WO2021068949A1 - 抗人Trop-2抗体及其应用 - Google Patents

抗人Trop-2抗体及其应用 Download PDF

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WO2021068949A1
WO2021068949A1 PCT/CN2020/120277 CN2020120277W WO2021068949A1 WO 2021068949 A1 WO2021068949 A1 WO 2021068949A1 CN 2020120277 W CN2020120277 W CN 2020120277W WO 2021068949 A1 WO2021068949 A1 WO 2021068949A1
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amino acid
acid sequence
seq
sequence shown
antibody
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French (fr)
Inventor
王双
王荣娟
焦莎莎
张畅
张姣
曾大地
张锦超
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Mabwell Shanghai Bioscience Co Ltd
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Mabwell Shanghai Bioscience Co Ltd
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Priority to EP20874923.4A priority Critical patent/EP4043495A4/en
Priority to US17/768,189 priority patent/US12558577B2/en
Priority to AU2020362827A priority patent/AU2020362827A1/en
Priority to CN202080071403.9A priority patent/CN114585649B/zh
Priority to JP2022521211A priority patent/JP2022553645A/ja
Priority to CA3154454A priority patent/CA3154454A1/en
Publication of WO2021068949A1 publication Critical patent/WO2021068949A1/zh
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Priority to JP2025128509A priority patent/JP2025163135A/ja
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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Definitions

  • the present invention belongs to the field of biomedicine, and relates to a new anti-human Trop-2 antibody or functional fragment thereof.
  • the present invention also relates to the application of the antibody or its functional fragment.
  • Trop-2 Tumor-associated calcium signal transducer 2
  • TACSTD2 tumor-associated calcium signal transducer 2
  • epithelial glycoprotein-1 antigen epithelial glycoprotein-1, EGP-1
  • gastrointestinal Tumor-associated antigen 1 gastrointestinal tumor-associated antigen, GA733-1
  • TACSTD2 tumor-associated calcium signal transducer 2
  • epithelial glycoprotein-1 antigen epithelial glycoprotein-1, EGP-1
  • gastrointestinal Tumor-associated antigen 1 gastrointestinal tumor-associated antigen, GA733-1
  • Trop-2 is a transmembrane glycoprotein. Unlike other proto-oncogenes, Trop2 has no mutations, which means that it does not cause changes in the genetic makeup of overexpression. Trop-2 stimulates cell growth through the ERK/MAPK and cyclin D1 pathways, thereby promoting tumor invasion, angiogenesis, tumor progression, and drug resistance. It has been found that Trop-2 is highly expressed in a variety of tumors, especially triple-negative breast cancer, non-small cell lung cancer, etc., and is related to prognosis. In contrast, Trop-2 is extremely low in normal tissues, making it an ideal target for ADC drugs.
  • Antibody drugs targeting trop-2 are currently mainly developed as ADC drugs. Incomplete statistics. There are more than 3 varieties under clinical research.
  • the small molecule conjugates mainly include irinotecan derivatives and tubulin inhibitors. . At present, it is believed that it is ideal to use the novel low-toxic topoisomerase inhibitor metabolite SN-38.
  • SN-38 is different from existing microtubule inhibitors and DNA alkylating agents in tumor suppression, and is especially suitable for tumors with high heterogeneity and multi-drug resistance, such as triple-negative breast cancer, pancreatic cancer, gastric cancer and other tumors .
  • the project with the fastest clinical progress is the IMMU-132 project of Immunomedics.
  • Phase III clinical trials for relapsed and metastatic triple-negative breast cancer have been carried out, single-use or combined with carboplatin to treat triple-negative breast cancer (NCT02161679)
  • Other antibody-conjugated drug projects with similar technologies include Daiichi Sankyo, Pfizer, and major ADC-based drug companies.
  • the advantage is that the immunogen is easy to purify, the foreign antigen has a single structure, and it is easy to screen and obtain monoclonal antibodies against the recombinant protein.
  • the modification such as glycosylation
  • folding mode of the recombinantly expressed protein are often affected by the recombinant expression system.
  • the purified free recombinant Trop-2 or its extracellular domain is different from the natural conformation of the Trop-2 extracellular domain bound to the cell membrane surface. This difference in protein conformation or spatial structure results in the specificity of the antibody produced by its immunity and the effective affinity for the natural conformation of the extracellular region of Trop-2 in vivo.
  • Trop-2 positive cells on the cell membrane surface are used as immunogens and screening antigens, the advantage is that the Trop-2 extracellular region has a natural conformation, and it has a high affinity for the Trop-2 extracellular region in vitro.
  • the monoclonal antibody is expected to have high affinity for the natural conformation of the extracellular region of Trop-2 in vivo.
  • Trop-2 positive cells on the cell membrane surface are complex as immunogenic antigens, which can easily shield the immunogenicity of the extracellular region of Trop-2; in addition, the cost of flow cytometric screening using Trop-2 positive cells on the cell membrane surface as the screening antigen High and low screening efficiency.
  • the present invention is based on the advantages and disadvantages of the Trop-2 recombinant protein and Trop-2 positive cells in the preparation of monoclonal antibodies.
  • Using recombinant Trop-2 protein as immunogen to immunize animals to prepare hybridoma cells avoids the shielding effect of Trop-2 when using complex antigens as immunogens; using recombinant Trop-2 protein as coating antigens, preliminary screening is carried out by ELISA screening strategy In order to make the screening high-throughput; the Trop-2 positive cells on the membrane surface are used to re-screen the hybridomas that are initially screened to ensure that the obtained positive antibodies can bind to the natural conformation of the extracellular region of Trop-2.
  • the further technical problem to be solved by the present invention is to make the anti-Trop-2 monoclonal antibody in the pre-clinical animal test stage closer to real human in vivo test results. Therefore, based on the common structure of human Trop-2 extracellular domain and monkey Trop-2 extracellular domain, the common structure that can specifically bind to human Trop-2 extracellular domain and monkey Trop-2 extracellular domain was obtained through species cross-reaction screening. Of monoclonal antibodies. In order to ensure the high specificity of anti-Trop-2 monoclonal antibodies, antibodies with specific binding ability to the extracellular region of mouse Trop-2 are excluded.
  • the present invention provides an anti-Trop-2 antibody through hybridoma screening and humanization technology.
  • the antibody has high affinity to human Trop-2 and has a specific killing effect on cancer cells; at the same time, the Antibodies have high internalization ability and are particularly suitable for the development of ADC drugs.
  • the present invention provides the following technical solutions.
  • the present invention provides a method for preparing an anti-Trop-2 monoclonal antibody, which includes the following steps:
  • step (3) Re-screen the positive hybridomas in step (2) with Trop-2 positive cells on the membrane surface;
  • the anti-Trop-2 monoclonal antibody specifically recognizes and binds to the natural epitope of the extracellular region of Trop-2.
  • the method for preparing the anti-Trop-2 monoclonal antibody of the present invention wherein the Trop-2 positive cells on the membrane surface in the step (3) are recombinant animal cells, and the animal cells are derived from the step (1) The same species as the animal immunized when preparing the hybridoma cells.
  • the method for preparing the anti-Trop-2 monoclonal antibody of the present invention wherein, in the step (1), the mouse is immunized to prepare hybridoma cells, and in the step (3), the Trop-2 positive cells on the membrane surface are expressed Recombinant mouse cells of exogenous Trop-2 protein.
  • the method for preparing the anti-Trop-2 monoclonal antibody of the present invention wherein the step (2) adopts enzyme-linked immunosorbent assay (ELISA) to screen positive hybridoma cells that secrete the anti-Trop-2 monoclonal antibody; Step (3) Re-screening using flow cytometry (FACS) to obtain hybridomas that secrete antibodies that specifically recognize and bind to the natural epitope of the extracellular region of Trop-2.
  • ELISA enzyme-linked immunosorbent assay
  • FACS flow cytometry
  • the preparation method of the anti-Trop-2 monoclonal antibody of the present invention further comprises (4) the step of identifying an antibody that specifically recognizes and binds to the natural epitope of the extracellular region of Trop-2. Including affinity identification and specificity identification.
  • the method for preparing the anti-Trop-2 monoclonal antibody of the present invention wherein the step (4) is selected to have specific binding ability to human Trop-2 and monkey Trop-2, but not to mouse Of monoclonal antibodies.
  • the present invention provides an antibody or fragment thereof, the antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) And the light chain variable region (VL) comprises a combination of CDRs selected from the following (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3):
  • the antibodies or fragments thereof bind to human Trop-2.
  • the heavy chain variable region comprises a sequence selected from:
  • variable region of the light chain comprises a sequence selected from:
  • the heavy chain variable region and light chain variable region contained in the antibody or fragment thereof of the present invention can be selected from the following combinations:
  • amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:1; and, the amino acid sequence shown in SEQ ID NO:18 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 18;
  • amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 3; and, the amino acid sequence shown in SEQ ID NO: 20 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 30;
  • 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: 27 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27;
  • 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: 28 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28;
  • amino acid sequence shown in SEQ ID NO: 14 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; 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;
  • amino acid sequence shown in SEQ ID NO: 14 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 35 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 35;
  • amino acid sequence shown in SEQ ID NO: 14 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 36 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 36;
  • the antibodies or fragments thereof are in any form such as monoclonal antibodies, single-chain antibodies, bifunctional antibodies, single-domain antibodies, nanobodies, fully or partially humanized antibodies, or chimeric antibodies, or the antibody Or a fragment thereof is a half antibody or an antigen-binding fragment of a half antibody, such as scFv, BsFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or Fv; the antibody or fragment thereof can be a mouse , Rats, humans or any other source;
  • the antibody or fragment thereof further comprises a human or murine constant region, preferably a human or murine light chain constant region (CL) and/or a heavy chain constant region (CH);
  • a human or murine constant region preferably a human or murine light chain constant region (CL) and/or a heavy chain constant region (CH);
  • the antibody or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and/or a kappa or lambda light chain constant region.
  • the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4. Type, the light chain constant region is ⁇ type;
  • the heavy chain constant region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 37 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 an amino acid sequence as shown in SEQ ID NO: 38 or an amino acid sequence having at least 75% identity with the amino acid sequence.
  • the above-mentioned at least 75% identity of the present invention is at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or even 99% identity, etc. Any percentage of identity ⁇ 75%.
  • the present invention also provides a nucleic acid molecule that encodes the heavy chain CDR, light chain CDR, heavy chain variable region, and light chain CDR of any antibody or fragment thereof Variable region, heavy chain or light chain.
  • 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.
  • the present invention provides a host cell comprising 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 antibodies or fragments thereof, nucleic acid molecules, vectors and/or host cells provided by the present invention can be obtained by using any conventional technical methods known in the art.
  • the antibodies or fragments thereof, nucleic acid molecules, vectors and/or host cells may be included in pharmaceutical compositions, more particularly in pharmaceutical preparations, so as to be used for various purposes according to actual needs.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or fragment thereof, nucleic acid molecule, vector and/or host cell of the present invention, and optionally pharmaceutically acceptable Accepted excipients.
  • the antibody or fragments thereof of the present invention can be used in combination with other antibody drugs with macrophage phagocytosis. Therefore, preferably, the antibody drug promotes the phagocytosis of the cell by macrophages by binding to the protein expressed on the cell surface. Therefore, the pharmaceutical composition provided by the present invention may also contain the other antibody drugs, preferably macrophage immune checkpoint antibodies; according to a specific embodiment of the present invention, the antibody is an anti-CD47 antibody.
  • the present invention also provides related applications of the above-mentioned subject matter.
  • the present invention provides the use of the antibody or fragments thereof, nucleic acid molecules, vectors, host cells, and/or pharmaceutical compositions in the preparation of medicines, which are preferably used for the treatment of Trop-2 High expression cancer; preferably, the Trop-2 high expression cancer is gastric cancer, pancreatic cancer, bowel cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple negative breast cancer or Cervical cancer.
  • the Trop-2 high expression cancer is gastric cancer, pancreatic cancer, bowel cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple negative breast cancer or Cervical cancer.
  • the use covers the use of the antibody or fragment thereof of the present invention in combination with other antibody drugs described above to prepare the drug.
  • the antibodies or fragments thereof provided by the present invention can also be fused or conjugated with other parts.
  • the present invention provides a fusion protein or conjugate comprising the antibody or fragment thereof of the present invention.
  • the fusion protein may include any other part that modifies the antibody or fragment thereof of the present invention, such as amino acids, polypeptides, or proteins.
  • the conjugate may include the antibody of the present invention or a fragment thereof and a drug conjugated therewith, wherein the drug is, for example, a cytotoxic agent.
  • the conjugate is an antibody drug conjugate (ADC) represented by the following formula: (antibody or fragment thereof of the present invention)-(linker)-(cytotoxic agent);
  • ADC antibody drug conjugate
  • the cytotoxic agent is a tubulin inhibitor (such as paclitaxel, docetaxel, etc.) or a DNA replication inhibitor (such as irinotecan or its metabolic active substance SN-38, etc.).
  • a tubulin inhibitor such as paclitaxel, docetaxel, etc.
  • a DNA replication inhibitor such as irinotecan or its metabolic active substance SN-38, etc.
  • the conjugate is an "anti-TROP-2 antibody-linker-SN-38 antibody drug conjugate".
  • the present invention also provides the use of the antibody or fragments thereof, nucleic acid molecules, vectors, host cells and/or pharmaceutical compositions in the preparation of antibody drug conjugates (ADC), and the antibody drug conjugates are preferably used in therapy Trop-2 high expression cancer; preferably, the Trop-2 high expression cancer is gastric cancer, pancreatic cancer, bowel cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple negative Breast cancer or cervical cancer.
  • ADC antibody drug conjugates
  • the present invention provides a method for preventing and/or treating diseases, the method comprising administering the antibody or fragment thereof, nucleic acid molecule, vector, host cell, pharmaceutical composition, fusion of the present invention to a subject in need thereof Protein or conjugate, and optionally other drugs or means.
  • the optional other drugs or means refer to other drugs or means that can be administered in combination with the antibody or fragments, nucleic acid molecules, vectors, host cells, pharmaceutical compositions, fusion proteins or conjugates of the present invention, such as small molecule Drugs, targeted drugs, antibodies and other recombinant protein drugs, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapy drugs, and radiotherapy.
  • the combined administration of the two can be carried out in any form, for example, simultaneously, continuously or at a certain time interval.
  • the disease is a cancer with high Trop-2 expression; further preferably, the cancer with high Trop-2 expression is gastric cancer, pancreatic cancer, bowel cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, and small cell lung cancer , Urothelial cancer, triple-negative breast cancer or cervical cancer.
  • the subject is a mammal, preferably, the subject is a human.
  • immunoglobulin sequence is used as a general term, including full-size antibodies, their individual chains, and all of their parts, domains or fragments (including but not limited to antigen-binding domains or fragments such as VHH, respectively). Domain or VH/VL domain).
  • antibody should be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (ie, "complete antibody molecules") and antigen-binding fragments thereof.
  • antigen-binding portion of an antibody, “antigen-binding fragment” of an antibody, and similar terms include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide that specifically binds to an antigen to form a complex Or glycoprotein.
  • the term “antigen-binding fragment” or “antibody fragment” of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to Trop-2.
  • Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, CDR-containing fragments, or isolated CDRs.
  • Antigen-binding fragments of antibodies can be derived from, for example, complete antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombination involving manipulation and expression of the variable and (optionally) constant domains of the DNA-encoding antibody Genetic engineering technology. Such DNA is known and/or is readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or may be synthetic.
  • DNA can be sequenced and manipulated chemically or by using molecular biology techniques, such as arranging one or more variable and/or constant domains into a suitable configuration, or introducing codons; forming cysteine residues; Modification, addition or removal of amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragment; and (vii) the smallest recognition unit (for example, isolated complementarity determining region (CDR), such as CDR3 peptide) composed of amino acid residues in the hypervariable region of the mimic antibody, or restricted FR3-CDR3- FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as the following are also encompassed in the expression "antigen-binding fragment” as used herein: domain-specific antibodies, single-domain antibodies, domain-deletion antibodies, chimeric antibodies, CDR-grafted antibodies , Bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, mini-antibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIP) and shark variable IgNAR domains.
  • SMIP small modular immunopharmaceuticals
  • the antigen-binding fragment of an antibody will typically contain at least one variable domain.
  • the variable domain can be of any size or amino acid composition, and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains can be positioned relative to each other in any suitable arrangement.
  • the variable region can be dimeric and contain VH-VH, VH-VL, or VL-VL dimers.
  • the antigen-binding fragment of the antibody may contain a monomeric VH or VL domain.
  • the antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • variable and constant domains that can be found in the antigen-binding fragments of the antibodies of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; ( iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; ( x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL.
  • variable and constant domains may be directly linked to each other or may be linked through a full or partial hinge or connector region .
  • the hinge region can be composed of at least 2 (for example, 5, 10, 15, 20, 40, 60 or more) amino acids, which produce flexible or semi-flexible bonds between adjacent variable and/or constant domains in a single polypeptide molecule United.
  • the antigen-binding fragment of the antibody of the present invention may comprise any of the variable and constant domain configurations listed above non-covalently associated with each other and/or with one or more monomeric VH or VL domains (e.g. Through disulfide bonds) homodimers or heterodimers (or other multimers).
  • the antigen-binding fragments can be monospecific or multispecific (e.g., bispecific).
  • Multispecific antigen-binding fragments of antibodies will typically contain at least two different variable domains, where each variable domain is capable of specifically binding to a separate antigen or different epitopes on the same antigen.
  • Any multispecific antibody format (including the exemplary bispecific antibody format disclosed herein) may be suitable for use in the context of the antigen-binding fragments of the antibodies of the present invention using conventional techniques available in the art.
  • chimeric antibody refers to an antibody in which (a) the constant region or part thereof is changed, substituted or exchanged so that the antigen binding site (variable region, CDR or part thereof) is different or changed from Types, effector functions and/or types of constant regions are connected; or (b) the variable region or part of it has a different or altered antigen-specific variable region (for example, CDRs from different species and Framework area) change, replace or exchange.
  • the chimeric antibody may comprise variable region fragments, for example, a recombinant antibody comprising two Fab or Fv regions or scFv. As indicated above, the chimera may also include an Fc region from a different source than the joined Fv region. In some cases, chimeric antibodies comprise chimeras located in the Fv region.
  • An example of such a chimeric antibody is a humanized antibody in which the Fvs and CDRs are from different sources.
  • humanized antibody is an antibody in which the antigen-binding loops obtained from the VH and VL regions of a non-human antibody, that is, CDRs, are grafted to human framework sequences. Humanization can be carried out according to the method described in the following documents, that is, the non-human CDR sequence is replaced with the corresponding sequence of the human antibody, for example, US Patent No. 5,545,806; No. 5,569,825; No. 5,633,425; No.
  • Transgenic mice or other organisms such as other mammals can also be used to express humanized or human antibodies, as disclosed in US Patent No. 6,673,986.
  • percent (%) identity means that after aligning sequences and introducing blanks, when necessary, to achieve the maximum percent identity (that is, the candidate and reference sequences can be aligned for optimal alignment). One or both of them introduce blanks, and can be used for comparison purposes regardless of non-homologous sequences), candidate sequences, such as the isolated anti-IL1-RAP antibody of the present invention, have the same amino acid (or nucleic acid) residues as the reference sequence The percentage of amino acid (or nucleic acid) residues.
  • the BLAST2.0 software can use standard settings to realize the comparison for the purpose of determining percent identity. Alignment can be performed to achieve maximum alignment over the entire length of the compared sequence.
  • the given candidate sequence and, and or for a given reference sequence (which can optionally be expressed as a given candidate sequence, which has or contains and, and or for a given reference sequence) is calculated as follows: A certain percentage of amino acid (or nucleic acid) sequence identity) The percentage of amino acid (or nucleic acid) sequence identity:
  • A is the number of amino acid (or nucleic acid) residues scored the same in the alignment of the candidate sequence and the reference sequence
  • B is the total number of amino acid (or nucleic acid) residues in the reference sequence.
  • the percent amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence will not be equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence Sex.
  • a reference sequence aligned for comparison with a candidate sequence can be shown to exhibit 50% over the full length of the candidate sequence or a selected portion of consecutive amino acid (or nucleic acid) residues of the candidate sequence.
  • Candidate sequence with 100% identity The length of the candidate sequence aligned for comparison purposes is at least 30%, for example, at least 40%, for example, at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence.
  • antibody target refers to molecules, compounds, or complexes that can be recognized by antibodies, that is, can be specifically bound by antibodies.
  • the term can refer to any molecule that can be specifically recognized by antibodies, for example, polypeptides, polynucleotides, carbohydrates, lipids, chemical moieties, or combinations of the above (e.g., phosphorylated or glycosylated polypeptides, etc.).
  • polypeptides polynucleotides
  • carbohydrates e.g., phosphorylated or glycosylated polypeptides, etc.
  • chemical moieties e.g., phosphorylated or glycosylated polypeptides, etc.
  • isolated refers to those obtained from the natural state by artificial means. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same high-purity polynucleotide or polypeptide isolated from this natural state is called isolation. of.
  • isolated or “isolated” does not exclude the mixing of artificial or synthetic materials, nor does it exclude the presence of other impure materials that do not affect the activity of the material.
  • host cell refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, insect cells such as S2 fruit fly cells or Sf9, or Fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells and other animal cells.
  • prokaryotic cells such as Escherichia coli
  • fungal cells such as yeast cells
  • insect cells such as S2 fruit fly cells or Sf9
  • Fibroblasts CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells and other animal cells.
  • KD refers to the dissociation equilibrium constant (KD) of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • KD dissociation equilibrium constant
  • the antibody binds to the antigen with a dissociation equilibrium constant of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less, for example , As measured in a BIACORE instrument using surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • Antibodies include but are not limited to polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab' and F(ab') 2 fragments, Fv, scFv and Fab expression libraries.
  • Monoclonal antibody (mAb) is an antibody obtained from a single cloned cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage cloned cell lines.
  • Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, and synthesis technology such as CDR grafting or other existing technologies.
  • TROP2 belongs to the TACSTD family, is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), stomach Intestinal tumor-associated antigen (GA733-1), surface marker 1 (M1S1). Trop-2 is overexpressed in a variety of malignant tumors and is an oncogene related to the occurrence, invasion and metastasis of malignant tumors.
  • TACSTD2 tumor-associated calcium signal transducer 2
  • EGP-1 epidermal glycoprotein 1
  • G733-1 stomach Intestinal tumor-associated antigen
  • M1S1 surface marker 1
  • the Trop-2 gene is located on the short arm of chromosome 1, and is specifically located as 1p32.1 [3]. The full length of the gene is 9072bp, with no introns and only one exon. The sequence similarity between mouse Trop-2 and human homologous gene is 87.4%.
  • the primary structure of Trop-2 protein is a 36kD polypeptide composed of 323 amino acids, which is a single-pass surface glycoprotein.
  • Trop-2 consists of a hydrophobic leader peptide (AA1-26), an extracellular domain (AA27-274), a transmembrane domain (AA275-297) and a cytoplasmic tail (AA298-323).
  • TROP2 protein The N-terminus of TROP2 protein is the extracellular domain (ECD), which is connected to the intracellular short tail (IC) through a unidirectional transmembrane helix (TM), thereby being fixed to the cell membrane.
  • ECD extracellular domain
  • IC intracellular short tail
  • TM transmembrane helix
  • PIP2 phosphatidylinositol 4,5-bisphosphate
  • the mutation of Serine residue 303 eliminated the ability of Trop-2 to stimulate tumor growth. The phosphorylation of this residue is responsible for protein kinase C (PKC).
  • PPC protein kinase C
  • ADC antibody-drug conjugate
  • the antibody molecules in ADC drugs often use humanized monoclonal antibodies to modify the crystallizable fragment (Fc) segment to reduce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependence Cytotoxicity (complement dependent cytotoxicity, CDC), etc.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement dependence Cytotoxicity
  • the most important role of antibody molecules is targeting, that is, the targeted delivery of small molecule compounds to the antigen-antibody binding site.
  • the antibody selectivity is poor or the antigen is present in normal tissues, it will cause the delivery of cytotoxic drugs to normal cells, resulting in targeted toxicity.
  • the shedding of small molecules in the circulation process can lead to a certain degree of off-target toxicity.
  • the Fc of an antibody molecule has the activity of binding to Fc receptors of immune cells such as Fc ⁇ Rs/FcRN, it is easy to bind to immune cells and cause the killing of immune cells.
  • ADC drugs as exogenous biological macromolecules, may also phagocytose cells in the circulation and enter the cell through pinocytosis to cause cell death.
  • linkers for ADC drugs mainly include hydrazone bonds, disulfide bonds and peptide bonds.
  • the hydrazone bond can be hydrolyzed under acidic conditions and is a relatively unstable linker.
  • Mylotarg uses the hydrazone key as a linker.
  • Disulfide bonds can be hydrolyzed in the high concentration of glutathione in cells, so they are not easy to fall off outside the cell.
  • the peptide bond is the most tightly bound, and it is only broken by the action of lysosomal proteolytic enzymes.
  • the stability of the linker directly affects the unanticipated dissociation of cytotoxic drugs, and this fragmentation causes exposure of small molecule cytotoxic drugs in the body, that is, off-target toxicity.
  • cytotoxic drugs for ADC drugs are chemotherapeutics routinely used in clinical practice, which determine the main toxic effect spectrum of ADC drugs. Because it has been widely used clinically, its toxicity characteristics are generally clear. According to the type of drug, such as tubulin polymerization inhibitor or DNA damaging agent/DNA replication inhibitor, the toxicity risk can be better grasped.
  • tubulin inhibitors tubulin inhibitors include dorestin and its auristatin derivatives auristatins (MMAE, MMAF, MMAD), maytansinoids and maytansinoids (DM1, DM2 , DM3, DM4), paclitaxel and taxol derivatives (docetaxel), docetaxel, vincristine, etc., the DNA damaging agent/DNA replication inhibitor such as irinotecan or its metabolic active substance SN-38 Wait.
  • MMAE auristatin derivatives auristatins
  • DM1, DM2 , DM3, DM4 maytansinoids and maytansinoids
  • paclitaxel and taxol derivatives docetaxel
  • docetaxel vincristine, etc.
  • the DNA damaging agent/DNA replication inhibitor such as irinotecan or its metabolic active substance SN-38 Wait.
  • composition means a mixture containing one or more of the compounds of the present invention 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 the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • the therapeutic composition should generally be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high antibody concentration.
  • Sterile injectable solutions can be prepared by incorporating the active compound (ie antibody or antibody portion) in the required amount together with one of the ingredients or combinations of ingredients listed above in a suitable solvent, as required, followed by filtration and sterilization. .
  • the method, composition, and combination therapy described in the present invention can be combined with other active agents or treatment methods.
  • the method includes administering the anti-Trop inhibitor described in the present invention to a subject in an amount effective to treat or prevent a disease (e.g., cancer).
  • a disease e.g., cancer
  • -2 antibody molecule optionally, with PD-1, PD-L1, PD-L2, LAG-3, CTLA-4, Tim-3 antibody (immunotherapy) or other tumor treatment antibodies, Her-2, EGFR, Combinations of one or more inhibitors of VEGF, VEGFR antibodies, etc., as well as ADC (antibody drug conjugates, such as T-DM1), bispecific antibodies, chemotherapeutic drugs, etc., including the administration of additional active agents or all of them can be Is administered in an amount or dose that is higher, lower or equal to the amount or dose of each active agent used alone (e.g., as a monotherapy).
  • the additional active agent or the total administered amount or dose is lower than
  • the present invention provides a new type of anti-human Trop-2 antibody, which has good biological activity: whether it is a Trop-2 recombinant protein or a Trop-2 antigen expressed on the cell surface, the antibody provided by the present invention (including the chimeric antibody) Both synthetic antibody and humanized antibody) can effectively bind, which is similar to the control antibody Sacituzumab.
  • the antibody provided by the present invention has a high affinity for human Trop-2: Compared with the control antibody Sacituzumab, the humanized antibody of the present invention has even higher specific binding ability to human Trop-2 protein, and the affinity is higher than that of Sacituzumab. . Therefore, the antibody of the present invention has a good pharmacological effect.
  • the antibody of the present invention also has good internalization ability: the internalization rate of the humanized antibody is similar to that of the control antibody Sacituzumab; the internalization ability is significantly enhanced after being labeled as ADC. Therefore, the antibody of the present invention has the potential to be used in the development of ADC drugs.
  • the Trop-2 antibody of the present invention can also have a synergistic effect with other antibodies.
  • the antibody of the present invention can be used in combination with CD47 to further promote the phagocytosis of tumor cells by macrophages.
  • the antibodies of the present invention also proved to have good in vivo efficacy.
  • the antibody of the present invention to prepare ADC, it is found that the anti-Trop2-ADC antibody has a dose-dependent inhibitory effect on tumor growth.
  • each ADC antibody has the same efficacy as the control antibody Sacituzumab, and it is not observed
  • the significant toxic effect of ADC small molecule SN38, the weight of each experimental group of animals increased steadily, and there was no significant difference from the control.
  • Figure 1 shows the results of the screening of positive hybridoma supernatant and Trop-2 binding on the surface of CHO cells.
  • Figure 2 shows the results of screening of positive hybridoma supernatant and Trop-2 binding on the surface of CHO cells.
  • Figure 3 shows the cross-reaction results of ELISA detection of positive hybridoma clone supernatant and different species of recombinant Trop-2.
  • Figure 4 shows the results of ELISA detecting the binding activity of anti-human Trop-2 chimeric antibody to Trop-2 recombinant protein, where Figure 4A: ch3-11; Figure 4B: ch4-3; Figure 4C: ch23-12; Figure 4D: ch11-4; Figure 4E: ch17-1.
  • Figure 5 shows the results of FACS detection of the binding activity of the anti-human Trop-2 chimeric antibody to the Trop-2 recombinant protein on the cell surface, where Figure 5A: ch3-11; Figure 5B: ch23-12; Figure 5C: ch11-4; Figure 5A: ch3-11; Figure 5B: ch23-12; Figure 5C: ch11-4; 5D: ch4-3; Figure 5E: ch17-1.
  • Figure 6 shows the species-specific results of the binding of anti-human Trop-2 antibodies to Trop-2 by ELISA, where Figure 6A: h23-12; Figure 6B: h4-3; Figure 6C: Sacituzumab.
  • Figure 7 shows the results of the affinity analysis of the anti-human Trop-2 antibody to the recombinant protein of the extracellular domain of human Trop-2, where Figure 7A: Sacituzumab; Figure 7B: h23-12; Figure 7C: h4-3.
  • Figure 8 shows the internalization observation results of anti-Trop-2 humanized antibody binding to Trop-2 on the surface of N87 cells.
  • Figure 9 shows the drug-time curve of a single administration of anti-Trop-2 humanized antibody in nude mice (detected by Trop-2), where Figure 9A: h23-12; Figure 9B: h4-3.
  • Figure 10 shows the inhibition rate of anti-Trop-2-ADC antibody on cell growth.
  • Figure 11 shows the change curve of body weight of Balb/C nu rumen cancer-bearing N87 mouse model mice, where Figure 11A: after ch4-3-SN38 administration; Figure 11B: after h23-12-SN38 administration; Figure 11C: After administration of isotype control antibody; Figure 11D: After high dose administration of ch4-3-SN38, h23-12-SN38.
  • Figure 12 shows the tumor volume change curve of the Balb/C nu rumen cancer N87 mouse model, where Figure 12A: after ch4-3-SN38 administration; Figure 12B: after h23-12-SN38 administration; Figure 12C: Isotype After administration of control antibody; Figure 12D: After high-dose administration of ch4-3-SN38 and h23-12-SN38.
  • Figure 13 shows the body weight change curve of SKOV3 subcutaneously transplanted tumor mouse model mice using anti-Trop2 antibody and anti-CD47 antibody in combination.
  • Figure 14 shows the tumor volume change curve of the SKOV3 subcutaneously transplanted tumor mouse model in which anti-Trop2 antibody and anti-CD47 antibody are used in combination.
  • Figure 15 shows the tumor volume change curve of the Balb/C nu rumen-bearing NCI-N87 subcutaneously transplanted tumor mouse model.
  • Figure 16 shows the weight change curve of the Balb/C nu rumen cancer NCI-N87 subcutaneously transplanted tumor mouse model mouse.
  • mice were immunized with human Trop-2 recombinant protein (serial number: NP_002344.2, 1aa-274aa), and human Trop-2-his recombinant protein (serial number: NP_002344.2, 1aa-274aa) was coated )
  • 96-well ELISA plate was used to detect serum titer by ELISA; mice whose serum titer reached the fusion requirement were used for the next step of cell fusion.
  • Cell fusion and hybridoma preparation select the mice with the required titer for shock immunization, take the mouse spleen aseptically after 3 days, prepare a B lymphocyte suspension, and mix it with SP2/0 myeloma cells at a ratio of 4:1 , The two kinds of cells fuse under the action of PEG After the fused cells were resuspended in HAT medium, they were divided into 96-well cell culture plates. Place 37°C, 5% CO 2 incubator for culture.
  • the top 21 clones with high reading values were selected according to the principle of high to low readings: m1-1, m3-11, m4-3, m5-5, m6-6, m7-13, m11 -4, m12-2, m12-4, m13-2, m14-2, m15-3, m16-7, m17-1, m18-4, m19-5, m20-4, m21-1, m22-1 , M23-12, m24-3 for the next step of FACS binding screening.
  • the reading frame of the Trop-2 gene was cloned from the vector containing Trop-2 cDNA (Cat.:HG10428-M, Beijing Yiqiao Shenzhou) by PCR, and cloned into the glutamylamine synthetase (GS )
  • electrotransfect Nucleofector IIb, Lonza
  • CD CHO AGTTM containing 50 ⁇ M MSX (Cat.:M5379, Sigma).
  • the hybridoma supernatants of the 21 clones were selected after 100-fold dilution and incubated with the constructed CHO cell (CHO/Trop-2 cell) suspension at 37°C for 30 min.
  • the following controls were set: (1) Positive control ( PC): mouse IgG constant region form of Sacituzumab, 1ug/ml; (2) Negative control (NC): irrelevant mouse antibody, 1ug/ml.
  • PC Positive control
  • NC negative control
  • F9006 goat anti-mouse IgG-FITC
  • the average fluorescence intensity (MFI) of the cells was measured by a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER) to verify whether the antibody secreted by the hybridoma can bind to Trop-2 on the surface of the CHO cell. The results are shown in the figure. 1.
  • the antibodies were diluted to 13nM and 0.66nM, and then incubated with a suspension of recombinant human Trop-2 expressing CHO cells (CHO/Trop-2 cells) at 37°C for 30 minutes, and the following controls were set: (1) Positive control (PC): The mouse IgG constant region form of Sacituzumab, 1ug/ml; (2) Negative control (NC): irrelevant mouse antibody, 1ug/ml. After washing the cells 3 times with PBS, goat anti-mouse IgG-FITC (Cat.: F9006, Sigma) diluted 1:200 was added and incubated for 30 min.
  • PC Positive control
  • NC Negative control
  • the average fluorescence intensity (MFI) of the cells was measured by a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER) to verify whether the antibody secreted by the hybridoma can bind to Trop-2 on the surface of CHO cells, as shown in Figure 2. As shown, the antibodies from the supernatants of 15 clones bound well to Trop-2 on the surface of CHO cells.
  • the human Trop-2-His recombinant protein (sequence number: NP_002344.2, 1aa-274aa), the cynomolgus Trop-2-His recombinant protein (sequence number: UniProtKB-A0A2K5UE71, 1aa-272aa), mouse Trop-2 -His recombinant protein (Cat.:50922-M08H, Beijing Yiqiao Shenzhou) was coated overnight at 4°C, and the coating concentration was 0.2, 1, and 1 ⁇ g/mL respectively; after washing the plate 3 times with PBS, add 5% BSA PBS, 37 Block at °C for 60 minutes, wash the plate with PBST 3 times; add PBS to dilute the 15 purified mouse antibodies to 1 ⁇ g/mL, set the following controls: (1) Positive control (PC): Sacituzumab (WHO Drug Information (Vol.
  • Blank control PBS.
  • the hybridoma cells m3-11, m4-3, m11-4, m17-1, and m23-12 secreting anti-human Trop-2 antibodies were expanded and cultured, and then used Mouse Monoclonal Antibody IgG Subclass Test Card (Cat.: A12403, VicNovo ) And Mouse Monoclonal Antibody Light/Heavy Chain Test Card (Cat.:A12401, VicNovo) according to the reagent operating procedures for subtype detection, the subtype identification is: heavy chain is IgG1, light chain is Kappa chain, m3-11, m4 -3.
  • the cloning of antibody genes of m11-4, m17-1, and m23-12 provides a basis.
  • the m3-11, m4-3, m11-4, m17-1, m23-12 hybridoma cells were extracted according to the instructions of the TRIzol kit (Cat.: 15596026, Invitrogen) to extract total cellular RNA; using M-MuLV reverse transcriptase (Cat.:M0253S, NEB) Reverse transcription of total RNA from hybridoma cells into cDNA; use degenerate primers (refer to the book [Dong Zhiwei, Wang Yan. Antibody Engineering (Second Edition).
  • Phusion kit Cat.: E0553L, NEB
  • Phusion kit Cat.: E0553L, NEB
  • gel recovery kit Cat.: AP- GX-250, Axygen
  • purify the PCR amplification product according to the instructions of the T vector cloning kit (Cat.: ZC205, ZC205)
  • the amplified PCR product is connected to the T vector and transformed into E. coli competent cells, the strain is amplified, After extracting the plasmid, DNA sequencing was performed to obtain the monoclonal antibody variable region sequence.
  • the nucleotide sequence of the heavy chain variable region DNA of the mouse antibody of clone m3-11 is shown in SEQ ID NO: 41, and the amino acid sequence of the heavy chain variable region of the mouse antibody of clone m3-11 is inferred from the DNA sequence, and the amino acid sequence of the heavy chain variable region of the mouse antibody of clone m3-11 is shown in SEQ ID NO: 1;
  • the nucleotide sequence of the light chain variable region DNA of the mouse antibody of clone m3-11 is shown in SEQ ID NO: 42, and the amino acid sequence of the light chain variable region of the clone m3-11 mouse antibody is inferred from the DNA sequence. See SEQ ID NO :18.
  • the nucleotide sequence of the heavy chain variable region DNA of the mouse antibody of clone m4-3 is shown in SEQ ID NO: 43, and the amino acid sequence of the heavy chain variable region of the mouse antibody of clone m4-3 is inferred from the DNA sequence, and the amino acid sequence of the heavy chain variable region of the mouse antibody of clone m4-3 is shown in SEQ ID NO: 2;
  • the nucleotide sequence of the light chain variable region DNA of the mouse antibody of clone m4-3 is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the mouse antibody of clone m4-3 is inferred from the DNA sequence. See SEQ ID NO:19.
  • the nucleotide sequence of the heavy chain variable region DNA of the mouse antibody of clone m11-4 is shown in SEQ ID NO: 47, and the amino acid sequence of the heavy chain variable region of the mouse antibody of clone m11-4 is inferred from the DNA sequence, and the amino acid sequence of the heavy chain variable region of the mouse antibody of clone m11-4 is shown in SEQ ID NO: 5;
  • the nucleotide sequence of the light chain variable region DNA of the mouse antibody of clone m11-4 is shown in SEQ ID NO: 48, and the amino acid sequence of the light chain variable region of the mouse antibody of clone m11-4 is inferred from the DNA sequence. See SEQ ID NO :twenty four.
  • the nucleotide sequence of the variable region DNA of the murine antibody heavy chain of clone m17-1 is shown in SEQ ID NO: 51, and the amino acid sequence of the variable region of the murine antibody heavy chain of clone m17-1 is inferred from the DNA sequence, and the amino acid sequence of the variable region of the murine antibody heavy chain of clone m17-1 is shown in SEQ ID NO: 11;
  • the nucleotide sequence of the light chain variable region DNA of the cloned m17-1 mouse antibody is shown in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region of the cloned m17-1 mouse antibody is inferred from the DNA sequence. See SEQ ID NO :30.
  • the nucleotide sequence of the variable region DNA of the murine antibody heavy chain of clone m23-12 is shown in SEQ ID NO: 53, and the amino acid sequence of the variable region of the murine antibody heavy chain of clone m23-12 is inferred from the DNA sequence, and the amino acid sequence of the variable region of the murine antibody heavy chain of clone m23-12 is shown in SEQ ID NO: 12;
  • the nucleotide sequence of the light chain variable region DNA of the murine antibody of clone m23-12 is shown in SEQ ID NO:54, and the amino acid sequence of the light chain variable region of the murine antibody of clone m23-12 is inferred from the DNA sequence. See SEQ ID NO: 31.
  • the light and heavy chain sequences of the control antibody (Sacituzumab) were fully synthesized, and the light and heavy chain sequences were cloned into the eukaryotic transient expression vector to obtain the control antibody light chain and heavy chain expression plasmids, which were transferred to E. coli for amplification, and a large number of controls were isolated and obtained
  • the antibody light chain and heavy chain plasmids using these plasmids, and according to the operating instructions of the transfection reagent 293fectin (Cat.:12347019, Gibco), respectively transfer the light and heavy chain plasmids of the control antibody into HEK293 cells for recombinant expression.
  • control antibody Sacituzumab amino acid sequence is derived from WHO Drug Information (Vol. 31, No. 1, 2017), the heavy chain amino acid sequence is shown in SEQ ID NO: 39, and the light chain amino acid sequence is shown in SEQ ID NO: 40.
  • the light chain variable region and heavy chain variable region genes of the corresponding murine antibody 3-11, 4-3, 11-4, 17-1, 23-12 obtained from each clone were introduced into the restriction site by PCR , Cloned into the eukaryotic transient expression vector upstream of the human-kappa light chain constant region and human IgG1 heavy chain constant region coding genes, respectively, to obtain the human-mouse chimeric light chain (pKN019-ch3-11L, pKN019-ch4-3L , PKN019-ch11-4L, pKN019-ch17-1L, pKN019-ch23-12L) and human-mouse chimeric heavy chain (pKN041-ch3-11H, pKN019-ch4-3H, pKN019-ch11-4H, pKN019-ch17- 1H, pKN019-ch23-12H) expression plasmid, transformed into E.
  • the transfection reagent 293fectin Cat.: 12347019, Gibco
  • Example 5 ELISA to detect the binding activity of anti-human Trop-2 chimeric antibody and Trop-2 recombinant protein
  • Human Trop-2-his recombinant protein (sequence number: NP_002344.2, 1aa-274aa), concentration 0.2 ⁇ g/mL, coated overnight at 4°C, sealed with 5% BSA in a constant temperature incubator at 37°C for 60 minutes.
  • Example 6 FACS detection of the binding activity of anti-human Trop-2 chimeric antibody to human Trop-2 recombinant protein on the surface of CHO cells
  • the binding ability of ch3-11, ch4-3, ch17-1, ch11-4, ch23-12 and the control antibody Sacituzumab to human Trop-2 recombinant protein on the surface of CHO cells was determined by FACS, and its half effective binding concentration (EC50) The values were 0.993nM, 3.326nM, 2.918nM, 1.154nM, 2.748nM and 2.316nM, respectively ( Figure 5). Compared with the control antibody Sacituzumab, the binding activity of ch3-11, ch11-4 is better, and the binding activity of ch4-3, ch17-1, and ch23-12 are similar. The results show that the anti-human Trop-2 chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12 can effectively bind to human Trop-2 recombinant protein on the surface of CHO cells.
  • Example 7 Anti-human Trop-2 chimeric antibody binds to the internalization activity of cell surface Trop-2
  • Each antibody is divided into four groups (1h, 3h, 5h experimental group and control group), each with 2 tubes.
  • the experimental group was placed in a 37°C electric constant temperature incubator, incubated for 1h, 3h, and 5h, and then placed on ice.
  • the control group was kept on ice as a negative control; after all samples were incubated, centrifuged at 1,500rpm, 4°C for 3min, and discarded the supernatant.
  • the chimeric antibodies ch3-11, ch4-3, ch11-4, and ch23-12 were placed in PBS containing 10% N,N-dimethylacetamide (DMA) (Cat.:ARK2190) at a concentration of 5 mg/mL. , Shanghai Feibo Chemical) in PBS and 20% DMA-containing PBS, placed at 37°C for 2 hours, use an ultrafiltration centrifuge tube to remove DMA from the sample, replace the buffer with PBS, and use G3000Wxl liquid chromatography analysis column (Cat. : SEC-0046, Tosoh), the purity of the sample was analyzed by high performance molecular exclusion chromatography (SEC-HPLC), and the results of the purity analysis are shown in Table 2.
  • DMA N,N-dimethylacetamide
  • SEC-HPLC high performance molecular exclusion chromatography
  • a comprehensive analysis of the murine antibody heavy chain sequence is performed to determine the antigen complementarity determinant (CDR) region where the antibody binds to the antigen and the framework that supports the conservative three-dimensional conformation of the antibody. Then, according to the result of homology comparison, search for the most similar human antibody template in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX), and combine the full sequence
  • humanized heavy chain variable region h23-12_VH1 nucleotide sequence of 23-12 antibody CDR grafting is shown in SEQ ID NO: 55, and the amino acid sequence is shown in SEQ ID NO: 13; humanized light chain variable region h23-12_VL1 nucleus See SEQ ID NO:56 for the nucleotide sequence and SEQ ID NO:32 for the amino acid sequence.
  • the sequence of the CDR region of the humanized light and heavy chain variable region of the CDR transplantation was designed for mutation, and the mutation sites are shown in Table 3 below.
  • h23-12_VL1, h23-12_VH1 The humanized design of h23-12 antibody light and heavy chain variable region (h23-12_VL1, h23-12_VH1) sequences were fully synthesized, and the humanized h23-12_VH1 was cloned into the eukaryotic transient expression vector pKN041 by restriction enzyme digestion.
  • the upstream of the human light chain C ⁇ coding gene of the transient expression vector pKN019, the light chain constant region nucleotide sequence is shown in SEQ ID NO: 60, and the amino acid sequence is shown in SEQ ID NO: 38, to construct humanized 23-12 light and heavy chains Expression vector, obtain light chain (pKN019-h23-12L1) and heavy chain (pKN041-h23-12H1) expression plasmids, transfer them into E. coli for amplification, and isolate and obtain h23-12 antibody light chain and heavy chain plasmids h23-12L1 h23-12H1.
  • the StarMut gene site-directed mutagenesis kit (GenStar, Cat.: T111-01) was used to express plasmids in the light chain (pKN019-h23-12L1) and heavy chain (pKN041-h23-12H1). Site-directed mutagenesis was carried out on the above and transformed into Escherichia coli for amplification to obtain expression plasmids with mutations in the CDR regions of the light and heavy chains of the h23-12 antibody (h23-12H2 ⁇ h23-12H7, h23-12L2 ⁇ h23-12L7), respectively corresponding to those in Table 3.
  • h23-12-1 means that the antibody is composed of a 23-12 humanized antibody light chain h23-12L1 and a humanized heavy chain h23-12H1, others analogy.
  • Human Trop-2-His recombinant protein (sequence number: NP_002344.2, 1aa-274aa) was used as the mobile phase, and the concentration of Trop-2-His recombinant protein was 60 nM. The binding time is 100s, and the dissociation time is 300s. After the experiment was completed, the blank control response value was subtracted, and the software was used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding.
  • the combined antibody affinity is 5.02E-10M, named h23-12, for further functional verification, the nucleotide sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 57, amino acid For the sequence, see SEQ ID NO: 14; for the nucleotide sequence of the light chain variable region, see SEQ ID NO: 58, and for the amino acid sequence, see SEQ ID NO: 33.
  • a comprehensive analysis of the murine antibody heavy chain sequence is performed to determine the antigen complementarity determinant (CDR) region where the antibody binds to the antigen and the framework that supports the conservative three-dimensional conformation of the antibody. Then, according to the result of homology comparison, search for the most similar human antibody template in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX), and combine the full sequence
  • SEQ ID NO: 45 for the nucleotide sequence of the humanized heavy chain variable region h4-3_VH1 of the 4-3 antibody CDR grafting, and SEQ ID NO: 3 for the amino acid sequence; see the humanized light chain variable region h4-3_VL1 nucleus See SEQ ID NO: 46 for the nucleotide sequence and SEQ ID NO: 20 for the amino acid sequence.
  • the CDR region sequence of the humanized light and heavy chain variable region of the CDR grafted was designed for mutation, and the mutation sites are shown in Table 6 below.
  • h4-3 antibody light and heavy chain variable region (h4-3_VL1, h4-3_VH1) sequence was fully synthesized, and the humanized h4-3_VH1 was cloned into the eukaryotic transient expression vector pKN041 by restriction enzyme digestion.
  • the upstream of the human light chain C ⁇ coding gene of the transient expression vector pKN019, the light chain constant region nucleotide sequence is shown in SEQ ID NO: 60, and the amino acid sequence is shown in SEQ ID NO: 38, to construct humanized 4-3 light and heavy chains Expression vector to obtain the light chain (pKN019-h4-3L1) and heavy chain (pKN041-h4-3H1) expression plasmids, transfer them into E. coli for amplification, and isolate the h4-3 antibody light chain and heavy chain plasmid h4-3L1 h4-3H1.
  • the StarMut gene site-directed mutagenesis kit (Cat.: T111-01, GenStar) was used to express plasmids in the light chain (pKN019-h4-3L1) and heavy chain (pKN041-h4-3H1) respectively.
  • Site-directed mutagenesis was carried out on the above and transformed into Escherichia coli for amplification, and the expression plasmids (h4-3H2 ⁇ h4-3H4, h4-3L2 ⁇ h4-3L5) of the h4-3 antibody light and heavy chain CDR region mutations were obtained, respectively corresponding to the ones in Table 6.
  • h4-3-1 means that the antibody is composed of 4-3 humanized antibody light chain h4-3L1 and humanized heavy chain h4-3H1, others analogy.
  • AHC Anti-human antibody Fc segment capture antibody
  • Human Trop-2-His recombinant protein (sequence number: NP_002344.2, 1aa-274aa) was used as the mobile phase, and the concentration of Trop-2-His recombinant protein was 60 nM.
  • the binding time is 100s, and the dissociation time is 300s.
  • the blank control response value is subtracted, and the software is used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding.
  • Antibody combination KD value (M) Antibody combination KD value (M) Sacituzumab 7.23E-10 h4-3-10 7.63E-10 ch4-3 3.00E-10 h4-3-11 8.02E-10 h4-3-1 3.04E-10 h4-3-12 1.03E-09 h4-3-2 4.11E-10 h4-3-13 2.81E-10 h4-3-3 5.01E-10 h4-3-14 5.71E-10 h4-3-4 6.36E-10 h4-3-15 6.87E-10 h4-3-5 2.73E-10 h4-3-16 1.53E-09 h4-3-6 3.97E-10 h4-3-17 5.42E-10 h4-3-7 4.66E-10 h4-3-18 6.71E-10 h4-3-8 8.62E-10 h4-3-19 5.99E-10 h4-3-9 3.17E-10 h4-3-20 8.92E-10
  • h4-3-1 the combined antibody affinity (KD) is 3.04E-10M, named h4-3, for further functional verification, the antibody heavy chain variable region nucleotide sequence is shown in SEQ ID NO: 45, amino acid For the sequence, see SEQ ID NO: 3; for the nucleotide sequence of the light chain variable region, see SEQ ID NO: 46, and for the amino acid sequence, see SEQ ID NO: 20.
  • a comprehensive analysis of the murine antibody heavy chain sequence is performed to determine the antigen complementarity determinant (CDR) region where the antibody binds to the antigen and the framework that supports the conservative three-dimensional conformation of the antibody. Then, according to the result of homology comparison, search for the most similar human antibody template in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX), and combine the full sequence
  • the humanized heavy chain variable region h11-4_VH1 nucleotide sequence of 11-4 antibody CDR grafting is shown in SEQ ID NO: 49, and the amino acid sequence is shown in SEQ ID NO: 6; the light chain variable region h11-4_VL1 nucleotide sequence See SEQ ID NO: 50, and the amino acid sequence see SEQ ID NO: 25.
  • the CDR grafted humanized light and heavy chain variable region sequence was subjected to mutation design, and the mutation sites are shown in Table 9 below.
  • h11-4_VL1, h11-4_VH1 The humanized design of the h11-4 antibody light and heavy chain variable region (h11-4_VL1, h11-4_VH1) sequence was fully synthesized, and the humanized h11-4_VH1 was cloned into the eukaryotic transient expression vector pKN041 by restriction enzyme digestion.
  • the upstream of the human light chain C ⁇ coding gene of the transient expression vector pKN019, the light chain constant region nucleotide sequence is shown in SEQ ID NO: 60, and the amino acid sequence is shown in SEQ ID NO: 38, to construct the humanized 11-4 light and heavy chains Expression vector, obtain light chain (pKN019-h11-4L1) and heavy chain (pKN041-h11-4H1) expression plasmids, transfer them into E. coli for amplification, and isolate and obtain h11-4 antibody light chain and heavy chain plasmids h11-4L1 h11-4H1.
  • the StarMut gene site-directed mutagenesis kit (Cat.: T111-01, GenStar) was used to express plasmids in the light chain (pKN019-h11-4L1) and heavy chain (pKN041-h11-4H1).
  • h11-4 antibody light and heavy chain mutant expression plasmids (h11-4H2 ⁇ h11-4H7, h11-4L2 ⁇ h11-4L5), respectively corresponding to 11- in Table 9 4 Humanized sequence; according to the operating instructions of the transfection reagent 293fectin (Cat.:12347019, Gibco), combine the light and heavy chain plasmids of the 11-4 humanized antibody, see Table 10 for the combination, and transfer it into HEK293 cells Recombinant expression.
  • h11-4-1 means that the antibody is composed of 11-4 humanized antibody light chain h11-4L1 and humanized heavy chain h11-4H1, others analogy.
  • the 11-4 antibody and the control antibody Sacituzumab were diluted to 4 ⁇ g/mL with PBS buffer, and flowed over the surface of the AHC probe (Cat.:18-0015, PALL) for 120s.
  • Human Trop-2-His recombinant protein (sequence number: NP_002344.2, 1aa-274aa) was used as the mobile phase, and the concentration of Trop-2-His recombinant protein was 60 nM. The binding time is 100s, and the dissociation time is 300s. After the experiment was completed, the blank control response value was subtracted, and the software was used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding.
  • Antibody combination KD value (M) Antibody combination KD value (M) Sacituzumab 7.84E-10 h11-4-10 3.51E-10 ch11-4 2.64E-10 h11-4-11 3.75E-10 h11-4-1 2.16E-09 h11-4-12 2.82E-10 h11-4-2 9.91E-10 h11-4-13 2.11E-10 h11-4-3 8.68E-10 h11-4-14 3.52E-10 h11-4-4 8.11E-10 h11-4-15 3.89E-10 h11-4-5 1.08E-09 h11-4-16 2.31E-10 h11-4-6 9.52E-10 h11-4-17 2.38E-10 h11-4-7 8.03E-10 h11-4-18 3.94E-10 h11-4-8 2.22E-10 h11-4-19 2.54E-10 h11-4-9 3.01E-10 h11-4-20 2.10E-10
  • Example 10 ELISA to detect the species specificity of anti-Trop-2 humanized antibody binding to Trop-2
  • the human Trop-2-his recombinant protein (sequence number: NP_002344.2, 1aa-274aa), the cynomolgus Trop-2-His recombinant protein (sequence number: UniProtKB-A0A2K5UE71, 1aa-272aa), mouse Trop-2 -His recombinant protein (Cat.:50922-M08H, Beijing Yiqiao Shenzhou) coated overnight at 4°C with a coating concentration of 1 ⁇ g/mL; after washing the plate 3 times with PBS, add 5% BSA PBS, block at 37°C for 60 min, wash with PBST Plate 3 times; add h23-12 of different dilution multiples (initial concentration is 10 ⁇ g/mL, 3 times gradient is diluted 14 concentrations), h4-3 (initial concentration is 3 ⁇ g/mL, 3 times gradient is sequentially diluted 12 Concentration), Sacituzumab (initial concentration 3 ⁇ g/m
  • the anti-human antibody Fc segment capture antibody (AHC) biological probe is used to capture the Fc segment of the antibody to determine the affinity of the antibody.
  • the antibodies (h23-12, h4-3 and control antibody Sacituzumab) were diluted to 4ug/mL with PBS buffer, and flowed over the surface of the AHC probe (Cat.:18-0015, PALL) for 120s.
  • Human Trop-2-his recombinant protein (serial number: NP_002344.2, 1aa-274aa) is used as the mobile phase, and the corresponding Trop-2-his concentration of each antibody is h23-12: 23, 30, 45, 75 nM; h4 -3: 23, 30, 45, 60 nM; Sacituzumab: 23, 30, 45, 75 nM.
  • the binding time is 100s, and the dissociation time is 300s.
  • the blank control response value was subtracted, and the software was used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding.
  • the reaction curves of h23-12, h4-3 and control antibody Sacituzumab with human Trop-2 recombinant protein are shown in Figure 7.
  • the curve is fitted and the affinity is calculated.
  • the affinity (KD) for h23-12 is 6.40E-10M, h4-3
  • the affinity (KD) is 5.45E-10M
  • the affinity (KD) of sacituzumab is 9.41E-10M.
  • the detailed kinetic parameters are shown in Table 13. The results show that h23-12 and h4-3 have high affinity with human Trop-2, which is comparable to the control antibody Sacituzumab, and the dissociation value of h23-12 is better than that of Sacituzumab.
  • Example 12 Anti-human Trop-2 humanized antibody binds to the internalization activity of cell surface Trop-2
  • the h23-12 labeled with Mix-n-Stain TM CF TM 488A (Cat.: MX488AS100, Sigma) and the control antibody Sacituzumab were diluted with RPMI 1640 (containing 10% FBS) to 15 ⁇ g/mL, and added to NCI-N87 cells
  • RPMI 1640 containing 10% FBS
  • One group was placed in a 37°C electric thermostat incubator, and the other group was placed in a 4°C refrigerator as a negative control; the negative control was incubated for 30 minutes and washed with PBS three times, observed and photographed with a fluorescence microscope, and the experimental group was incubated at 37°C for 5 hours and then observed with a fluorescence microscope And take pictures.
  • Example 7 The method shown in Example 7 was used to detect the internalization rate on BxPC cells at 3 h by FACS. The results are shown in Table 14, suggesting that the internalization rate after humanization is equivalent to Sac.
  • mice Four healthy female 5-week-old Balb/C nude mice were divided into a group, and a single single dose (20mg/kg) was injected intraperitoneally with h4-3 at 4h, 8h, 24h, 48h, 96h, and 144h. Serum was collected at 192h, 240h and stored at -20°C to observe its pharmacokinetic properties.
  • the anti-human antibody Fc segment capture antibody (AHC) biological probe is used to capture the Fc segment of the antibody to determine the affinity of the antibody.
  • ADC drug SN38-labeled antibody First, prepare ADC drug SN38-labeled antibody. Use 20 equivalents of dithiothreitol (DTT) to reduce the antibody in a sodium phosphate buffer within the pH range of 7.0 ⁇ 0.5 for 2 hours, and then use an ultrafiltration centrifuge tube to purify the reduced antibody to remove excess DTT. The reduced antibody is replaced with a sodium phosphate solution with a pH of 7.0 ⁇ 0.5. Using 7-15% v/v DMSO as a co-solvent, the reduced antibody was incubated with CL2A-SN-38 at ambient temperature for 30 minutes. Finally, the excess small molecules are removed by ultrafiltration centrifuge tubes. Use mass spectrometry to analyze the molecular weight of the antibody-conjugated drug, and calculate the antibody-conjugated ratio (DAR) value of the antibody. In the end, each antibody carries an average of 7.5 SN38 molecules.
  • DTT dithiothreitol
  • the SN38-labeled antibodies prepared as described above: h23-12-SN38, ch4-3-SN38, ch11-4-SN38, positive control antibody Sacituzumab-SN38, and naked antibodies h23-12, ch4-3, ch11 -4.
  • the positive control antibody Sacituzumab is diluted to 4 ⁇ g/mL with PBS buffer, and flows over the surface of the AHC probe (Cat.:18-0015, PALL) for 120s.
  • Human Trop-2-his recombinant protein serial number: NP_002344.2, 1aa-274aa
  • the binding time is 300s, and the dissociation time is 300s.
  • the blank control response value was subtracted, and the software was used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding.
  • the antibodies to be tested include the ADC drug SN38-labeled antibodies prepared as described above: h23-12-SN38, ch4-3-SN38, ch11-4-SN38, positive control antibody Sacituzumab-SN38, and naked anti-h232-12, ch4- 3. ch11-4, positive control antibody Sacituzumab and negative isotype control antibody NC-IgG1, 10ug/ml.
  • the internalization ratio of h23-12 naked antibody and SN38-labeled ADC antibody is similar, and the degree of internalization is similar to that of the control antibody; while ch4-3 and ch11-4-labeled SN38 ADC antibody are more The internalization ratio of naked antibodies is high.
  • mice Take five-week-old, female BALB/c nude mice, subcutaneously inoculate 3 ⁇ 10 6 human gastric cancer cells (NCI-N87), and randomly group them when the tumor grows to about 150 mm 3 , 6 mice per group, grouping and dosage, The frequency is shown in Table 21.
  • Each group is administered intravenously twice a week, and the tumor volume and mouse body weight are measured at the same time. When the weight of the mouse drops by more than 15%, or the tumor volume of a single animal exceeds 3000mm 3 or a group of animals The experiment was stopped when the average tumor volume exceeded 2000 mm 3 and the mice were euthanized.
  • the anti-Trop2-ADC antibody has a dose-dependent inhibitory effect on tumor growth.
  • high doses (10mg/kg) no difference in efficacy between the ADC antibodies is observed, and No obvious toxic effect of ADC small molecule SN38 was observed, and the weight of animals in each experimental group increased steadily, which was not significantly different from the control.
  • Example 18 Pharmacodynamic evaluation of anti-Trop2 antibody and anti-CD47 antibody in combination with SKOV3 subcutaneous xenograft tumor model
  • mice Five-week-old, female BALB/c nude mice were inoculated subcutaneously in the right flank of each mouse with 3 ⁇ 10 6 human ovarian cancer cells (SKOV3). When the tumor grew to about 150 mm 3 , they were randomly divided into groups, 6 mice/ The group, grouping, dosage and frequency of administration are shown in Table 22. Each group was administered by intraperitoneal injection twice a week for a total of 5 times. The tumor volume and the weight of the mice were measured at the same time, and the state of the mice was observed. After the last dose, the mice were euthanized.
  • the anti-CD47 antibody refer to the patent application publication US20150183874A1, which is humanized 5F9version 2.
  • the h23-12+Anti-CD47 combination group showed certain anti-tumor activity, while the Anti-CD47 and h23-12 single-drug groups had no obvious anti-tumor effect.
  • the Trop2 antibody and the anti-CD47 antibody of the present invention can synergistically promote the phagocytosis of tumor cells by macrophages, and therefore have a synergistic anti-tumor effect.
  • mice Take five-week-old, female BALB/c nude mice, subcutaneously inoculate 3 ⁇ 10 6 human gastric cancer cells (NCI-N87), and randomly group them when the tumor grows to about 100 mm 3 , 6 mice/group, grouping and dosage, The frequency is shown in Table 23.
  • Each group was administered intravenously twice a week for a total of 6 weeks.
  • the tumor volume and the weight of the mouse were measured at the same time. When the weight of the mouse fell by more than 15%, or the tumor volume of a single animal exceeded The experiment was stopped when 3000 mm 3 or the average tumor volume of a group of animals exceeded 2000 mm 3 and the mice were euthanized.
  • anti-Trop2-ADC antibody has a dose-dependent inhibitory effect on tumor growth.
  • ADC antibody ch3-11-SN38 and ch11-4-SN38 are slightly more effective than Sacituzumab -SN38, and no obvious toxic effect of ADC small molecule SN38 has been observed.

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Abstract

本发明提供了一种结合人肿瘤相关钙信号传感器2(Trop-2)蛋白的抗体或其片段，以及所述抗体或其片段用于预防或治疗疾病的用途。本发明的抗体或其片段能够有效结合人Trop-2蛋白，而且具有内化活性，并在ADC药物标记后内化活性增强，且在小鼠模型的体内药效和安全性上不低于对照抗体。

Description

抗人Trop-2抗体及其应用
本专利申请要求于2019年10月11日提交的申请号为CN201910962965.1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
本发明属于生物医药领域，涉及一种新的抗人Trop-2抗体或其功能性片段。本发明还涉及所述抗体或其功能性片段的应用。
背景技术
Trop-2(Tumor-associated calcium signal transducer 2)，又名肿瘤相关钙信号传感器(tumor-associated calcium signal transducer，TACSTD2)、上皮糖蛋白-1抗原(epithelial glycoprotein-1，EGP-1)、胃肠肿瘤相关抗原1(gastrointestinal tumor-associated antigen，GA733-1)，是一类由TACSTD2基因编码的细胞表面糖蛋白，全长323个氨基酸，胞外区由3个结构域组成，证实可以二聚体形式存在。
Trop-2是跨膜糖蛋白，与其它原癌基因不同，Trop2没有突变，这意味着它没有导致过表达的遗传组成改变。Trop-2通过ERK/MAPK和cyclin D1通路刺激细胞生长，从而促进肿瘤的侵袭、血管生成、肿瘤进展以及耐药等机制。已发现Trop-2在多种肿瘤中高表达，尤其是三阴性乳腺癌、非小细胞肺癌等，且与预后相关。相对地，Trop-2在正常组织中表达极低，为ADC药物比较理想的靶点。
靶向trop-2的抗体药物目前主要以ADC药物进行开发，不完全统计，临床在研品种在3个以上，其中的小分子偶联物主要包括伊利替康的衍生物和微管蛋白抑制剂。目前认为，选用新颖的低毒性的拓扑异构酶抑制剂代谢产物SN-38比较理想。SN-38与现有微管抑制剂、DNA烷化剂的肿瘤抑制存在差异，特别适用于存在较高异质性、多耐药机制的肿瘤，如三阴乳腺癌、胰腺癌、胃癌等肿瘤。临床进展最快的项目为Immunomedics的IMMU-132项目，已经进行了针对复发和转移的三阴乳腺癌(ASCENT-Study)的三期临床试验、单用或与卡铂联用治疗三阴乳腺癌(NCT02161679)的二期临床试验、治疗尿路上皮癌(NCT03547973)的二期临床试验、治疗实体瘤包括胃癌、 宫颈癌、小细胞肺癌(NCT01631552)的一期/二期临床试验等。具有类似技术的抗体偶联药物项目还有第一三共、辉瑞公司以及各大具有ADC基础的药物公司等。
目前抗Trop-2的抗体临床在研品种较少。因此，本领域仍需要寻找新型的、特别适合开发ADC药物的抗Trop-2抗体。尽管前期研究中Trop-2作为肿瘤治疗性靶点展示出良好的应用前景，但由于Trop-2表位序列结构及生物学功能不明确，因此在抗Trop-2单克隆抗体药物研发中，前期体外筛选中获得的高亲和力单抗，在体内试验中却往往表现出较低的肿瘤靶向活性和细胞毒素递送能力，这也直接导致迄今为止尚没有抗Trop-2单克隆抗体药物完成临床有效性试验。
发明内容
基于现有技术中Trop-2作为肿瘤治疗靶点的机理、抗Trop-2抗体药物的临床试验进展，发明人经过深入研究分析发现抗Trop-2抗体临床前试验、临床试验的有效性未达到理论预期的原因不仅在于单纯的对Trop-2亲和力不足；而是由于抗体结合的最佳表位通常为Trop-2的重组蛋白或其片段，而并非Trop-2的天然构象，因而尽管体外阶段能够筛选获得抗Trop-2的高亲和力抗体，然而其在体内靶向Trop-2胞外区天然构象的有效亲和力仍然不足。
一方面，如果采用重组蛋白作为免疫原和包被抗原，其优点在于免疫原易纯化、外源抗原结构单一，容易筛选获得针对所述重组蛋白的单抗。然而，重组表达蛋白的修饰(例如糖基化)、折叠方式等往往受到重组表达系统的影响。即使采用哺乳动物表达系统，纯化获得的游离重组Trop-2或其胞外区也与细胞膜表面结合的Trop-2胞外区天然构象有所差异。这种蛋白构象或空间结构的差异导致其免疫产生的抗体的特异性以及在体内针对Trop-2胞外区天然构象的有效亲和力。
另一方面，如果采用细胞膜表面Trop-2阳性的细胞作为免疫原和筛选抗原，其优点在于所述Trop-2胞外区具有天然构象，体外对所述Trop-2胞外区具有高亲和力的单抗预期在体内也将具有针对Trop-2胞外区天然构象的高亲和力。然而，细胞膜表面Trop-2阳性的细胞作为免疫原抗原种类复杂，容易屏蔽Trop-2胞外区的免疫原性；此外，以细胞膜表面Trop-2阳性的细 胞作为筛选抗原进行流式细胞筛选成本高、筛选效率低。
基于上述前期研究报道，本发明根据Trop-2重组蛋白、Trop-2阳性细胞在制备单克隆抗体中各技术路线的优缺点。以重组Trop-2蛋白为免疫原免疫动物制备杂交瘤细胞避免了使用复杂抗原作为免疫原时对Trop-2的屏蔽作用；以重组Trop-2蛋白作为包被抗原，通过ELISA筛选策略进行初筛以使筛选具有高通量；以膜表面Trop-2阳性细胞对初筛阳性的杂交瘤进行复筛以确保获得的阳性抗体能够结合Trop-2胞外区的天然构象。
本发明进一步要解决的技术问题是使抗Trop-2单克隆抗体在临床前的动物试验阶段能够更接近真实的人体内试验结果。为此基于人Trop-2胞外区与猴Trop-2胞外区的共同结构，通过种属交叉反应筛选获得能够特异性结合人Trop-2胞外区和猴Trop-2胞外区共同结构的单克隆抗体。为了确保抗Trop-2单抗的高度特异性，排除对鼠Trop-2胞外区具有特异性结合能力的抗体。
针对上述技术问题，本发明通过杂交瘤筛选和人源化技术提供一种抗Trop-2抗体，所述抗体对人Trop-2具有高亲和力，具有癌细胞的特异性杀伤作用；同时，所述抗体具有高内化能力，特别适合于开发ADC药物。
具体而言，本发明提供如下技术方案。
一方面，本发明提供一种抗Trop-2单克隆抗体的制备方法，其包括以下步骤：
(1)以重组Trop-2蛋白为免疫原免疫动物制备杂交瘤细胞；
(2)以重组Trop-2蛋白为包被抗原筛选分泌抗Trop-2单克隆抗体的阳性杂交瘤细胞；
(3)以膜表面Trop-2阳性细胞对步骤(2)中的阳性杂交瘤进行复筛；
所述抗Trop-2单克隆抗体特异性识别并结合Trop-2胞外区天然表位。
优选的，本发明所述抗Trop-2单克隆抗体的制备方法，其中，所述步骤(3)中膜表面Trop-2阳性细胞为重组动物细胞，且所述动物细胞来自与步骤(1)制备杂交瘤细胞时免疫的动物相同的物种。
更优选的，本发明所述抗Trop-2单克隆抗体的制备方法，其中，所述步骤(1)中免疫小鼠制备杂交瘤细胞，步骤(3)中膜表面Trop-2阳性细胞为表达外源Trop-2蛋白的重组小鼠细胞。
优选的，本发明所述抗Trop-2单克隆抗体的制备方法，其中，所述步骤 (2)采用酶联免疫吸附测定(ELISA)筛选分泌抗Trop-2单克隆抗体的阳性杂交瘤细胞；步骤(3)采用流式细胞分析(FACS)复筛获得分泌特异性识别并结合Trop-2胞外区天然表位的抗体的杂交瘤。
优选的，本发明所述抗Trop-2单克隆抗体的制备方法，其中，还包括(4)对特异性识别并结合Trop-2胞外区天然表位的抗体进行鉴定的步骤，所述鉴定包括亲和力鉴定和特异性鉴定。
优选的，本发明所述抗Trop-2单克隆抗体的制备方法，其中，步骤(4)中选择对人Trop-2、猴Trop-2具有特异性结合能力，而对鼠没有特异性结合能力的单克隆抗体。
另一方面，本发明提供一种抗体或其片段，所述抗体或其片段包含重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)包含选自以下的CDR组合(HCDR1、HCDR2、HCDR3；LCDR1、LCDR2、LCDR3)：
  HCDR1 HCDR2 HCDR3 HCDR1 HCDR2 HCDR3
1 SYWMY EINPSNGRTNYNEKFKS EGHNYDGSLGAMDH RSSQSLTNSYGNTFLS GISNRFS FQSTHQPYT
2 DYVIG EIYLGSGTIYYTEKFKG GSIFPFDY SASSSVSYMY DTSTLAS QQWSSYPYT
3 DYVIG EIYLGSGTIYYAEKFKG GSIFPFDY SASSSVSYMY DTSTLAS QQWSSYPYT
4 DYVIG EIYLGSGTIYYTEKFKG GSIFPFDY RASSSVSYMY DTSTLAS QQWSSYPYT
5 DYVIG EIYLGSGTIYYAEKFKG GSIFPFDY RASSSVSYMY DTSTLAS QQWSSYPYT
6 DYVIG EIYLGSGTIYYTEKFKG GSIFPFDY SASSSVSYMY DASTLAS QQWSSYPYT
7 DYVIG EIYLGSGTIYYTEKFKG GSIFPFDY SASSSVSYMY DTSTLQS QQWSSYPYT
8 SYWIN NIYPSNSYTNYNQKFKD YRSDGFAY RASQNIGTSIH FASESIS QQSNSWPFT
9 SYWIN NIYPSNSYTNYNQKFKD YRSDGFAY RASQNIGTSIE FASESIS QQSNSWPFT
10 SYWIN NIYPSNSYTNYNQKFKD YRSEGFAY RASQNIGTSIE FASESIS QQSNSWPFT
11 SYWIN NIYPSNSYTNYNQKFKD YRSGGFAY RASQNIGTSIE FASESIS QQSNSWPFT
12 SYWIN NIYPSNSYTNYNQKFKD YRSDAFAY RASQNIGTSIE FASESIS QQSNSWPFT
13 SYWIN NIYPSNSYTNYNQKFKD YRSDGFAY RASQNIGTSIS FASESIS QQSNSWPFT
14 SYWIN NIYPSNSYTNYNQKFKD YRSEGFAY RASQNIGTSIS FASESIS QQSNSWPFT
15 SYWIN NIYPSNSYTNYNQKFKD YRSGGFAY RASQNIGTSIS FASESIS QQSNSWPFT
16 SYWIN NIYPSNSYTNYNQKFKD YRSDAFAY RASQNIGTSIS FASESIS QQSNSWPFT
17 SYWIN NIYPSNSYTNYNQKFKD YRSDGFAY RASQNIGTSIA FASESIS QQSNSWPFT
18 SYWIN NIYPSNSYTNYNQKFKD YRSEGFAY RASQNIGTSIA FASESIS QQSNSWPFT
19 SYWIN NIYPSNSYTNYNQKFKD YRSGGFAY RASQNIGTSIA FASESIS QQSNSWPFT
20 SYWIN NIYPSNSYTNYNQKFKD YRSDAFAY RASQNIGTSIA FASESIS QQSNSWPFT
21 DSAMS SISRGDDTYYPDSVKG DRFGFAY KSGQSLLDSDGKTYFN LVSMLDS WQGTHFPFT
22 SYWMH EITPSDNYTSYNQKFKG GHGNYVSFDY RASQDISNYLN YTSRLHS QQGYTLPPYT
23 SYWMH EITPSDNYTSYNQKFKG GEGNYVSFDY RASQDISNYLN YTSRLHS QQGYTLPPYT
24 SYWMH EITPSDNYGSYNQKFKG GHGNYVSFDY RASQDISNYLN YTSRLES QQGYTLPPYT
25 SYWMH EITPSDNYTSYNQKFKG GEGNYVSFDY RASQDISNYLN YTSRLES QQGYTLPPYT
26 SYWMH EITPSDNYGSYNQKFKG GHGNYVSFDY RASQDISNYLN YTSRLQS QQGYTLPPYT
27 SYWMH EITPSDNYGSYNQKFKG GHGNYVSFDY RASQDISNYLN YTSRLHS QQYYTLPPYT
28 SYWMH EITPGDNYTSYNQKFKG GHGNYVSFDY RASQDISNYLN YTSRLHS QQGYSLPPYT
29 SYWMH EITPSDNYGSYNQKFKG GHGNYVSFDY RASQDISNYLN YTSRLHS QQGYSLPPYT
30 SYWMH EITPSDNYTSYNQKFKG GEGNYVSFDY RASQDISNYLN YTSRLHS QQGYSLPPYT
所述抗体或其片段的结合人Trop-2。
优选地，所述重链可变区包含选自以下的序列：
SEQ ID NO:1至SEQ ID NO:17中任一个所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的氨基酸序列；和/或
所述轻链可变区包含选自以下的序列：
SEQ ID NO:18至SEQ ID NO:36中任一个所示的氨基酸序列或与所示的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，本发明的抗体或其片段包含的重链可变区和轻链可变区可以选自以下组合：
(1)如SEQ ID NO:1所示的氨基酸序列或与如SEQ ID NO:1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:18所示的氨基酸序列或与如SEQ ID NO:18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:2所示的氨基酸序列或与如SEQ ID NO:2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: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；
(3)如SEQ ID NO:3所示的氨基酸序列或与如SEQ ID NO:3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:20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；
(4)如SEQ ID NO:4所示的氨基酸序列或与如SEQ ID NO:4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:20所示的氨基酸序列或与如SEQ ID NO:20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:3所示的氨基酸序列或与如SEQ ID NO:3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: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；
(6)如SEQ ID NO:4所示的氨基酸序列或与如SEQ ID NO:4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: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；
(7)如SEQ ID NO:3所示的氨基酸序列或与如SEQ ID NO:3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:3所示的氨基酸序列或与如SEQ ID NO:3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:5所示的氨基酸序列或与如SEQ ID NO:5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:24所示的氨基酸序列或与如SEQ ID NO:24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:6所示的氨基酸序列或与如SEQ ID NO:6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: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:26所示的氨基酸序列或与如SEQ ID NO:26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: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: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；
(13)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；和，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；
(14)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: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；
(15)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: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；
(16)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: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；
(17)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；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；
(18)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: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；
(19)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: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；
(20)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: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；
(21)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；和，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；
(22)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: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；
(23)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: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；
(24)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；和，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；
(25)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；和，如SEQ ID NO:31所示的氨基酸序列或与如SEQ ID NO:31所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(26)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: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；
(27)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: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；
(28)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；和，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；
(29)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: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；
(30)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；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；
(31)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；和，如SEQ ID NO:35所示的氨基酸序列或与如SEQ ID NO:35所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(32)如SEQ ID NO:15所示的氨基酸序列或与如SEQ ID NO:15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:36所示的氨基酸序列或与如SEQ ID NO:36所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(33)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；和，如SEQ ID NO:36所示的氨基酸序列或与如SEQ ID NO:36所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(34)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:36所示的氨基酸序列或与如SEQ ID NO:36所示的氨基酸序列具有至少75％同一性的氨基酸序列。
一般地，所述抗体或其片段为单克隆抗体、单链抗体、双功能抗体、单域抗体、纳米抗体、完全或部分人源化的抗体或者嵌合抗体等任意形式，或者，所述抗体或其片段为半抗体或半抗体的抗原结合片段，例如scFv、BsFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv；所述抗体或其片段可为小鼠、大鼠、人或任何其它来源的；
优选地，所述抗体或其片段还包含人或鼠的恒定区，优选包含人或鼠的轻链恒定区(CL)和/或重链恒定区(CH)；
更优选地，所述抗体或其片段包含选自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，所述抗体为单克隆抗体，优选为鼠源、嵌合或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或IgG4亚型，轻链恒定区为κ型；
根据本发明的具体实施方式，所述单克隆抗体的重链恒定区包含如SEQ ID NO:37所示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；
优选地，所述单克隆抗体的轻链恒定区包含如SEQ ID NO:38所示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。
本发明上文所述的至少75％同一性为至少80％、优选至少85％、更优选至少90％、进一步优选至少91％、92％、93％、94％、95％、96％、97％、98％或甚至99％同一性等≥75％的任何百分比的同一性。
基于本发明的抗体或其片段，另一方面，本发明还提供一种核酸分子，其编码本发明任意抗体或其片段中的重链CDR、轻链CDR、重链可变区、轻链可变区、重链或轻链。
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，另一方面，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。
基于本发明的公开内容，本发明提供的抗体或其片段、核酸分子、载体和/或宿主细胞可以通过使用本领域已知的任何常规技术方法获得。所述抗体或其片段、核酸分子、载体和/或宿主细胞可以被包含在药物组合物中，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，本发明还提供一种药物组合物，所述药物组合物包含本发明所述的抗体或其片段、核酸分子、载体和/或宿主细胞，以及任选地药学上可接受的辅料。
本发明的抗体或其片段能够与具有巨噬细胞吞噬作用的其他抗体类药物联合使用。因此优选地，所述抗体类药物通过与细胞表面表达蛋白的结合促进巨噬细胞对所述细胞的吞噬。因此，本发明提供的药物组合物还可包含所述其他抗体类药物，优选巨噬细胞类免疫检查点抗体；根据本发明的具体实施方式，所述抗体为抗CD47抗体。
作为结合人Trop-2或其任何部分的抗体，本发明还提供上述主题的相关应用。
具体而言，再一方面，本发明提供所述的抗体或其片段、核酸分子、载体、宿主细胞和/或药物组合物在制备药物中的用途，所述药物优选地用于治疗Trop-2高表达癌症；优选地，所述Trop-2高表达癌症为胃癌、胰腺癌、肠癌、卵巢癌、鳞状肺癌、非小细胞肺癌、小细胞肺癌、尿路上皮癌、三阴性乳腺癌或宫颈癌。
在这一方面，所述用途涵盖本发明的抗体或其片段与上文所述的其他抗体类药物联合制备所述药物的用途。
本发明提供的抗体或其片段还可以与其他部分融合或缀合。例如，本发明提供一种融合蛋白或缀合物，其包含本发明的抗体或其片段。
关于融合蛋白，所述融合蛋白可包含对本发明所述抗体或其片段进行修饰的任何其它部分，例如氨基酸、多肽或蛋白。
关于缀合物，所述缀合物可包含本发明的抗体或其片段以及与其缀合的药物，其中所述药物例如为细胞毒剂。
优选地，所述缀合物为由下式所示的抗体药物缀合物(ADC)：(本发明 的抗体或其片段)-(接头)-(细胞毒剂)；
优选地，所述细胞毒剂为微管蛋白抑制剂(如紫杉醇、多西他赛等)或DNA复制抑制剂(如伊立替康或其代谢活性物SN-38等)。
根据本发明的具体实施方式，所述缀合物为“抗TROP-2抗体-接头-SN-38抗体药物缀合物”。
本发明还提供所述抗体或其片段、核酸分子、载体、宿主细胞和/或药物组合物在制备抗体药物缀合物(ADC)中的用途，所述抗体药物缀合物优选地用于治疗Trop-2高表达癌症；优选地，所述Trop-2高表达癌症为胃癌、胰腺癌、肠癌、卵巢癌、鳞状肺癌、非小细胞肺癌、小细胞肺癌、尿路上皮癌、三阴性乳腺癌或宫颈癌。
另外，本发明提供一种预防和/或治疗疾病的方法，所述方法包括给有此需要的受试者施用本发明的抗体或其片段、核酸分子、载体、宿主细胞、药物组合物、融合蛋白或缀合物，以及任选的其他药物或手段。该任选的其他药物或手段是指可以与本发明的抗体或其片段、核酸分子、载体、宿主细胞、药物组合物、融合蛋白或缀合物联合施用的其他药物或手段，例如小分子化药、靶向药、抗体等重组蛋白药、疫苗、ADC、溶瘤病毒、基因和核酸治疗药物和放射疗法。二者的联合施用可以采取任意形式进行，例如同时、连续或间隔一定时间进行。
优选地，所述疾病为Trop-2高表达癌症；进一步优选地，所述Trop-2高表达癌症为胃癌、胰腺癌、肠癌、卵巢癌、鳞状肺癌、非小细胞肺癌、小细胞肺癌、尿路上皮癌、三阴性乳腺癌或宫颈癌。所述受试者为哺乳类动物，优选地，所述受试者为人。
除非另外指出，术语“免疫球蛋白序列”均用作通用术语，包括全尺寸抗体，其单独的链，以及其所有部分、结构域或片段(分别包括但不限于抗原结合结构域或片段如VHH结构域或VH/VL结构域)。
术语“抗体”应理解是涵盖包含两个免疫球蛋白重链和两个免疫球蛋白轻链的抗体分子(即，“完全抗体分子”)以及其抗原结合片段。如本文所用，术语抗体的“抗原结合部分”、抗体的“抗原结合片段”和类似术语，包括特异性结合抗原以形成复合物的任何天然存在、酶促可获得、合成或基因工程改造的多肽或糖蛋白。如本文所用，术语抗体的“抗原结合片段”或“抗体片段”是指抗体中保留特异性结合于Trop-2的能力的一个或多个片段。抗 体片段可以包括Fab片段、F(ab′)2片段、Fv片段、dAb片段、含有CDR的片段或经分离的CDR。抗体的抗原结合片段可以使用任何适合的标准技术衍生自例如完全抗体分子，所述适合的标准技术是如蛋白水解消化或涉及DNA编码抗体可变和(视情况)恒定域的操纵和表达的重组基因工程改造技术。这类DNA是已知的和/或是从例如商业来源、DNA文库(包括例如噬菌体-抗体文库)轻易可获得的或可以是合成的。可以化学方式或通过使用分子生物学技术对DNA进行测序和操纵，例如将一个或多个可变和/或恒定域布置成适合的组态，或引入密码子；形成半胱氨酸残基；修饰、添加或去除氨基酸等。
抗原结合片段的非限制性实例包括：(i)Fab片段；(ii)F(ab′)2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；(vi)dAb片段；和(vii)由模拟抗体的高变区的氨基酸残基组成的最小识别单位(例如经分离的互补决定区(CDR)，如CDR3肽)，或限制性FR3-CDR3-FR4肽。如以下的其它工程改造分子也涵盖于如本文所用的表述“抗原结合片段”内：结构域-特异性抗体、单-结构域抗体、结构域-缺失抗体、嵌合抗体、CDR-接枝抗体、双功能抗体、三功能抗体、四功能抗体、微型抗体、纳米抗体(nanobody)(例如单价纳米抗体、二价纳米抗体等)、小模块化免疫药物(SMIP)和鲨鱼可变IgNAR结构域。
抗体的抗原结合片段将典型地包含至少一个可变域。可变域可以具有任何尺寸或氨基酸组成，且通常将包含至少一个CDR，其邻近一个或多个构架序列或与其同框。在VH结构域与VL结构域相缔合的抗原结合片段中，VH和VL结构域可以任何适合的排列相对于彼此定位。举例来说，可变区可以是二聚的且含有VH-VH、VH-VL或VL-VL二聚体。或者，抗体的抗原结合片段可以含有单聚VH或VL结构域。
在某些实施例中，抗体的抗原结合片段可以含有共价键联到至少一个恒定域的至少一个可变域。本发明的抗体的抗原结合片段内可以发现的可变和恒定域的非限制性、示例性组态包括：(i)VH-CH1；(ii)VH-CH2；(iii)VH-CH3；(iv)VH-CH1-CH2；(v)VH-CH1-CH2-CH3；(vi)VH-CH2-CH3；(vii)VH-CL；(viii)VL-CH1；(ix)VL-CH2；(x)VL-CH3；(xi)VL-CHl-CH2；(xii)VL-CH1-CH2-CH3；(xiii)VL-CH2-CH3；和(xiv)VL-CL。在可变和恒定域的包括上文所列的示例性组态中的任一个的任何组态中，可变和恒定域可彼此直接键联或可以通过完全或部分铰链或连接符区键联。铰链区可以由至少2(例如5、10、15、20、40、60或更多)个氨基酸组成，其在单一多肽分子 中在邻近可变和/或恒定域之间产生柔性或半柔性键联。此外，本发明抗体的抗原结合片段可以包含上文所列的可变和恒定域组态中的任一个彼此非共价缔合和/或与一个或多个单聚VH或VL结构域(例如通过二硫键)的同二聚体或异二聚体(或其它多聚体)。
就完全抗体分子来说，抗原结合片段可以是单特异性或多特异性的(例如双特异性)。抗体的多特异性抗原结合片段将典型地包含至少两个不同的可变域，其中各可变域能够特异性结合于单独的抗原或相同抗原上不同的表位。任何多特异性抗体格式(包括本文所公开的示例性双特异性抗体格式)均可适合于使用所属领域中可用的常规技术在本发明的抗体的抗原结合片段的背景下使用。
术语“嵌合抗体”是指这样的抗体，其中(a)恒定区或其一部分被改变、取代或交换，使得抗原结合位点(可变区、CDR或其一部分)与具有不同的或改变的类型、效应子功能和/或种类的恒定区连接；或(b)可变区或或其一部分被以具有不同的或改变的抗原特异性的可变区(例如，来自不同的物种的CDR和框架区)改变、取代或交换。嵌合的抗体可包含可变区片段，例如，包含两个Fab或Fv区或scFv的重组抗体。如上文所表明，嵌合体还可包含来自与接合的Fv区不同的来源的Fc区。在一些情形下，嵌合的抗体包含位于Fv区内的嵌合体。这样的嵌合抗体的实例为人源化的抗体，其中Fvs和CDR来自不同的来源。
术语“人源化抗体”是这样的抗体，其中从非人抗体的VH和VL区获得的抗原结合环，即CDR被移植至人框架序列。可根据以下文献中描述的方法进行人源化，即将非人CDR序列取代为人抗体的对应序列，例如，美国专利第5,545,806号；第5,569,825号；第5,633,425号；第5,661,016号；Riechmann et al.,Nature 332:323-327(1988)；Marks et al.,Bio/Technology 10:779-783(1992)；Morrison,Nature 368:812-13(1994)；Fishwild et al.,Nature Biotechnology 14:845-51(1996)。转基因小鼠或其他生物体如其他哺乳动物，也可用于表达人源化的或人抗体，如在第6,673,986号美国专利中公开的。
如本文中使用的，术语“百分比(％)同一性”是指在对齐序列和引入空白后，在必要时，以实现最大百分比同一性(即，可以为最佳对齐而在候选和参照序列的其中之一或两者中引入空白，且可以为比较目的而不管非同源序列)，候选序列例如本发明的分离的抗IL1-RAP抗体的与参照序列的氨基酸 (或核酸)残基相同的氨基酸(或核酸)残基的百分比。可BLAST2.0软件，使用标准设置实现为确定百分比同一性目的的比对。可进行比对来实现在比较序列的全长上的最大对齐。在一些实施方案中，按下式计算给定候选序列与、和或对给定参照序列(其可以可选地表述为给定的候选序列，其具有或包含与、和或对给定参照序列的某一百分比氨基酸(或核酸)序列同一性)的百分比氨基酸(或核酸)序列同一性：
100×(A/B的分数)
其中A为在候选序列和参照序列的对齐中评分为相同的氨基酸(或核酸)残基的数目，且其中B为参照序列中的氨基酸(或核酸)残基的总数目。在其中候选序列的长度不等于参照序列的长度的一些实施方案中，候选序列与参照序列的百分比氨基酸(或核酸)序列同一性将不等于参照序列与候选序列的百分比氨基酸(或核酸)序列同一性。
在特定的实施方案中，对齐以用于与候选序列进行比较的参照序列可显示出在候选序列或候选序列的连续的氨基酸(或核酸)残基的选定部分的全长上显示50％-100％的同一性的候选序列。为比较目的而对齐的候选序列的长度为参照序列长度的至少30％，例如，至少40％，例如，至少50％、60％、70％、80％、90％或100％。当候选序列中的位置被与参照序列中的对应的位置相同的氨基酸(或核酸)残基占据时，则分子在该位置处是相同的。
本文中使用的术语“抗原”、“免疫原”、“抗体靶标”、“靶标分析物”等术语是指可被抗体识别，即，可被抗体特异性结合的分子、化合物或复合物。术语可以指可被抗体，例如，多肽、多核苷酸、碳水化合物、脂类、化学部分或以上的组合(例如，磷酸化的或糖基化的多肽等)特异性识别的任何分子。技术人员将理解该术语不暗示分子在每种情形下均为免疫原性的，但简单地指其可被抗体靶向。
术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核昔酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核昔酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌等原核细胞，如酵母细胞等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
术语“KD”是指特定抗体一抗原相互作用的解离平衡常数(KD)，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体一抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数结合抗原，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。例如用KINEXA方法KINEXA 400仪器上检测到的抗体和细胞结合的亲和力。
术语“特异性结合”指抗体与抗原的一种或多种抗原决定簇反应而不与其他多肽反应或以很低的亲和性(Kd>10-6)结合其他多肽。抗体包括但不限于多克隆、单克隆、嵌合、dAb(结构域抗体)、单链、Fab、Fab’和F(ab’)2片段、Fv、scFv及Fab表达文库。单克隆抗体(mAb)是由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术及合成技术如CDR grafting或其它现有技术进行重组得到。
术语“Trop-2”，TROP2属于TACSTD家族，是由TACSTD2基因编码表达的细胞表面糖蛋白，又名肿瘤相关钙离子信号转导子2(TACSTD2)、表皮糖蛋白1(EGP-1)、胃肠肿瘤相关抗原(GA733-1)、表面标志物1(M1S1)。Trop-2在多种恶性肿瘤中过表达，是一种与恶性肿瘤发生、侵袭和转移有关的癌基因。
Trop-2基因位于第1号染色体的短臂上，具体定位为1p32.1[3]。基因全长为9072bp，无内含子，只有1个外显子。小鼠Trop-2与人类同源基因序列相似度为87.4％。Trop-2蛋白初级结构是由323个氨基酸组成的36kD的多肽，是一种单次跨膜的表面糖蛋白。Trop-2由疏水性前导肽(AA1-26)，细胞外结构域(AA27-274)，一个跨膜结构域(AA275-297)和一个胞质尾部(AA298-323)组成。TROP2蛋白N-端为胞外域(ECD)，该胞外域通过一个单向跨膜螺旋(TM)与胞内短尾(IC)连接，从而固定于胞膜。其细胞质尾部有高度保守的磷脂酰肌醇4，5-二磷酸(PIP2)结合序列，表明PIP2在TROP2的信号转导 中起重要作用。除了PIP2结合基序外，它还含有保守的酪氨酸和丝氨酸磷酸化位点。303位丝氨酸残基的突变消除了Trop-2刺激肿瘤生长的能力。该残基的磷酸化由蛋白激酶C(PKC)负责。
术语“抗体药物偶联物”(antibody-drug conjugate，ADC)，是通过一个化学链接将具有生物活性的小分子药物连接到单抗上，单抗作为载体将小分子药物靶向运输到目标细胞中。
ADC药物中的抗体分子常采用人源化单抗，对可结晶片段(Fc)段进行修饰，以降低抗体依赖性细胞介导细胞毒性(anti-body-dependentcell-mediatedcytotoxicity，ADCC)和补体依赖性细胞毒性(complement dependent cytotoxicity，CDC)等。首先抗体分子作为生物大分子，存在一般生物大分子的毒性风险，如免疫原性和免疫毒性，以及单抗可能的ADCC作用、CDC作用、肾基底膜免疫复合物沉积等。其次，在ADC药物中，抗体分子最重要的作用是靶向作用，即将小分子化合物定向投递到抗原抗体结合位点。如果抗体选择性较差或正常组织存在该抗原，则会造成细胞毒药物投递到正常细胞中，造成靶向毒性。第三，除靶向毒性外，循环过程中的小分子脱落可导致一定程度的脱靶毒性。抗体分子的Fc如存在与免疫细胞Fc受体如FcγRs/FcRN结合的活性，易与免疫细胞结合，造成免疫细胞的杀伤。最后，ADC药物做为外源性生物大分子，还可能在循环中吞噬细胞，通过胞饮作用进入胞内造成细胞死亡。
ADC药物常用的连接子主要包括腙键、二硫键和肽键。腙键在酸性条件下即可发生水解，是相对不稳定的连接子。Mylotarg即采用腙键作为连接子，研究人员认为这是导致Mylotarg失败的一个重要原因。二硫键在细胞内高浓度的谷胱甘肽中可发生水解，因此不易在胞外脱落。肽键的结合最为紧密，仅在溶酶体蛋白水解酶的作用才发生断裂。连接子的稳定性直接影响细胞毒药物的非预期解离情况，这种断裂造成小分子细胞毒药物在体内的暴露，即脱靶毒性。
ADC药物常用的细胞毒药物为临床上常规使用的化疗药物，决定ADC药物主要的毒效应谱。由于已在临床上广泛使用，其毒性特征一般都比较清楚。根据药物类型如微管蛋白聚合抑制剂或DNA损伤剂/DNA复制抑制剂即能较好地把握其毒性风险。其中，所述微管蛋白抑制剂微管蛋白抑制剂包括海兔毒素及其奥瑞他汀类衍生物auristatins(MMAE、MMAF、MMAD)， 美登素及美登素类衍生物maytansinoids(DM1、DM2、DM3、DM4)，紫杉醇及紫杉醇类衍生物(多西紫杉醇)、多西他赛、长春新碱等，所述DNA损伤剂/DNA复制抑制剂如伊立替康或其代谢活性物SN-38等。
“任选”、“任选地”、“任意”或“任一”意味着随后所描述地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，“任选包含1个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
术语“药物组合物”表示含有一种或多种本发明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。治疗性组合物一般应当是无菌的并且在制造和储存条件下稳定。可以将组合物配制为溶液、微乳液、分散剂、脂质体或适合高抗体浓度的其他有序结构。可以通过将活性化合物(即抗体或抗体部分)以要求的量连同上文所列举的一种成分或成分组合在适宜的溶剂中并入，根据需要，随后过滤消毒，制备无菌可注射溶液剂。
本发明所述的方法、组合物、联合治疗可以与其他活性剂或治疗方式，所述的方法包括向对象以有效治疗或预防疾病(例如，癌症)的量，施用本发明所述的抗Trop-2抗体分子，任选地，与PD-1、PD-L1、PD-L2、LAG-3、CTLA-4、Tim-3抗体(免疫治疗)或其它肿瘤治疗抗体，Her-2、EGFR、VEGF、VEGFR抗体等，以及ADC(抗体药物偶联，如T-DM1)，双特异抗体，化疗药物等的一种或多种抑制剂的组合，还包括施用额外的活性剂或全部可以按这样的量或剂量施用，所述量或剂量高于、低于或等于单独(例如，作为单一疗法)使用的每种活性剂的量或剂量。额外的活性剂或全部的施用量或剂量比单独(例如，作为单一疗法)使用的每种活性剂的量或剂量低(例如，至少20％、至少30％、至少40％或至少50％)。
本发明提供了一种新型的抗人Trop-2抗体，所述抗体具有良好的生物学活性：无论是Trop-2重组蛋白还是细胞表面表达的Trop-2抗原，本发明提供的抗体(包括嵌合抗体和人源化抗体)均可以有效结合，与对照抗体Sacituzumab相近。同时，本发明提供的抗体对人Trop-2具有高亲和力：与对照抗体Sacituzumab相比，本发明的人源化抗体甚至具有更高的人Trop-2蛋白的特异性结合能力，亲和力高于Sacituzumab。因此，本发明的抗体具 有良好的药效。
实验证明，本发明的抗体还具有良好的内化能力：人源化抗体的内化率与对照抗体Sacituzumab相近；在标记成ADC后内化能力明显增强。因此，本发明的抗体具有用于开发ADC药物的潜力。本发明的Trop-2抗体还可与其他抗体具有协同作用，例如，本发明的抗体可以与CD47联用可以进一步促进巨噬细胞对肿瘤细胞的吞噬作用。
此外，本发明抗体还证明具有良好的体内药效。采用本发明的抗体制成ADC，发现该抗Trop2-ADC抗体对肿瘤生长具有剂量依赖地抑制作用，在高剂量下(10mg/kg)各ADC抗体与对照抗体Sacituzumab药效相当，且未观察到ADC小分子SN38的明显的毒性作用，各实验组动物体重平稳增长，与对照无明显差别。
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显示了阳性杂交瘤上清与CHO细胞表面Trop-2结合筛选结果。
图2显示了阳性杂交瘤上清与CHO细胞表面Trop-2结合筛选结果。
图3显示了ELISA检测阳性杂交瘤克隆上清与不同种属重组Trop-2的交叉反应结果。
图4显示了ELISA检测抗人Trop-2嵌合抗体与Trop-2重组蛋白的结合活性结果，其中图4A：ch3-11；图4B：ch4-3；图4C：ch23-12；图4D：ch11-4；图4E：ch17-1。
图5显示了FACS检测抗人Trop-2嵌合抗体与细胞表面Trop-2重组蛋白的结合活性结果，其中图5A：ch3-11；图5B：ch23-12；图5C：ch11-4；图5D：ch4-3；图5E：ch17-1。
图6显示了ELISA检测抗人Trop-2抗体与Trop-2结合的种属特异性结果，其中图6A：h23-12；图6B：h4-3；图6C：Sacituzumab。
图7显示了抗人Trop-2抗体对人Trop-2胞外区重组蛋白的亲和力分析结果，其中图7A：Sacituzumab；图7B：h23-12；图7C：h4-3。
图8显示了抗Trop-2人源化抗体结合N87细胞表面Trop-2后的内化观察结果。
图9显示了抗Trop-2人源化抗体在裸小鼠体内单次给药的药-时曲线 (Trop-2检测的)，其中图9A：h23-12；图9B：h4-3。
图10显示了抗Trop-2-ADC抗体对细胞生长的抑制率。
图11显示了Balb/C nu荷瘤胃癌N87小鼠模型小鼠体重的变化曲线，其中图11A：ch4-3-SN38给药后；图11B：h23-12-SN38给药后；图11C：同型对照抗体给药后；图11D：ch4-3-SN38、h23-12-SN38高剂量给药后。
图12显示了Balb/C nu荷瘤胃癌N87小鼠模型的肿瘤体积变化曲线，其中图12A：ch4-3-SN38给药后；图12B：h23-12-SN38给药后；图12C：同型对照抗体给药后；图12D：ch4-3-SN38、h23-12-SN38高剂量给药后。
图13显示了抗Trop2抗体与抗CD47抗体联用的SKOV3皮下移植瘤小鼠模型小鼠体重的变化曲线。
图14显示了抗Trop2抗体与抗CD47抗体联用的SKOV3皮下移植瘤小鼠模型的肿瘤体积变化曲线。
图15显示了Balb/C nu荷瘤胃癌NCI-N87皮下移植瘤小鼠模型的肿瘤体积变化曲线。
图16显示了Balb/C nu荷瘤胃癌NCI-N87皮下移植瘤小鼠模型小鼠体重的变化曲线。
实施发明的最佳方式
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原料、试剂材料等，如无特殊说明，均为市售购买产品。
实施例1 抗人Trop-2抗体杂交瘤细胞制备
免疫：采用人Trop-2重组蛋白(序列号：NP_002344.2，1aa-274aa)免疫Balb/c小鼠，用包被人Trop-2-his重组蛋白(序列号：NP_002344.2，1aa-274aa)的96孔酶标板以ELISA法检测血清滴度；血清滴度达到融合要求的小鼠用于下一步的细胞融合。
细胞融合及杂交瘤制备：选取滴度达到要求的小鼠，进行冲击免疫，3天后无菌取小鼠脾脏，制备B淋巴细胞悬液，与SP2/0骨髓瘤细胞以4:1的比例混合，在PEG作用下使两种细胞融合。融合后的细胞用HAT培养基重 悬后，分装96孔细胞培养板。置37℃，5％CO 2培养箱内培养。
实施例2 抗人Trop-2抗体阳性杂交瘤细胞株的筛选
1.阳性杂交瘤结合筛选
融合后10-14天，以人Trop-2-his重组蛋白(序列号：NP_002344.2，1aa-274aa)(20ng/ml)包被酶标板，4℃，过夜；PBS洗三次后，用4％脱脂奶粉-PBS封闭，室温，1hr；用PBS洗三遍，加入杂交瘤克隆培养上清，室温，1hr。设以下对照：(1)阳性对照(PC)：免疫后小鼠血清(用PBS 1:1000稀释)；(2)阴性对照(NC)：无细胞生长的融合孔。经PBST(0.05％Tween-PBS)洗三遍，PBS洗两遍，加入HRP-羊抗小鼠IgG(Fcγ)，37℃，0.5hr；再经PBST(0.05％Tween20-PBS)洗3遍，加入TMB显色液，避光显色15-30min，加入ELISA终止液，终止反应；酶标仪读A450值。
按从高到低原则选择读值高的前95个克隆进行二次ELISA确认，选取25个抗体分泌阳性细胞pool采用有限稀释法进行亚克隆，于铺板10天后，挑选单克隆细胞上清用ELISA方法进一步筛选阳性克隆，ELISA方法同上，按从高到低原则选择读值高的前21个克隆m1-1、m3-11、m4-3、m5-5、m6-6、m7-13、m11-4、m12-2、m12-4、m13-2、m14-2、m15-3、m16-7、m17-1、m18-4、m19-5、m20-4、m21-1、m22-1、m23-12、m24-3进行下一步FACS结合筛选。
2.阳性杂交瘤与CHO细胞表面Trop-2结合筛选
通过PCR从含有Trop-2 cDNA的载体(Cat.:HG10428-M，北京义翘神州)中克隆出Trop-2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(Cat.:M5379，Sigma)的CD CHO AGTTM培养基(Cat.:12490-025，Gibco)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，预筛获得的22个细胞生长孔，并将其放大至24孔细胞培养板，最终通过流式细胞分析(FACS)挑选出1-T-21号克隆(CHO/Trop-2细胞)，进行放大培养和冻存以及用于FACS检测。
根据上述ELISA结果，挑选该21个克隆的杂交瘤上清100倍稀释后与构建的CHO细胞(CHO/Trop-2细胞)悬液在37℃孵育30min，设以下对照： (1)阳性对照(PC)：Sacituzumab的鼠IgG恒定区形式，1ug/ml；(2)阴性对照(NC)：无关鼠抗体，1ug/ml。以PBS洗涤细胞3次后，加入1:200稀释的羊抗鼠IgG-FITC(Cat.:F9006，Sigma)并孵育30min。PBS洗涤细胞3次后通过流式细胞仪(型号B49007AD，SNAW31211，BECKMAN COULTER)检测细胞的平均荧光强度(MFI)以验证杂交瘤分泌的抗体是否可以与CHO细胞表面Trop-2结合，结果见图1。
根据图1及细胞状态，挑选克隆m1-1、m3-11、m4-3、m6-6、m7-13、m11-4、m12-2、m12-4、m13-2、m14-2、m16-7、m17-1、m19-5、m21-1、m23-12，取杂交瘤上清利用ProA亲和层析柱进行纯化，将纯化得到的鼠抗体再次进行结合确认。将抗体分别稀释至13nM和0.66nM，然后与重组表达人Trop-2的CHO细胞(CHO/Trop-2细胞)悬液在37℃孵育30min，设以下对照：(1)阳性对照(PC)：Sacituzumab的鼠IgG恒定区形式，1ug/ml；(2)阴性对照(NC)：无关鼠抗体，1ug/ml。以PBS洗涤细胞3次后，加入1:200稀释的羊抗鼠IgG-FITC(Cat.:F9006，Sigma)并孵育30min。PBS洗涤细胞3次后通过流式细胞仪(型号B49007AD，SNAW31211，BECKMAN COULTER)检测细胞的平均荧光强度(MFI)以验证杂交瘤分泌的抗体是否可以与CHO细胞表面Trop-2结合，如图2所示，来自15个克隆上清的抗体与CHO细胞表面Trop-2都有很好的结合。
选择m3-11、m4-3、m11-4、m17-1、m23-12作为候选克隆进行下一步筛选。
3.阳性杂交瘤克隆的种属交叉的ELISA筛选
将人Trop-2-His重组蛋白(序列号：NP_002344.2，1aa-274aa)、食蟹猴Trop-2-His重组蛋白(序列号：UniProtKB-A0A2K5UE71，1aa-272aa)、小鼠Trop-2-His重组蛋白(Cat.:50922-M08H，北京义翘神州)4℃包被过夜，包被浓度分别为0.2、1、1μg/mL；PBS洗板3次后，加入5％BSA PBS，37℃封闭60min，PBST洗板3次；加入PBS将上述15株纯化后的鼠抗体稀释至1μg/mL，设以下对照：(1)阳性对照(PC)：Sacituzumab(WHO Drug Information(Vol.31,No.1,2017)，SEQ ID NO:39和SEQ ID NO:40)的鼠IgG恒定区形式，1ug/ml；(2)阴性对照(NC)：无关杂交瘤抗体，1μg/mL；(3)空白对照：PBS。37℃孵育60min，PBST洗板4次；加入1:5000稀释的HRP-羊抗小鼠IgG(Fcr)(Cat:115-035-071，Jackson Immuno Research)，37℃孵 育30min，PBST洗板4次；加入TMB底物显色，37℃孵育10min后，加入2M HCl终止反应；以630nm为参比波长，读取并记录波长450nm下孔板的吸光度A450nm-630nm。除克隆m12-4、m17-1、m19-5、m21-1的抗体与小鼠Trop-2有交叉外，其余与鼠无交叉，但所有杂交瘤抗体均可以特异性地与重组人、食蟹猴Trop-2结合(图3)。
实施例3 鼠源抗人Trop-2抗体的序列测定
将分泌抗人Trop-2抗体的杂交瘤细胞m3-11、m4-3、m11-4、m17-1、m23-12扩大培养后，用Mouse Monoclonal Antibody IgG Subclass Test Card(Cat.：A12403，VicNovo)及Mouse Monoclonal Antibody Light/Heavy Chain Test Card(Cat.:A12401，VicNovo)按照试剂操作规程进行亚型检测，亚型鉴定为：重链为IgG1，轻链为Kappa链，为m3-11、m4-3、m11-4、m17-1、m23-12的抗体基因的克隆提供依据。
将m3-11、m4-3、m11-4、m17-1、m23-12杂交瘤细胞按照TRIzol试剂盒(Cat.:15596026，Invitrogen)说明书步骤提取细胞总RNA；利用M-MuLV反转录酶(Cat.:M0253S，NEB)将杂交瘤细胞总RNA反转录成cDNA；使用简并引物(可参照图书[董志伟，王琰。抗体工程(第二版)。北京医科大学出版社，2001，313-314])和Phusion试剂盒(Cat.:E0553L，NEB)扩增抗体轻链可变区IgVL(κ)和重链可变区V H序列；利用胶回收试剂盒(Cat.:AP-GX-250，Axygen)纯化PCR扩增产物；按照T载体克隆试剂盒(Cat.:ZC205，庄盟生物)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。
测序结果显示：
克隆m3-11的鼠抗体重链可变区DNA的核苷酸序列见SEQ ID NO:41，由该DNA序列推测得到克隆m3-11的鼠抗体重链可变区氨基酸序列见SEQ ID NO:1；克隆m3-11的鼠抗体轻链可变区DNA的核苷酸序列见SEQ ID NO:42，由该DNA序列推测得到克隆m3-11鼠抗体轻链可变区氨基酸序列见SEQ ID NO:18。
SEQ ID NO:1：
Figure PCTCN2020120277-appb-000001
Figure PCTCN2020120277-appb-000002
SEQ ID NO:18：
Figure PCTCN2020120277-appb-000003
克隆m4-3的鼠抗体重链可变区DNA的核苷酸序列见SEQ ID NO:43，由该DNA序列推测得到克隆m4-3的鼠抗体重链可变区氨基酸序列见SEQ ID NO:2；克隆m4-3的鼠抗体轻链可变区DNA的核苷酸序列见SEQ ID NO:44，由该DNA序列推测得到克隆m4-3的鼠抗体轻链可变区氨基酸序列见SEQ ID NO:19。
SEQ ID NO:2：
Figure PCTCN2020120277-appb-000004
SEQ ID NO:19：
Figure PCTCN2020120277-appb-000005
克隆m11-4的鼠抗体重链可变区DNA的核苷酸序列见SEQ ID NO:47，由该DNA序列推测得到克隆m11-4的鼠抗体重链可变区氨基酸序列见SEQ ID NO:5；克隆m11-4的鼠抗体轻链可变区DNA的核苷酸序列见SEQ ID NO:48，由该DNA序列推测得到克隆m11-4鼠抗体轻链可变区氨基酸序列见SEQ ID NO:24。
SEQ ID NO:5：
Figure PCTCN2020120277-appb-000006
SEQ ID NO:24：
Figure PCTCN2020120277-appb-000007
Figure PCTCN2020120277-appb-000008
克隆m17-1的鼠抗体重链可变区DNA的核苷酸序列见SEQ ID NO:51，由该DNA序列推测得到克隆m17-1的鼠抗体重链可变区氨基酸序列见SEQ ID NO:11；克隆m17-1鼠抗体轻链可变区DNA的核苷酸序列见SEQ ID NO:52，由该DNA序列推测得到克隆m17-1的鼠抗体轻链可变区氨基酸序列见SEQ ID NO:30。
SEQ ID NO:11：
Figure PCTCN2020120277-appb-000009
SEQ ID NO:30：
Figure PCTCN2020120277-appb-000010
克隆m23-12的鼠抗体重链可变区DNA的核苷酸序列见SEQ ID NO:53，由该DNA序列推测得到克隆m23-12的鼠抗体重链可变区氨基酸序列见SEQ ID NO:12；克隆m23-12的鼠抗体轻链可变区DNA的核苷酸序列见SEQ ID NO:54，由该DNA序列推测得到克隆m23-12的鼠抗体轻链可变区氨基酸序列见SEQ ID NO:31。
SEQ ID NO:12：
Figure PCTCN2020120277-appb-000011
SEQ ID NO:31：
Figure PCTCN2020120277-appb-000012
实施例4 抗人Trop-2嵌合抗体及对照抗体的制备
将对照抗体(Sacituzumab)轻重链序列进行全合成，将轻重链序列分别克隆至真核瞬时表达载体中，获得对照抗体轻链和重链表达质粒，转入大肠 杆菌扩增，分离获得大量含对照抗体轻链和重链的质粒，利用这些质粒，并根据转染试剂293fectin(Cat.:12347019，Gibco)的操作说明，分别将对照抗体的轻、重链质粒转入HEK293细胞中重组表达。细胞转染后5-6天，取培养上清，利用ProA亲和层析柱对表达上清进行纯化，获得对照抗体。其中，对照抗体Sacituzumab氨基酸序列来源于WHO Drug Information(Vol.31,No.1,2017)，重链氨基酸序列见SEQ ID NO:39，轻链氨基酸序列见SEQ ID NO:40。
将从各克隆获得的对应的鼠源抗体3-11、4-3、11-4、17-1、23-12的轻链可变区和重链可变区基因通过PCR引入酶切位点，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瞬时表达载体中，获得人-鼠嵌合轻链(pKN019-ch3-11L、pKN019-ch4-3L、pKN019-ch11-4L、pKN019-ch17-1L、pKN019-ch23-12L)和人-鼠嵌合重链(pKN041-ch3-11H、pKN019-ch4-3H、pKN019-ch11-4H、pKN019-ch17-1H、pKN019-ch23-12H)表达质粒，转入大肠杆菌扩增，分离获得大量含人-鼠嵌合抗体轻链和重链的质粒，利用该质粒，并根据转染试剂293fectin(Cat.:12347019，Gibco)的操作说明，将嵌合抗体ch3-11、ch4-3、ch11-4、ch17-1、ch23-12的轻、重链质粒分别转入HEK293细胞中重组表达。细胞转染后5-6天，取培养上清，利用ProA亲和层析柱对表达上清进行纯化，获得嵌合抗体ch3-11、ch4-3、ch17-1、ch11-4、ch23-12。
实施例5 ELISA检测抗人Trop-2嵌合抗体与Trop-2重组蛋白的结合活性
人Trop-2-his重组蛋白(序列号：NP_002344.2，1aa-274aa)，浓度0.2μg/mL，4℃包被过夜，用5％BSA于37℃恒温培养箱封闭60min。分别加入ch3-11、ch4-3、ch17-1、ch11-4、ch23-12和对照抗体Sacituzumab(起始浓度为2μg/mL，3倍连续稀释，8个梯度)，37℃恒温培养箱反应60min后。PBST洗板4次；然后加入1:5000稀释的HRP-anti-human Fc(Cat.:109-035-098，Jackson Immuno Research)，反应45min，加入TMB(Cat.:ME142，北京泰天河生物)底物显色15min，2M HCl终止后读板。以630nm为参比波长，读取并记录波长450nm下孔板的吸光度值A450nm-630nm。
通过ELISA测定了ch3-11、ch4-3、ch17-1、ch11-4、ch23-12和对照抗体 Sacituzumab对人Trop-2重组蛋白的结合能力，其半数有效结合浓度(EC50)值分别为0.3147nM、0.3195nM、0.3278nM、0.2366nM、0.4581nM和0.271nM(图4)，基本相当。结果表明ch3-11、ch4-3、ch17-1、ch11-4、ch23-12嵌合抗体与人Trop-2重组蛋白具有高亲和力，鼠抗体3-11、4-3、11-4、17-1、23-12的序列克隆正确。
实施例6 FACS检测抗人Trop-2嵌合抗体与CHO细胞表面人Trop-2重组蛋白的结合活性
将重组表达人Trop-2的CHO细胞(CHO/Trop-2细胞)悬液分别与嵌合抗体(ch3-11、ch4-3、ch17-1、ch11-4、ch23-12)(浓度为30μg/mL，10μg/mL，5μg/mL起始3倍连续稀释9个梯度，共11个梯度)在37℃孵育30min，设以下对照：(1)阳性对照(PC)：对照抗体Sacituzumab；(2)阴性对照(NC)：IgG1同型对照抗体NC-IgG1。以PBS洗涤细胞3次后，加入1:100稀释的羊抗人IgG-FITC(Cat.:F9512，Sigma)并孵育30min。PBS洗涤细胞3次后通过流式细胞仪(型号B49007AD，SNAW31211，BECKMAN COULTER)检测细胞的平均荧光强度(MFI)以检测嵌合抗体与CHO细胞表面的人Trop-2结合能力。
通过FACS测定了ch3-11、ch4-3、ch17-1、ch11-4、ch23-12和对照抗体Sacituzumab对CHO细胞表面的人Trop-2重组蛋白的结合能力，其半数有效结合浓度(EC50)值分别为0.993nM、3.326nM、2.918nM、1.154nM、2.748nM和2.316nM(图5)。与对照抗体Sacituzumab相比，ch3-11、ch11-4结合活性更好，ch4-3、ch17-1、ch23-12结合活性相近。结果显示抗人Trop-2嵌合抗体ch3-11、ch4-3、ch17-1、ch11-4、ch23-12可以有效结合CHO细胞表面的人Trop-2重组蛋白。
实施例7 抗人Trop-2嵌合抗体结合细胞表面Trop-2的内化活性
取人胰腺癌细胞BxPC-3，5×10 5个细胞/管，分别加入稀释至10μg/ml的嵌合抗体ch3-11、ch4-3、ch11-4、ch23-12、阳性对照抗体Sacituzumab，每个抗体分为四组(孵育1h、3h、5h实验组及对照组)，每组2管。实验组放入37℃电热恒温培养箱，分别孵育1h、3h、5h后放置冰上，对照组一直冰育作为阴性对照；所有样品孵育完成后，1,500rpm，4℃离心3min，弃上 清，冰冷PBS洗涤1遍，加入二抗即抗人IgG(Fc specific)-FITC抗体(Cat.:F9512，Sigma)，冰育30分钟后，1,500rpm离心3min，弃上清，冰冷PBS洗涤，取200ul冰冷PBS重悬细胞，进行FACS检测平均荧光强度MFI，并通过公式计算内化效率：tx时间点的％MFI＝37℃孵育样品的MFI×100％/4℃孵育对照样品的MFI；tx时间点的内化百分比＝100％-tx时间点的％MFI。
结果如表1所示，ch4-3、ch23-12与对照抗体Sacituzumab内化比例相近，而ch3-11、ch11-4无明显内化。
表1.细胞表面Trop-2介导的抗人Trop-2嵌合抗体的内化百分比
Figure PCTCN2020120277-appb-000013
实施例8 抗人Trop-2嵌合抗体耐受破坏的稳定性
将嵌合抗体ch3-11、ch4-3、ch11-4、ch23-12以5mg/mL的浓度分别置于PBS、含10％N,N-二甲基乙酰胺(DMA)(Cat.:ARK2190，上海沸柏化工)的PBS和含20％DMA的PBS中，37℃放置2h后，使用超滤离心管除去样品中的DMA，置换缓冲液为PBS，利用G3000Wxl液相色谱分析柱(Cat.:SEC-0046，东曹)，通过高效分子排阻色谱法(SEC-HPLC)分析样品纯度，纯度分析结果见表2。
结果显示，4株抗体均可以较好耐受DMA，10％时纯度下降不明显，20％时纯度略有下降，提示抗体对后续ADC工艺可能具有较好的耐受性。
表2.DMA处理前后抗体HPLC纯度分析
  ch23-12 ch3-11 ch4-3 ch11-4
PBS 97.52％ 97.57％ 99.47％ 99.25％
10％DMA 97.18％ 96.29％ 98.44％ 99.18％
20％DMA 93％ 96.27％ 94％ 98.96％
实施例9 抗人Trop-2单克隆抗体的人源化及重组表达
1.鼠源单克隆抗体23-12的人源化
(1)CDR移植
首先对鼠源抗体重链序列进行综合分析，确定抗体与抗原结合的抗原互补决定簇(CDR)区域及支撑抗体保守三维构象的框架区(framework)。随后根据同源性比对结果，在人抗体germline库(http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX)寻找最相似人源抗体模板，结合全序列blast结果和重链CDR3序列特点，进行CDR移植，实现了23-12重链可变区(VH)在框架区的全人源化。根据同源性比对结果，在人抗体germline库(http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX)寻找最相似人源抗体模板，结合全序列blast结果和轻链CDR3序列特点，进行CDR移植，实现了轻链框架区的高度人源化。
23-12抗体CDR移植的人源化重链可变区h23-12_VH1核苷酸序列见SEQ ID NO:55，氨基酸序列见SEQ ID NO:13；人源化轻链可变区h23-12_VL1核苷酸序列见SEQ ID NO:56，氨基酸序列见SEQ ID NO:32。
SEQ ID NO:13：
Figure PCTCN2020120277-appb-000014
SEQ ID NO:32：
Figure PCTCN2020120277-appb-000015
(2)CDR区突变设计
根据鼠源抗体23-12序列特点，对CDR移植的人源化轻重链可变区CDR区序列进行突变设计，突变位点见下表3。
表3. 23-12人源化序列设计
Figure PCTCN2020120277-appb-000016
Figure PCTCN2020120277-appb-000017
注：氨基酸残基位点编号依照Kabat编号系统。
2.人源化单克隆抗体23-12的重组表达
将人源化设计的h23-12抗体轻重链可变区(h23-12_VL1、h23-12_VH1)序列进行全合成，将人源化的h23-12_VH1通过酶切克隆入真核瞬时表达载体pKN041的人IgG1的重链恒定区编码基因的上游，重链恒定区核苷酸序列见SEQ ID NO:59，氨基酸序列见SEQ ID NO:37；将人源化的h23-12_VL1通过酶切克隆入真核瞬时表达载体pKN019的人轻链Cκ的编码基因的上游，轻链恒定区核苷酸序列见SEQ ID NO:60，氨基酸序列见SEQ ID NO:38，构建人源化23-12轻、重链表达载体，获得轻链(pKN019-h23-12L1)和重链(pKN041-h23-12H1)表达质粒，转入大肠杆菌扩增，分离获得h23-12抗体轻链和重链的质粒h23-12L1、h23-12H1。
根据表3所示的突变设计，利用StarMut基因定点突变试剂盒(GenStar，Cat.:T111-01)，分别在轻链(pKN019-h23-12L1)和重链(pKN041-h23-12H1)表达质粒上进行定点突变，转入大肠杆菌扩增，获得h23-12抗体轻、重链CDR区突变表达质粒(h23-12H2～h23-12H7、h23-12L2～h23-12L7)，分别对应表3中的23-12人源化序列；根据转染试剂293fectin(Cat.:12347019，Gibco)的操作说明，将23-12人源化抗体的轻、重链质粒进行组合，组合见表4，转入HEK293细胞中重组表达。
表4.人源化23-12轻、重链序列组合
Figure PCTCN2020120277-appb-000018
Figure PCTCN2020120277-appb-000019
注：该表表示各种23-12轻重链组合所得序列，如h23-12-1表示该抗体由23-12人源化抗体轻链h23-12L1和人源化重链h23-12H1组成，其他类推。
细胞转染后5-6天，取培养上清，利用ProA亲和层析柱对表达上清进行纯化，获得的23-12的不同人源化抗体，利用Fortebio公司的Octet QKe system仪器，采用抗人抗体Fc段的捕获抗体(AHC)生物探针捕获抗体Fc段的方法测定抗体亲和力。测定时将23-12抗体及对照抗体Sacituzumab用PBS缓冲液稀释至4μg/mL，流经AHC探针(Cat.:18-0015，PALL)表面，时间为120s。人Trop-2-His重组蛋白(序列号：NP_002344.2，1aa-274aa)作为流动相，Trop-2-His重组蛋白浓度为60nM。结合时间为100s，解离时间为300s。实验完毕，扣除空白对照响应值，用软件进行1:1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。
通过ForteBio测定h23-12突变体组合抗体、嵌合抗体ch23-12及对照抗体Sacituzumab与人Trop-2-His重组蛋白的亲和力(表5)。
表5. 23-12不同抗体与人Trop-2胞外区重组蛋白的亲和力测定结果
Figure PCTCN2020120277-appb-000020
Figure PCTCN2020120277-appb-000021
选择h23-12-25，该组合抗体亲和力(KD)为5.02E-10M，命名为h23-12，进行进一步功能验证，该抗体重链可变区核苷酸序列见SEQ ID NO:57，氨基酸序列见SEQ ID NO:14；轻链可变区核苷酸序列见SEQ ID NO:58，氨基酸序列见SEQ ID NO:33。
SEQ ID NO:14：
Figure PCTCN2020120277-appb-000022
SEQ ID NO:33：
Figure PCTCN2020120277-appb-000023
3.鼠源单克隆抗体4-3的人源化
(1)CDR移植
首先对鼠源抗体重链序列进行综合分析，确定抗体与抗原结合的抗原互补决定簇(CDR)区域及支撑抗体保守三维构象的框架区(framework)。随后根据同源性比对结果，在人抗体germline库 (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX)寻找最相似人源抗体模板，结合全序列blast结果和重链CDR3序列特点，进行CDR移植，实现了4-3重链可变区(VH)在框架区的全人源化。根据同源性比对结果，在人抗体germline库(http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX)寻找最相似人源抗体模板，结合全序列blast结果和轻链CDR3序列特点，进行CDR移植，实现了轻链框架区的全人源化。
4-3抗体CDR移植的人源化重链可变区h4-3_VH1核苷酸序列见SEQ ID NO:45，氨基酸序列见SEQ ID NO:3；人源化轻链可变区h4-3_VL1核苷酸序列见SEQ ID NO:46，氨基酸序列见SEQ ID NO:20。
SEQ ID NO:3：
Figure PCTCN2020120277-appb-000024
SEQ ID NO:20：
Figure PCTCN2020120277-appb-000025
(2)CDR区突变设计
根据鼠源抗体4-3序列特点，对CDR移植的人源化轻重链可变区CDR区序列进行突变设计，突变位点见下表6。
表6. 4-3人源化序列设计
Figure PCTCN2020120277-appb-000026
注：氨基酸残基位点编号依照Kabat编号系统。
4.人源化单克隆抗体4-3的重组表达
将人源化设计的h4-3抗体轻重链可变区(h4-3_VL1、h4-3_VH1)序列 进行全合成，将人源化的h4-3_VH1通过酶切克隆入真核瞬时表达载体pKN041的人IgG1的重链恒定区编码基因的上游，重链恒定区核苷酸序列见SEQ ID NO:59，氨基酸序列见SEQ ID NO:37；将人源化的h4-3_VL1通过酶切克隆入真核瞬时表达载体pKN019的人轻链Cκ的编码基因的上游，轻链恒定区核苷酸序列见SEQ ID NO:60，氨基酸序列见SEQ ID NO:38，构建人源化4-3轻、重链表达载体，获得轻链(pKN019-h4-3L1)和重链(pKN041-h4-3H1)表达质粒，转入大肠杆菌扩增，分离获得h4-3抗体轻链和重链的质粒h4-3L1、h4-3H1。
根据表6所示的突变设计，利用StarMut基因定点突变试剂盒(Cat.:T111-01，GenStar)，分别在轻链(pKN019-h4-3L1)和重链(pKN041-h4-3H1)表达质粒上进行定点突变，转入大肠杆菌扩增，获得h4-3抗体轻、重链CDR区突变表达质粒(h4-3H2～h4-3H4、h4-3L2～h4-3L5)，分别对应表6中的4-3人源化序列；根据转染试剂293fectin(Cat.:12347019，Gibco)的操作说明，将4-3人源化抗体的轻、重链质粒进行组合，组合见表7，转入HEK293细胞中重组表达。
表7.人源化4-3轻、重链序列组合
  h4-3H1 h4-3H2 h4-3H3 h4-3H4
h4-3L1 h4-3-1 h4-3-2 h4-3-3 h4-3-4
h4-3L2 h4-3-5 h4-3-6 h4-3-7 h4-3-8
h4-3L3 h4-3-9 h4-3-10 h4-3-11 h4-3-12
h4-3L4 h4-3-13 h4-3-14 h4-3-15 h4-3-16
h4-3L5 h4-3-17 h4-3-18 h4-3-19 h4-3-20
注：该表表示各种4-3轻重链组合所得序列，如h4-3-1表示该抗体由4-3人源化抗体轻链h4-3L1和人源化重链h4-3H1组成，其他类推。
细胞转染后5-6天，取培养上清，利用ProA亲和层析柱对表达上清进行纯化，获得的4-3的不同人源化抗体，利用Fortebio公司的Octet QKe system仪器，采用抗人抗体Fc段的捕获抗体(AHC)生物探针捕获抗体Fc段的方法测定抗体亲和力。测定时将4-3抗体及对照抗体Sacituzumab用PBS缓冲液稀释至4μg/mL，流经AHC探针(Cat.:18-0015，PALL)表面，时间为120s。人Trop-2-His重组蛋白(序列号：NP_002344.2，1aa-274aa)作为流动相，Trop-2-His重组蛋白浓度为60nM。结合时间为100s，解离时间为300s。实 验完毕，扣除空白对照响应值，用软件进行1:1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。
通过ForteBio测定h4-3突变体组合抗体、嵌合抗体ch4-3及对照抗体Sacituzumab与人Trop-2-His重组蛋白的亲和力(表8)。
表8. 4-3不同人源化抗体与人Trop-2胞外区重组蛋白的亲和力测定结果
抗体组合 KD值(M) 抗体组合 KD值(M)
Sacituzumab 7.23E-10 h4-3-10 7.63E-10
ch4-3 3.00E-10 h4-3-11 8.02E-10
h4-3-1 3.04E-10 h4-3-12 1.03E-09
h4-3-2 4.11E-10 h4-3-13 2.81E-10
h4-3-3 5.01E-10 h4-3-14 5.71E-10
h4-3-4 6.36E-10 h4-3-15 6.87E-10
h4-3-5 2.73E-10 h4-3-16 1.53E-09
h4-3-6 3.97E-10 h4-3-17 5.42E-10
h4-3-7 4.66E-10 h4-3-18 6.71E-10
h4-3-8 8.62E-10 h4-3-19 5.99E-10
h4-3-9 3.17E-10 h4-3-20 8.92E-10
选择h4-3-1，该组合抗体亲和力(KD)为3.04E-10M，命名为h4-3，进行进一步功能验证，该抗体重链可变区核苷酸序列见SEQ ID NO:45，氨基酸序列见SEQ ID NO:3；轻链可变区核苷酸序列见SEQ ID NO:46，氨基酸序列见SEQ ID NO:20。
SEQ ID NO:3：
Figure PCTCN2020120277-appb-000027
SEQ ID NO:20：
Figure PCTCN2020120277-appb-000028
5.鼠源单克隆抗体11-4的人源化
(1)CDR移植
首先对鼠源抗体重链序列进行综合分析，确定抗体与抗原结合的抗原互补决定簇(CDR)区域及支撑抗体保守三维构象的框架区(framework)。随后根据同源性比对结果，在人抗体germline库(http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX)寻找最相似人源抗体模板，结合全序列blast结果和重链CDR3序列特点，进行CDR移植，实现了11-4重链可变区(VH)在框架区的全人源化。根据同源性比对结果，在人抗体germline库(http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX)寻找最相似人源抗体模板，结合全序列blast结果和轻链CDR3序列特点，进行CDR移植，，实现了轻链框架区的全人源化。
11-4抗体CDR移植的人源化重链可变区h11-4_VH1核苷酸序列见SEQ ID NO:49，氨基酸序列见SEQ ID NO:6；轻链可变区h11-4_VL1核苷酸序列见SEQ ID NO:50，氨基酸序列见SEQ ID NO:25。
SEQ ID NO:6
Figure PCTCN2020120277-appb-000029
SEQ ID NO:25
Figure PCTCN2020120277-appb-000030
(2)CDR区突变设计
根据鼠源抗体11-4序列特点，对CDR移植的人源化轻重链可变区序列进行突变设计，突变位点见下表9。
表9. 11-4人源化序列设计
Figure PCTCN2020120277-appb-000031
Figure PCTCN2020120277-appb-000032
注：氨基酸残基位点编号依照Kabat编号系统。
6.人源化单克隆抗体11-4的重组表达
将人源化设计的h11-4抗体轻重链可变区(h11-4_VL1、h11-4_VH1)序列进行全合成，将人源化的h11-4_VH1通过酶切克隆入真核瞬时表达载体pKN041的人IgG1的重链恒定区编码基因的上游，重链恒定区核苷酸序列见SEQ ID NO:59，氨基酸序列见SEQ ID NO:37；将人源化的h11-4_VL1通过酶切克隆入真核瞬时表达载体pKN019的人轻链Cκ的编码基因的上游，轻链恒定区核苷酸序列见SEQ ID NO:60，氨基酸序列见SEQ ID NO:38，构建人源化11-4轻、重链表达载体，获得轻链(pKN019-h11-4L1)和重链(pKN041-h11-4H1)表达质粒，转入大肠杆菌扩增，分离获得h11-4抗体轻链和重链的质粒h11-4L1、h11-4H1。
根据表9所示的突变设计，利用StarMut基因定点突变试剂盒(Cat.:T111-01，GenStar)，分别在轻链(pKN019-h11-4L1)和重链(pKN041-h11-4H1)表达质粒上进行定点突变，转入大肠杆菌扩增，获得h11-4抗体轻、重链突变表达质粒(h11-4H2～h11-4H7、h11-4L2～h11-4L5)，分别对应表9中的11-4人源化序列；根据转染试剂293fectin(Cat.:12347019，Gibco)的操作说明，将11-4人源化抗体的轻、重链质粒进行组合，组合见表10，转入HEK293细胞中重组表达。
表10.人源化11-4轻、重链序列组合
Figure PCTCN2020120277-appb-000033
注：该表表示各种11-4轻重链组合所得序列，如h11-4-1表示该抗体由11-4人源化抗体轻链h11-4L1和人源化重链h11-4H1组成，其他类推。
细胞转染后5-6天，取培养上清，利用ProA亲和层析柱对表达上清进行纯化，获得的11-4的不同人源化抗体，利用Fortebio公司的Octet QKe system仪器，采用抗人抗体Fc段的捕获抗体(AHC)生物探针捕获抗体Fc段的方法测定抗体亲和力。测定时将11-4抗体及对照抗体Sacituzumab用PBS缓冲液稀释至4μg/mL，流经AHC探针(Cat.:18-0015，PALL)表面，时间为120s。人Trop-2-His重组蛋白(序列号：NP_002344.2，1aa-274aa)作为流动相，Trop-2-His重组蛋白浓度为60nM。结合时间为100s，解离时间为300s。实验完毕，扣除空白对照响应值，用软件进行1:1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。
通过ForteBio测定h11-4突变体组合抗体、嵌合抗体ch11-4及对照抗体Sacituzumab与人Trop-2-His重组蛋白的亲和力(表11)。
表11. 11-4不同人源化抗体与人Trop-2胞外区重组蛋白的亲和力测定结果
抗体组合 KD值(M) 抗体组合 KD值(M)
Sacituzumab 7.84E-10 h11-4-10 3.51E-10
ch11-4 2.64E-10 h11-4-11 3.75E-10
h11-4-1 2.16E-09 h11-4-12 2.82E-10
h11-4-2 9.91E-10 h11-4-13 2.11E-10
h11-4-3 8.68E-10 h11-4-14 3.52E-10
h11-4-4 8.11E-10 h11-4-15 3.89E-10
h11-4-5 1.08E-09 h11-4-16 2.31E-10
h11-4-6 9.52E-10 h11-4-17 2.38E-10
h11-4-7 8.03E-10 h11-4-18 3.94E-10
h11-4-8 2.22E-10 h11-4-19 2.54E-10
h11-4-9 3.01E-10 h11-4-20 2.10E-10
实施例10 ELISA检测抗Trop-2人源化抗体与Trop-2结合的种属特异性
将人Trop-2-his重组蛋白(序列号：NP_002344.2，1aa-274aa)、食蟹猴Trop-2-His重组蛋白(序列号：UniProtKB-A0A2K5UE71，1aa-272aa)、小鼠Trop-2-His重组蛋白(Cat.:50922-M08H，北京义翘神州)4℃包被过夜，包被浓度1μg/mL；PBS洗板3次后，加入5％BSA PBS，37℃封闭60min， PBST洗板3次；加入不同稀释倍数的h23-12(起始浓度为10μg/mL，3倍梯度依次稀释14个浓度)，h4-3(起始浓度为3μg/mL，3倍梯度依次稀释12个浓度)，Sacituzumab(起始浓度为3μg/mL，3倍梯度依次稀释12个浓度)，每个浓度一个平行孔，37℃孵育60min，PBST洗板4次；加入1:5000稀释的HRP-anti-human Fc(Cat.:109-035-098，Jackson Immuno Research)，37℃孵育30min，PBST洗板4次；加入TMB底物显色，37℃孵育10min后，加入2M HCl终止反应；以630nm为参比波长，读取并记录波长450nm下孔板的吸光度A450nm-630nm。
实验结果表明，h23-12，h4-3及对照抗体Sacituzumab均可以特异性地与重组人、食蟹猴Trop-2结合，而与重组小鼠Trop-2则无结合活性(图6，表12)，为人源化抗体进行药理毒理实验提供依据。
表12.抗Trop-2人源化抗体与不同种属Trop-2结合的EC50
Figure PCTCN2020120277-appb-000034
实施例11 抗Trop-2人源化抗体亲和力分析
利用Fortebio公司的Octet QKe system仪器，采用抗人抗体Fc段的捕获抗体(AHC)生物探针捕获抗体Fc段的方法测定抗体亲和力。
测定时将抗体(h23-12、h4-3和对照抗体Sacituzumab)用PBS缓冲液稀释至4ug/mL，流经AHC探针(Cat.:18-0015，PALL)表面，时间为120s。人Trop-2-his重组蛋白(序列号：NP_002344.2，1aa-274aa)作为流动相，每个抗体对应的Trop-2-his浓度分别为h23-12：23、30、45、75nM；h4-3：23、30、45、60nM；Sacituzumab：23、30、45、75nM。结合时间为100s，解离时间为300s。实验完毕，扣除空白对照响应值，用软件进行1:1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。
h23-12、h4-3及对照抗体Sacituzumab与人Trop-2重组蛋白的反应曲线如图7所示，拟合曲线并计算亲和力，h23-12亲和力(KD)为6.40E-10M，h4-3亲和力(KD)为5.45E-10M，Sacituzumab亲和力(KD)为9.41E-10M。 详细动力学参数如表13所示。结果表明h23-12、h4-3与人Trop-2具有高亲和力，与对照抗体Sacituzumab相当，且h23-12解离值优于Sacituzumab。
表13.抗Trop-2人源化抗体与人Trop-2胞外区重组蛋白的亲和力测定结果
  KD值(M) kon(1/Ms) kdis(1/s)
h23-12 6.40E-10 1.78E+05 1.14E-04
h4-3 5.45E-10 2.67E+05 1.46E-04
Sacituzumab 9.41E-10 2.08E+05 1.94E-04
实施例12 抗人Trop-2人源化抗体结合细胞表面Trop-2的内化活性
将天然表达人Trop-2的人胃癌细胞NCI-N87，以2×10 3个细胞/孔的密度接种于96孔细胞培养板，培养24小时。用PBS洗涤细胞1次，弃上清。将使用Mix-n-Stain TMCF TM488A(Cat.:MX488AS100，Sigma)标记的h23-12和对照抗体Sacituzumab，用RPMI 1640(含10％FBS)稀释至15μg/mL，加入NCI-N87细胞中，一组放37℃电热恒温培养箱，一组放4℃冰箱作为阴性对照；阴性对照孵育30分钟后PBS洗涤3次，用荧光显微镜观察并拍照，37℃实验组孵育5h后用荧光显微镜观察并拍照。
实验结果(图8)表明人源化h23-12和对照抗体Sacituzumab在37℃条件下都能够被Trop-2介导内吞，在细胞质内呈点状分布。提示抗体人源化后仍然能够保持内化活性。
利用实施例7所示方法通过FACS检测3h时在BxPC细胞上的内化率，结果如表14所示，提示人源化后内化率和Sac相当。
表14.BXPC-3细胞表面Trop-2介导的抗人Trop-2抗体的内化百分比
Figure PCTCN2020120277-appb-000035
实施例13 单次给予Balb/C裸小鼠的药代动力学研究
将健康雌性5周龄Balb/C裸小鼠，2只分为一组，单次单剂量(15mg/kg)腹腔注射h23-12后，分别于第5h、25h、48h、96h、168h、240h收集血清，并于-20℃保存，并设置对照组，与h23-12腹腔注射等同剂量的对照品Sacituzumab进行比较，观察其药代动力学特性。
将健康雌性5周龄Balb/C裸小鼠，4只分为一组，单次单剂量(20mg/kg)腹腔注射h4-3后，分别于第4h、8h、24h、48h、96h、144h、192h、240h收集血清，并于-20℃保存，观察其药代动力学特性。
使用包被人Trop-2-his(序列号：NP_002344.2，1aa-274aa)ELISA的方法检测血清中药物浓度，同时做标准曲线。以标准抗体的浓度为Y轴，OD值为X轴，拟合线性曲线，将检测血清的OD值带入公式可求得血清中抗体含量，并根据公式T 1/2＝|0.693/k|，计算药物半衰期T 1/2。
药-时曲线结果显示，h23-12、h4-3与对照抗体Sacituzumab在小鼠体内均具有较长的半衰期，药物代谢半衰期T 1/2表现相当(图9A，图9B，表15)，提示抗体在体内没有明显的失活现象，具有较好的结构稳定性。其代谢符合单抗药物的基本特征，T 1/2约为170h。
表15.抗Trop-2抗体在裸小鼠体内单次给药的药代动力学参数
  T 1/2(Trop-2检测)
Sacituzumab(n＝2) 168±14
h23-12(n＝2) 174±12
h4-3(n＝4) 180±57
实施例14 抗Trop-2裸抗及其ADC抗体的亲和力分析
利用Fortebio公司的Octet QKe system仪器，采用抗人抗体Fc段的捕获抗体(AHC)生物探针捕获抗体Fc段的方法测定抗体亲和力。
首先制备ADC药物SN38标记的抗体。使用20当量的二硫苏糖醇(DTT)将抗体在pH7.0±0.5范围内的磷酸钠缓冲液中还原2h，随后将还原后的抗体利用超滤离心管纯化除去多余的DTT，并将还原后的抗体置换到pH 7.0±0.5的磷酸盐钠冲液中。利用7-15％v/v的DMSO作为共溶剂，将还原后的抗体与CL2A-SN-38在环境温度下孵育30min。最后，将多余的小分子通过超滤离心管去除。利用质谱法对抗体偶联药物的分子量进行分析，计算得到抗体 的抗体偶联比例(DAR)值。最终每个抗体平均携带7.5个SN38分子。
测定时将如上所述制备的SN38标记的抗体：h23-12-SN38、ch4-3-SN38、ch11-4-SN38、阳性对照抗体Sacituzumab-SN38，以及裸抗h23-12、ch4-3、ch11-4、阳性对照抗体Sacituzumab用PBS缓冲液稀释至4μg/mL，流经AHC探针(Cat.:18-0015，PALL)表面，时间为120s。人Trop-2-his重组蛋白(序列号：NP_002344.2，1aa-274aa)作为流动相，浓度为60nM。结合时间为300s，解离时间为300s。实验完毕，扣除空白对照响应值，用软件进行1:1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。
如表16显示，标记SN38的ADC抗体与裸抗相比，与人Trop-2重组蛋白的亲和力未发生明显变化。
表16.抗Trop-2裸抗及其ADC抗体与人Trop-2重组蛋白的亲和力测定结果
Figure PCTCN2020120277-appb-000036
实施例15 FACS检测BXPC-3介导的抗Trop-2裸抗及其ADC抗体的内化
按实施例7所描述的方法，分别检测人胰腺癌细胞BXPC-3和人胃癌细胞NCI-N87上的内化率。待测抗体包括如上所述制备的ADC药物SN38标记的抗体：h23-12-SN38、ch4-3-SN38、ch11-4-SN38、阳性对照抗体Sacituzumab-SN38，以及裸抗h232-12、ch4-3、ch11-4、阳性对照抗体Sacituzumab和阴性同型对照抗体NC-IgG1，10ug/ml。
如表17，表18所示，h23-12的裸抗和标记SN38的ADC抗体的内化比率相近，且与对照抗体内化程度相近；而ch4-3，ch11-4标记SN38的ADC抗体较裸抗的内化比率高。
表17.NCI-N87细胞表面Trop-2介导的抗人Trop-2抗体的内化百分比
Figure PCTCN2020120277-appb-000037
Figure PCTCN2020120277-appb-000038
表18.BXPC-3细胞表面Trop-2介导的抗人Trop-2抗体的内化百分比
Figure PCTCN2020120277-appb-000039
实施例16 抗Trop-2-ADC抗体对细胞的杀伤活性检测
取人胰腺癌细胞(BxPC-3)，按2×10 3个/孔接种于96孔细胞培养板中，置于37℃5％CO 2孵箱中培养过夜后，按照表19加入不同浓度的SN38标记的抗Trop-2抗体样本(每个浓度设置两个平行孔并设置空白细胞孔(未经任何处理))置于37℃5％CO 2孵箱培养3h后，更换为新鲜的完全培养基。第二天重复前一天的处理方式，连续处理4天后用Cell Counting Kit-8(CCK-8)试剂盒检测SN38标记的抗Trop-2抗体对细胞的杀伤活性。
表19.抗Trop-2-ADC抗体及作用浓度
Figure PCTCN2020120277-appb-000040
Figure PCTCN2020120277-appb-000041
结果显示(图10，表20)，各抗Trop-2ADC抗体均特异性杀伤靶细胞，且杀伤活性上与对照抗体Sacituzumab-SN38无明显差别。
表20.抗Trop-2ADC抗体对细胞生长的的抑制活性
名称 IC 50(ug/ml)
空白细胞 /
h23-12-SN38 1.409
Sacituzumab-SN38 4.311
ch4-3-SN38 3.299
ch11-4-SN38 2.879
实施例17 抗Trop2-ADC抗体在N87皮下移植瘤模型中的药效学评价
取五周龄、雌性BALB/c裸鼠，皮下接种3×10 6个人胃癌细胞(NCI-N87)，待肿瘤生长至150mm 3左右时进行随机分组，6只/组，分组及给药剂量、频率如表21，每组每周静脉注射给药两次，给药同时测量瘤体积及小鼠体重，当小鼠体重下降超过15％时，或单只动物瘤体积超过3000mm 3或一组动物平均瘤体积超过2000mm 3时停止实验，给予小鼠安乐死。
表21.裸小鼠分组及给药剂量、频率
Figure PCTCN2020120277-appb-000042
如图11，图12所示，抗Trop2-ADC抗体对肿瘤生长具有剂量依赖的抑制作用，在高剂量下(10mg/kg)，各ADC抗体之间未观察到药效优劣的差 别，且未观察到ADC小分子SN38的明显的毒性作用，各实验组动物体重平稳增长，与对照无明显差别。
实施例18 抗Trop2抗体与抗CD47抗体联用在SKOV3皮下移植瘤模型中的药效学评价
取五周龄、雌性BALB/c裸鼠，每只小鼠右侧胁肋部皮下接种3×10 6个人卵巢癌细胞(SKOV3)，待肿瘤生长至150mm 3左右时进行随机分组，6只/组，分组及给药剂量、频率如表22，每组每周腹腔注射给药两次，共给药5次，给药同时测量瘤体积及小鼠体重，并对小鼠状态进行观察，实验末次给药后，给予小鼠安乐死。抗CD47抗体参见专利申请公开文件US20150183874A1，为人源化5F9version 2。
表22.裸小鼠分组及给药剂量、频率
组别 药物 给药剂量 给药频率
1 Anti-CD47 10mg/kg Biw
2 h23-12 2mg/kg Biw
3 h23-12+Anti-CD47 2mg/kg+10mg/kg Biw
4 阴性对照hIgG4 2mg/kg Biw
如图13，图14所示，与阴性对照hIgG4相比，h23-12+Anti-CD47联合组表现出一定的抑瘤活性，同时Anti-CD47与h23-12单药组无明显抑瘤效果。由此说明，本发明的Trop2抗体与抗CD47抗体可协同促进巨噬细胞对肿瘤细胞的吞噬，因此具有协同抑瘤作用。
实施例19 抗Trop2-ADC抗体在N87皮下移植瘤模型中的药效学评价
取五周龄、雌性BALB/c裸鼠，皮下接种3×10 6个人胃癌细胞(NCI-N87)，待肿瘤生长至100mm 3左右时进行随机分组，6只/组，分组及给药剂量、频率如表23，每组每周静脉注射给药两次，共给药6周，给药同时测量瘤体积及小鼠体重，当小鼠体重下降超过15％时，或单只动物瘤体积超过3000mm 3或一组动物平均瘤体积超过2000mm 3时停止实验，给予小鼠安乐死。
表23.裸小鼠分组及给药剂量、频率
组别 药物 给药剂量 给药频率
1 ch3-11-SN38 5mg/kg Biw×6
2 ch11-4-SN38 5mg/kg Biw×6
3 Sacituzumab-SN38 5mg/kg Biw×6
4 阴性对照hIgG1 5mg/kg Biw×6
5 ADC对照hIgG1-SN38 5mg/kg Biw×6
如图15，图16所示，抗Trop2-ADC抗体对肿瘤生长具有剂量依赖的抑制作用，在5mg/kg剂量下，ADC抗体ch3-11-SN38，ch11-4-SN38药效略优于Sacituzumab-SN38，且未观察到ADC小分子SN38的明显的毒性作用。
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。

Claims (25)

  1. 一种抗Trop-2单克隆抗体的制备方法，其包括以下步骤：
    (1)以重组Trop-2蛋白为免疫原免疫动物制备杂交瘤细胞；
    (2)以重组Trop-2蛋白为包被抗原筛选分泌抗Trop-2单克隆抗体的阳性杂交瘤细胞；
    (3)以膜表面Trop-2阳性细胞对步骤(2)中的阳性杂交瘤进行复筛；
    所述抗Trop-2单克隆抗体特异性识别并结合Trop-2胞外区天然表位。
  2. 根据权利要求1所述抗Trop-2单克隆抗体的制备方法，其特征在于所述步骤(3)中膜表面Trop-2阳性细胞为重组动物细胞，且所述动物细胞来自与步骤(1)制备杂交瘤细胞时免疫的动物相同的物种。
  3. 根据权利要求2所述抗Trop-2单克隆抗体的制备方法，其特征在于步骤(1)中免疫小鼠制备杂交瘤细胞，步骤(3)中膜表面Trop-2阳性细胞为表达外源Trop-2蛋白的重组小鼠细胞。
  4. 根据权利要求1所述抗Trop-2单克隆抗体的制备方法，其特征在于所述步骤(2)采用酶联免疫吸附测定(ELISA)筛选分泌抗Trop-2单克隆抗体的阳性杂交瘤细胞；步骤(3)采用流式细胞分析(FACS)复筛获得分泌特异性识别并结合Trop-2胞外区天然表位的抗体的杂交瘤。
  5. 根据权利要求1所述抗Trop-2单克隆抗体的制备方法，其特征在于还包括(4)对特异性识别并结合Trop-2胞外区天然表位的抗体进行鉴定的步骤，所述鉴定包括亲和力鉴定和特异性鉴定。
  6. 根据权利要求5所述抗Trop-2单克隆抗体的制备方法，其特征在于选择对人Trop-2、猴Trop-2具有特异性结合能力，而对鼠没有特异性结合能力的单克隆抗体。
  7. 根据权利要求1至6中任一项所述抗Trop-2单克隆抗体的制备方法获得的抗Trop-2单克隆抗体或其片段。
  8. 根据权利要求7所述抗Trop-2单克隆抗体或其片段，其特征在于所述单克隆抗体与人Trop-2胞外区重组蛋白的亲和力常数KD值小于1×10 -8、5×10 -9、1×10 -9、或5×10 -10。
  9. 一种抗体或其片段，所述抗体或其片段包含重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)包含选 自以下的CDR组合(HCDR1、HCDR2、HCDR3；LCDR1、LCDR2、LCDR3)：
      HCDR1 HCDR2 HCDR3 HCDR1 HCDR2 HCDR3 1 SYWMY EINPSNGRTNYNEKFKS EGHNYDGSLGAMDH RSSQSLTNSYGNTFLS GISNRFS FQSTHQPYT 2 DYVIG EIYLGSGTIYYTEKFKG GSIFPFDY SASSSVSYMY DTSTLAS QQWSSYPYT 3 DYVIG EIYLGSGTIYYAEKFKG GSIFPFDY SASSSVSYMY DTSTLAS QQWSSYPYT 4 DYVIG EIYLGSGTIYYTEKFKG GSIFPFDY RASSSVSYMY DTSTLAS QQWSSYPYT 5 DYVIG EIYLGSGTIYYAEKFKG GSIFPFDY RASSSVSYMY DTSTLAS QQWSSYPYT 6 DYVIG EIYLGSGTIYYTEKFKG GSIFPFDY SASSSVSYMY DASTLAS QQWSSYPYT 7 DYVIG EIYLGSGTIYYTEKFKG GSIFPFDY SASSSVSYMY DTSTLQS QQWSSYPYT 8 SYWIN NIYPSNSYTNYNQKFKD YRSDGFAY RASQNIGTSIH FASESIS QQSNSWPFT 9 SYWIN NIYPSNSYTNYNQKFKD YRSDGFAY RASQNIGTSIE FASESIS QQSNSWPFT 10 SYWIN NIYPSNSYTNYNQKFKD YRSEGFAY RASQNIGTSIE FASESIS QQSNSWPFT 11 SYWIN NIYPSNSYTNYNQKFKD YRSGGFAY RASQNIGTSIE FASESIS QQSNSWPFT 12 SYWIN NIYPSNSYTNYNQKFKD YRSDAFAY RASQNIGTSIE FASESIS QQSNSWPFT 13 SYWIN NIYPSNSYTNYNQKFKD YRSDGFAY RASQNIGTSIS FASESIS QQSNSWPFT 14 SYWIN NIYPSNSYTNYNQKFKD YRSEGFAY RASQNIGTSIS FASESIS QQSNSWPFT 15 SYWIN NIYPSNSYTNYNQKFKD YRSGGFAY RASQNIGTSIS FASESIS QQSNSWPFT 16 SYWIN NIYPSNSYTNYNQKFKD YRSDAFAY RASQNIGTSIS FASESIS QQSNSWPFT 17 SYWIN NIYPSNSYTNYNQKFKD YRSDGFAY RASQNIGTSIA FASESIS QQSNSWPFT 18 SYWIN NIYPSNSYTNYNQKFKD YRSEGFAY RASQNIGTSIA FASESIS QQSNSWPFT 19 SYWIN NIYPSNSYTNYNQKFKD YRSGGFAY RASQNIGTSIA FASESIS QQSNSWPFT 20 SYWIN NIYPSNSYTNYNQKFKD YRSDAFAY RASQNIGTSIA FASESIS QQSNSWPFT 21 DSAMS SISRGDDTYYPDSVKG DRFGFAY KSGQSLLDSDGKTYFN LVSMLDS WQGTHFPFT 22 SYWMH EITPSDNYTSYNQKFKG GHGNYVSFDY RASQDISNYLN YTSRLHS QQGYTLPPYT 23 SYWMH EITPSDNYTSYNQKFKG GEGNYVSFDY RASQDISNYLN YTSRLHS QQGYTLPPYT 24 SYWMH EITPSDNYGSYNQKFKG GHGNYVSFDY RASQDISNYLN YTSRLES QQGYTLPPYT 25 SYWMH EITPSDNYTSYNQKFKG GEGNYVSFDY RASQDISNYLN YTSRLES QQGYTLPPYT 26 SYWMH EITPSDNYGSYNQKFKG GHGNYVSFDY RASQDISNYLN YTSRLQS QQGYTLPPYT 27 SYWMH EITPSDNYGSYNQKFKG GHGNYVSFDY RASQDISNYLN YTSRLHS QQYYTLPPYT 28 SYWMH EITPGDNYTSYNQKFKG GHGNYVSFDY RASQDISNYLN YTSRLHS QQGYSLPPYT
    29 SYWMH EITPSDNYGSYNQKFKG GHGNYVSFDY RASQDISNYLN YTSRLHS QQGYSLPPYT 30 SYWMH EITPSDNYTSYNQKFKG GEGNYVSFDY RASQDISNYLN YTSRLHS QQGYSLPPYT
  10. 根据权利要求9所述的抗体或其片段，其特征在于，所述重链可变区包含选自以下的序列：
    SEQ ID NO:1至SEQ ID NO:17中任一个所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的氨基酸序列；和/或
    所述轻链可变区包含选自以下的序列：
    SEQ ID NO:18至SEQ ID NO:36中任一个所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的氨基酸序列。
  11. 根据权利要求9或10所述的抗体或其片段，其特征在于，所述抗体或其片段包含的重链可变区和轻链可变区选自以下组合：
    (1)如SEQ ID NO:1所示的氨基酸序列或与如SEQ ID NO:1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:18所示的氨基酸序列或与如SEQ ID NO:18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:2所示的氨基酸序列或与如SEQ ID NO:2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: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；
    (3)如SEQ ID NO:3所示的氨基酸序列或与如SEQ ID NO:3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:20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；
    (4)如SEQ ID NO:4所示的氨基酸序列或与如SEQ ID NO:4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:20所示的氨基酸序列或与如SEQ ID NO:20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:3所示的氨基酸序列或与如SEQ ID NO:3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: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；
    (6)如SEQ ID NO:4所示的氨基酸序列或与如SEQ ID NO:4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: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；
    (7)如SEQ ID NO:3所示的氨基酸序列或与如SEQ ID NO:3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:3所示的氨基酸序列或与如SEQ ID NO:3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:5所示的氨基酸序列或与如SEQ ID NO:5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:24所示的氨基酸序列或与如SEQ ID NO:24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:6所示的氨基酸序列或与如SEQ ID NO:6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: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:26所示的氨基酸序列或与如SEQ ID NO:26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: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: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；
    (13)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；和，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；
    (14)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: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；
    (15)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: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；
    (16)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: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；
    (17)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；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；
    (18)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: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；
    (19)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: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；
    (20)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: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；
    (21)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；和，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；
    (22)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: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；
    (23)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: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；
    (24)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；和，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；
    (25)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；和，如SEQ ID NO:31所示的氨基酸序列或与如SEQ ID NO:31所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (26)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: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；
    (27)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: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；
    (28)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；和，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；
    (29)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: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；
    (30)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；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；
    (31)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；和，如SEQ ID NO:35所示的氨基酸序列或与如SEQ ID NO:35所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (32)如SEQ ID NO:15所示的氨基酸序列或与如SEQ ID NO:15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:36所示的氨基酸序列或与如SEQ ID NO:36所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (33)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；和，如SEQ ID NO:36所示的氨基酸序列或与如SEQ ID NO:36所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (34)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:36所示的氨基酸序列或与如SEQ ID NO:36所示的氨基酸序列具有至少75％同一性的氨基酸序列。
  12. 根据权利要求1至11中任一项所述的抗体或其片段，其特征在于，所述抗体或其片段为单克隆抗体、单链抗体、双功能抗体、单域抗体、纳米抗体、完全或部分人源化的抗体或者嵌合抗体等任意形式，或者，所述抗体或其片段为半抗体或半抗体的抗原结合片段，例如scFv、BsFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv；
    优选地，所述抗体或其片段还包含人或鼠的恒定区，优选包含人或鼠的轻链恒定区(CL)和/或重链恒定区(CH)；
    更优选地，所述抗体或其片段包含选自IgG、IgA、IgM、IgD或IgE的 重链恒定区和/或κ或λ型轻链恒定区。
  13. 根据权利要求1至12中任一项所述的抗体或其片段，其特征在于，所述抗体为单克隆抗体，优选为鼠源、嵌合或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或IgG4亚型，轻链恒定区为κ型；
    优选地，所述单克隆抗体的重链恒定区包含如SEQ ID NO:37所示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；
    优选地，所述单克隆抗体的轻链恒定区包含如SEQ ID NO:38所示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。
  14. 一种核酸分子，其编码权利要求1至13中任一项所述的抗体或其片段或者编码所述抗体或其片段中包含的重链CDR、轻链CDR、轻链可变区、重链可变区、重链或轻链。
  15. 一种载体，其包含权利要求14所述的核酸分子。
  16. 一种宿主细胞，所述宿主细胞包含权利要求14所述的核酸分子和/或权利要求15所述的载体，或者所述宿主细胞被权利要求14所述的核酸分子和/或权利要求15所述的载体转化或转染。
  17. 一种药物组合物，其包含权利要求1至13中任一项所述的抗体或其片段、权利要求14所述的核酸分子、权利要求15所述的载体或权利要求16所述的宿主细胞，以及任选地药学上可接受的辅料。
  18. 根据权利要求17所述的药物组合物，其特征在于，所述药物组合物还包含其他抗体类药物；
    优选地，所述抗体类药物为巨噬细胞类免疫检查点抗体，更优选为抗CD47抗体。
  19. 权利要求1至13中任一项所述的抗体或其片段、权利要求14所述的核酸分子、权利要求15所述的载体、权利要求16所述的宿主细胞或权利要求17或18所述的药物组合物在制备药物中的用途；
    优选地，所述药物用于治疗Trop-2高表达癌症；
    优选地，所述Trop-2高表达癌症为胃癌、胰腺癌、肠癌、卵巢癌、鳞状肺癌、非小细胞肺癌、小细胞肺癌、尿路上皮癌、三阴性乳腺癌或宫颈癌。
  20. 一种试剂盒，所述试剂盒包括权利要求1至13中任一项所述的抗体分子或其片段、权利要求14所述的核酸分子、权利要求15所述的载体、权利要求16所述的宿主细胞或权利要求17或18所述的药物组合物。
  21. 一种融合蛋白，所述融合蛋白包含权利要求1至13中任一项所述的抗体或其片段。
  22. 一种缀合物，所述缀合物包含权利要求1至13中任一项所述的抗体或其片段以及与其缀合的药物，其中所述药物为细胞毒剂。
  23. 根据权利要求22所述的缀合物，所述缀合物为由下式所示的抗体药物缀合物(ADC)：(权利要求1至13中任一项所述的抗体或其片段)-(接头)-(细胞毒剂)；
    优选地，所述细胞毒剂为微管蛋白抑制剂(如紫杉醇、多西他赛等)或DNA复制抑制剂(如伊立替康或其代谢活性物SN-38等)。
  24. 权利要求1至13中任一项所述的抗体或其片段、权利要求14所述的核酸分子、权利要求15所述的载体或权利要求16所述的宿主细胞在制备抗体药物缀合物(ADC)中的用途；
    优选地，所述抗体药物缀合物用于治疗Trop-2高表达癌症；
    优选地，所述Trop-2高表达癌症为胃癌、胰腺癌、肠癌、卵巢癌、鳞状肺癌、非小细胞肺癌、小细胞肺癌、尿路上皮癌、三阴性乳腺癌或宫颈癌。
  25. 一种预防和/或治疗疾病的方法，所述方法包括给有此需要的受试者施用权利要求1至13中任一项所述的抗体或其片段、权利要求14所述的核酸分子、权利要求15所述的载体、权利要求16所述的宿主细胞、权利要求17或18所述的药物组合物、权利要求21所述的融合蛋白、权利要求22或23所述的缀合物，以及任选的其他药物或手段；
    优选地，所述疾病为Trop-2高表达癌症；
    优选地，所述Trop-2高表达癌症为胃癌、胰腺癌、肠癌、卵巢癌、鳞状肺癌、非小细胞肺癌、小细胞肺癌、尿路上皮癌、三阴性乳腺癌或宫颈癌。
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EP4438627A4 (en) * 2021-12-07 2026-02-11 Chia Tai Tianqing Pharmaceutical Group Co Ltd ANTI-TROP-2 ANTIBODY OR ANTIGEN-BINDING FRAGMENT OF IT
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