭液5％BSA+PBS
抗体稀释液5％BSA+PBS+20％空白小鼠血清
酶联板洗涤液0.1％Tween+PBS
3)包被好的酶联板PBS清洗三遍，300ul/孔；
4)加入封闭液，200ul/孔，37℃封闭1h；
5)起始血清使用封闭液稀释至合适浓度，用抗体稀释液稀释至合适的浓度范围，具体浓度的稀释倍数需根据预实验来调整，原则上使检测的血清最终显色值在标准品显色值范围内；
6)抗体稀释液稀释抗体标准品，标准品的稀释仍然根据预实验来调整，使标准品拟合出线性曲线(如有合适软件，也可拟合出S形曲线)。
7)倒掉酶联板中的封闭液，两种包被方法的酶联板分别加入稀释好的抗体标准品与待测血清，100ul/孔，于37℃孵育1h；
8)PBST洗板3遍；
9)1∶5000稀释二抗，加入洗涤好的酶联板中，100ul/孔，37℃孵育40min；
10)PBST洗板3遍；
11)TMB显色，100ul/孔，避光10min；
12)加入50ul 2M HCL终止，450nm读数。
结果见图8的图8A、8B和表20。
表20.抗体的小鼠体内代谢结果
Figure PCTCN2021088661-appb-000018
浓度：ug/ml
NA：低于检测下限，检测下限为9.77ng/ml。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。
Figure PCTCN2021088661-appb-000019
Figure PCTCN2021088661-appb-000020
Figure PCTCN2021088661-appb-000021
Figure PCTCN2021088661-appb-000022
Figure PCTCN2021088661-appb-000023
Figure PCTCN2021088661-appb-000024
Figure PCTCN2021088661-appb-000025
Figure PCTCN2021088661-appb-000026
Figure PCTCN2021088661-appb-000027

Claims (18)

  1. 一种抗体分子或其片段，所述抗体分子或其片段包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区和轻链可变区分别包含选自以下的重链CDR和轻链CDR的组合：
    (1)依次示于SEQ ID NO：35、39、43的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：45、47、49的CDR-L1、CDR-L2、CDR-L3；
    (2)依次示于SEQ ID NO：36、40、43的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：45、47、49的CDR-L1、CDR-L2、CDR-L3；
    (3)依次示于SEQ ID NO：37、41、43的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：45、47、49的CDR-L1、CDR-L2、CDR-L3；
    (4)依次示于SEQ ID NO：38、42、44的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：46、48、50的CDR-L1、CDR-L2、CDR-L3；
    (5)依次示于SEQ ID NO：51、55、59的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：61、63、65的CDR-L1、CDR-L2、CDR-L3；
    (6)依次示于SEQ ID NO：52、56、59的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：61、63、65的CDR-L1、CDR-L2、CDR-L3；
    (7)依次示于SEQ ID NO：53、57、59的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：61、63、65的CDR-L1、CDR-L2、CDR-L3；
    (8)依次示于SEQ ID NO：54、58、60的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：62、64、66的CDR-L1、CDR-L2、CDR-L3；
    (9)依次示于SEQ ID NO：51、55、59的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：67、63、65的CDR-L1、CDR-L2、CDR-L3；
    (10)依次示于SEQ ID NO：51、68、59的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：67、63、65的CDR-L1、CDR-L2、CDR-L3；
    (11)依次示于SEQ ID NO：51、69、59的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：67、63、65的CDR-L1、CDR-L2、CDR-L3；
    (12)依次示于SEQ ID NO：70、74、78的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：45、80、49的CDR-L1、CDR-L2、CDR-L3；
    (13)依次示于SEQ ID NO：71、75、78的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：45、80、49的CDR-L1、CDR-L2、CDR-L3；
    (14)依次示于SEQ ID NO：72、76、78的CDR-H1、CDR-H2、CDR-H3； 和，依次示于SEQ ID NO：45、80、49的CDR-L1、CDR-L2、CDR-L3；
    (15)依次示于SEQ ID NO：73、77、79的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：46、81、50的CDR-L1、CDR-L2、CDR-L3；
    (16)依次示于SEQ ID NO：82、86、90的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：92、94、96的CDR-L1、CDR-L2、CDR-L3；
    (17)依次示于SEQ ID NO：83、87、90的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：92、94、96的CDR-L1、CDR-L2、CDR-L3；
    (18)依次示于SEQ ID NO：84、88、90的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：92、94、96的CDR-L1、CDR-L2、CDR-L3；
    (19)依次示于SEQ ID NO：85、89、91的CDR-H1、CDR-H2、CDR-H3；和，依次示于SEQ ID NO：93、95、97的CDR-L1、CDR-L2、CDR-L3。
  2. 根据权利要求1所述的抗体分子或其片段，其特征在于，所述抗体分子或其片段中，所述重链可变区包含示于SEQ ID NO：7、9或10的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：8、11或12的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；或者，
    所述重链可变区包含示于SEQ ID NO：13、15、16、18、19、20或21的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：14、22、23或25的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；或者
    所述重链可变区包含示于SEQ ID NO：27、29或30的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：28、31或32的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；或者，
    所述重链可变区包含示于SEQ ID NO：33的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：34的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
  3. 根据权利要求1或2所述的抗体分子或其片段，其特征在于，所述抗体分子或其片段中的重链可变区和轻链可变区选自以下氨基酸序列的组合：
    (1)如SEQ ID NO：7所示的氨基酸序列或与如SEQ ID NO：7所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：8所示的 氨基酸序列或与如SEQ ID NO：8所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (2)如SEQ ID NO：9所示的氨基酸序列或与如SEQ ID NO：9所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：11所示的氨基酸序列或与如SEQ ID NO：11所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (3)如SEQ ID NO：9所示的氨基酸序列或与如SEQ ID NO：9所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：12所示的氨基酸序列或与如SEQ ID NO：12所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (4)如SEQ ID NO：10所示的氨基酸序列或与如SEQ ID NO：10所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：11所示的氨基酸序列或与如SEQ ID NO：11所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (5)如SEQ ID NO：10所示的氨基酸序列或与如SEQ ID NO：10所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：12所示的氨基酸序列或与如SEQ ID NO：12所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (6)如SEQ ID NO：13所示的氨基酸序列或与如SEQ ID NO：13所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：14所示的氨基酸序列或与如SEQ ID NO：14所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (7)如SEQ ID NO：16所示的氨基酸序列或与如SEQ ID NO：16所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：22所示的氨基酸序列或与如SEQ ID NO：22所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (8)如SEQ ID NO：16所示的氨基酸序列或与如SEQ ID NO：16所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：23所示的氨基酸序列或与如SEQ ID NO：23所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (9)如SEQ ID NO：16所示的氨基酸序列或与如SEQ ID NO：16所示的 氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：25所示的氨基酸序列或与如SEQ ID NO：25所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (10)如SEQ ID NO：19所示的氨基酸序列或与如SEQ ID NO：19所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：25所示的氨基酸序列或与如SEQ ID NO：25所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (11)如SEQ ID NO：21所示的氨基酸序列或与如SEQ ID NO：21所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：25所示的氨基酸序列或与如SEQ ID NO：25所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (12)如SEQ ID NO：27所示的氨基酸序列或与如SEQ ID NO：27所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：28所示的氨基酸序列或与如SEQ ID NO：28所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (13)如SEQ ID NO：29所示的氨基酸序列或与如SEQ ID NO：29所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：32所示的氨基酸序列或与如SEQ ID NO：32所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (14)如SEQ ID NO：30所示的氨基酸序列或与如SEQ ID NO：30所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：32所示的氨基酸序列或与如SEQ ID NO：32所示的氨基酸序列具有至少75％同一性的氨基酸序列；或者
    (15)如SEQ ID NO：33所示的氨基酸序列或与如SEQ ID NO：33所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO：34所示的氨基酸序列或与如SEQ ID NO：34所示的氨基酸序列具有至少75％同一性的氨基酸序列。
  4. 根据权利要求1至3中任一项所述的抗体分子或其片段，其特征在于，所述抗体分子或其片段结合脊髓灰质炎病毒受体样分子4(Nectin-4)，优选哺乳动物Nectin-4，更优选灵长类动物Nectin-4，进一步优选人或猴Nectin-4，特别是人Nectin-4。
  5. 根据权利要求1至4中任一项所述的抗体分子或其片段，其特征在于，所述抗体分子为鼠源抗体、嵌合抗体或者完全或部分人源化抗体；所述片段为所述抗体分子的scFv、dsFv、(dsFv) 2、Fab、Fab′、F(ab′) 2或Fv片段；
    优选地，所述抗体分子为单克隆抗体或单链抗体；
    优选地，所述抗体分子或其片段还包含恒定区，优选包含鼠或人的重链恒定区(CH)和/或轻链恒定区(CL)；优选地，所述抗体分子或其片段包含重链和轻链；
    更优选地，所述抗体分子或其片段包含IgG、IgA、IgM、IgD或IgE的重链恒定区和/或K或λ型轻链恒定区。
  6. 根据权利要求1至5中任一项所述的抗体分子或其片段，其特征在于，所述抗体分子为单克隆抗体，优选为人源化的单克隆抗体；
    优选地，所述单克隆抗体的重链恒定区为IgG1型，轻链恒定区为K型。
  7. 一种核酸分子，其包含编码权利要求1至6中任一项所述的抗体分子或其片段中包含的轻链可变区、重链可变区、重链或轻链的核苷酸序列。
  8. 一种载体，其包含权利要求7所述的核酸分子。
  9. 一种宿主细胞，其包含权利要求7所述的核酸分子和/或权利要求8所述的载体，或者所述宿主细胞被权利要求7所述的核酸分子和/或权利要求8所述的载体转化或转染。
  10. 一种组合物，其包含权利要求1至6中任一项所述的抗体分子或其片段、权利要求7所述的核酸分子、权利要求8所述的载体或权利要求9所述的宿主细胞；
    优选地，所述组合物为药物组合物，其可选地还包含药学上可接受的载体、辅料或赋形剂。
  11. 权利要求1至6中任一项所述的抗体分子或其片段、权利要求7所述的核酸分子、权利要求8所述的载体、权利要求9所述的宿主细胞或权利要求10所述的组合物在制备用于检测或诊断疾病或障碍的试剂中的用途。
  12. 一种检测或诊断疾病或障碍的方法，所述方法包括使权利要求1至6中任一项所述的抗体分子或其片段、权利要求7所述的核酸分子、权利要求8所述的载体、权利要求9所述的宿主细胞或权利要求10所述的组合物与来自受试者的样品相接触；
    其中，所述受试者为哺乳类动物，优选灵长类动物，更优选人。
  13. 权利要求1至6中任一项所述的抗体分子或其片段在制备抗体药物 偶联物中的用途。
  14. 一种抗体药物偶联物，其由权利要求1至6中任一项所述的抗体分子或其片段与细胞毒性部分偶联形成；
    优选地，所述细胞毒性部分为微管蛋白抑制剂、拓扑异构酶抑制剂或DNA结合剂；
    更优选地，所述微管蛋白抑制剂选自美登素衍生物、Monomethyl auristatin E(MMAE)、Monomethylauristatin F(MMAF)、Monomethyl Dolastatin 10、Tubulysin类衍生物、Cryptophycin类衍生物和Taltobulin；
    更优选地，所述拓扑异构酶抑制剂选自阿霉素(Doxorubicin)代谢产物PNU-159682衍生物和伊立替康(irinotecan，CPT-11)代谢产物SN38衍生物；
    更优选地，所述DNA结合剂选自PBD类衍生物和Duocarmycin类衍生物。
  15. 权利要求1至6中任一项所述的抗体分子或其片段、权利要求7所述的核酸分子、权利要求8所述的载体、权利要求9所述的宿主细胞、权利要求10所述的组合物或权利要求14所述的抗体药物偶联物在制备预防或治疗疾病或障碍的药物中的用途。
  16. 一种预防或治疗疾病或障碍的方法，所述方法包括给有此需要的受试者施用权利要求1至6中任一项所述的抗体分子或其片段、权利要求7所述的核酸分子、权利要求8所述的载体、权利要求9所述的宿主细胞、权利要求10所述的组合物或权利要求14所述的抗体药物偶联物；
    其中，所述受试者为哺乳类动物，优选灵长类动物，更优选人。
  17. 一种试剂盒，所述试剂盒包括权利要求1至6中任一项所述的抗体分子或其片段、权利要求7所述的核酸分子、权利要求8所述的载体、权利要求9所述的宿主细胞、权利要求10所述的组合物或权利要求14所述的抗体药物偶联物；
    优选地，所述试剂盒用于治疗、检测或诊断疾病或障碍。
  18. 根据权利要求11至17中任一项所述的用途或方法或试剂盒，其特征在于，所述疾病或障碍为Nectin-4高表达的肿瘤或癌症；
    优选地，所述疾病或障碍为实体肿瘤；
    优选地，所述疾病或障碍为膀胱癌、胰腺癌、乳腺癌(包括三阴性亚型和基底亚型)、非小细胞肺癌、胃癌、食管癌、卵巢癌等，特别是膀胱癌、乳腺癌、卵巢癌或肺癌。
PCT/CN2021/088661 2020-04-21 2021-04-21 一种抗Nectin-4的抗体及其应用 WO2021213434A1 (zh)

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JP2022563917A JP2023522229A (ja) 2020-04-21 2021-04-21 Ｎｅｃｔｉｎ－４に対する抗体及びその用途
CN202180030277.7A CN115427452A (zh) 2020-04-21 2021-04-21 一种抗Nectin-4的抗体及其应用
MX2022013163A MX2022013163A (es) 2020-04-21 2021-04-21 Anticuerpo contra nectin-4 y aplicacion del mismo.
US17/996,403 US20230265183A1 (en) 2020-04-21 2021-04-21 Antibody against nectin-4 and application thereof
KR1020227040582A KR20230007406A (ko) 2020-04-21 2021-04-21 넥틴-4 (nectin-4) 에 대한 항체 및 이의 응용
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CN115877008A (zh) * 2022-12-05 2023-03-31 中国科学院自动化研究所 用于膀胱癌检测的光学分子成像探针及其制备方法和应用
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