Anti-human Trop-2 antibody and application thereof

文档序号：795817 发布日期：2021-04-13 浏览：4次中文

阅读说明：本技术 抗人Trop-2抗体及其应用 (Anti-human Trop-2 antibody and application thereof ) 是由王双王荣娟焦莎莎张畅张姣曾大地张锦超于 2019-10-11 设计创作，主要内容包括：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。(The invention provides an antibody or a fragment thereof for binding human tumor-associated calcium signal sensor 2(Trop-2) protein, and application of the antibody or the fragment thereof in preventing or treating diseases. The antibody or the fragment thereof can effectively combine with human Trop-2 protein, has internalization activity, enhances the internalization activity after ADC drug labeling, and is not lower than a control antibody in the in vivo drug effect and safety of a mouse model.)

1. An 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 light chain variable region (VL) comprise a CDR combination (HCDR1, HCDR2, HCDR 3; LCDR1, LCDR2, LCDR3) selected from:

2. the antibody or fragment thereof of claim 1, wherein the heavy chain variable region comprises a sequence selected from the group consisting of:

1 to 17 or an amino acid sequence having at least 75% identity to the amino acid sequence shown; and/or

The light chain variable region comprises a sequence selected from:

18 to 36 or an amino acid sequence having at least 75% identity to the amino acid sequence shown.

3. The antibody or fragment thereof of claim 1 or 2, wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

(1) an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 1; and, the amino acid sequence as set forth in SEQ ID NO. 18 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 18;

(2) an amino acid sequence as shown in SEQ ID NO. 2 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 2; and, the amino acid sequence as set forth in SEQ ID NO. 19 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 19;

(3) an amino acid sequence as set forth in SEQ ID NO. 3 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 3; and, an amino acid sequence as set forth in SEQ ID NO. 20 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 30;

(4) an amino acid sequence as set forth in SEQ ID NO. 4 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 4; and, an amino acid sequence as set forth in SEQ ID NO. 20 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 20;

(5) an amino acid sequence as set forth in SEQ ID NO. 3 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 3; and, the amino acid sequence as set forth in SEQ ID NO:21 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 21;

(6) an amino acid sequence as set forth in SEQ ID NO. 4 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 4; and, the amino acid sequence as set forth in SEQ ID NO:21 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 21;

(7) an amino acid sequence as set forth in SEQ ID NO. 3 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 3; and, the amino acid sequence as set forth in SEQ ID NO. 22 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 22;

(8) an amino acid sequence as set forth in SEQ ID NO. 3 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 3; and, an amino acid sequence as set forth in SEQ ID NO. 23 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 23;

(9) an amino acid sequence as set forth in SEQ ID NO. 5 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 5; and, an amino acid sequence as set forth in SEQ ID NO. 24 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 24;

(10) an amino acid sequence as shown in SEQ ID NO. 6 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 6; and, an amino acid sequence as set forth in SEQ ID NO. 25 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 25;

(11) an amino acid sequence as set forth in SEQ ID NO. 7 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 7; and, the amino acid sequence as set forth in SEQ ID NO. 26 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 26;

(12) an amino acid sequence as set forth in SEQ ID NO. 7 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 7; and, the amino acid sequence as set forth in SEQ ID NO. 27 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 27;

(13) an amino acid sequence as shown in SEQ ID NO. 8 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 8; and, the amino acid sequence as set forth in SEQ ID NO. 27 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 27;

(14) an amino acid sequence as shown in SEQ ID NO. 9 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 9; and, the amino acid sequence as set forth in SEQ ID NO. 27 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 27;

(15) an amino acid sequence as shown in SEQ ID NO. 10 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 10; and, the amino acid sequence as set forth in SEQ ID NO. 27 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 27;

(16) an amino acid sequence as set forth in SEQ ID NO. 7 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 7; and, the amino acid sequence as set forth in SEQ ID NO 28 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 28;

(17) an amino acid sequence as shown in SEQ ID NO. 8 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 8; and, the amino acid sequence as set forth in SEQ ID NO 28 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 28;

(18) an amino acid sequence as shown in SEQ ID NO. 9 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 9; and, the amino acid sequence as set forth in SEQ ID NO 28 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 28;

(19) an amino acid sequence as shown in SEQ ID NO. 10 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 10; and, the amino acid sequence as set forth in SEQ ID NO 28 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 28;

(20) an amino acid sequence as set forth in SEQ ID NO. 7 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 7; and, the amino acid sequence as set forth in SEQ ID NO. 29 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 29;

(21) an amino acid sequence as shown in SEQ ID NO. 8 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 8; and, the amino acid sequence as set forth in SEQ ID NO. 29 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 29;

(22) an amino acid sequence as shown in SEQ ID NO. 9 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 9; and, the amino acid sequence as set forth in SEQ ID NO. 29 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 29;

(23) an amino acid sequence as shown in SEQ ID NO. 10 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 10; and, the amino acid sequence as set forth in SEQ ID NO. 29 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 29;

(24) an amino acid sequence as shown in SEQ ID NO. 11 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 11; and, an amino acid sequence as set forth in SEQ ID NO. 30 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 30;

(25) an amino acid sequence as shown in SEQ ID NO. 12 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 12; and, the amino acid sequence as set forth in SEQ ID NO. 31 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 31;

(26) an amino acid sequence as shown in SEQ ID NO. 13 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 13; and, an amino acid sequence as set forth in SEQ ID NO:32 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 32;

(27) an amino acid sequence as shown in SEQ ID NO. 16 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 16; and, an amino acid sequence as set forth in SEQ ID NO:32 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 32;

(28) an amino acid sequence as set forth in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 14; and, the amino acid sequence as set forth in SEQ ID NO 33 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 33;

(29) an amino acid sequence as shown in SEQ ID NO. 16 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 16; and, the amino acid sequence as set forth in SEQ ID NO 33 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 33;

(30) an amino acid sequence as set forth in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 14; and, the amino acid sequence as set forth in SEQ ID NO. 34 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 34;

(31) an amino acid sequence as set forth in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 14; and, the amino acid sequence as set forth in SEQ ID NO. 35 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 35;

(32) an amino acid sequence as shown in SEQ ID NO. 15 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 15; and, the amino acid sequence as set forth in SEQ ID NO. 36 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 36;

(33) an amino acid sequence as set forth in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 14; and, the amino acid sequence as set forth in SEQ ID NO. 36 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 36;

(34) an amino acid sequence as shown in SEQ ID NO. 16 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 16; and, the amino acid sequence as set forth in SEQ ID NO. 36 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 36.

4. The antibody or fragment thereof according to any one of claims 1 to 3, wherein the antibody or fragment thereof is in any form of a monoclonal antibody, a single chain antibody, a bifunctional antibody, a single domain antibody, a nanobody, a fully or partially humanized antibody, or a chimeric antibody, or alternatively, the anti-human antibody isThe body or fragment thereof is a half-antibody or antigen-binding fragment of a half-antibody, e.g., scFv, BsFv, dsFv, (dsFv)₂、Fab、Fab'、F(ab')₂Or Fv;

preferably, 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 heavy chain constant region (CH);

more preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and/or a light chain constant region of the kappa or lambda type.

5. The antibody or fragment thereof according to any one of claims 1 to 4, wherein 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 of the IgG1 or IgG4 subtype and the light chain constant region is of the kappa type;

preferably, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO 37 or an amino acid sequence having at least 75% identity to said amino acid sequence;

preferably, the light chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO 38 or an amino acid sequence having at least 75% identity to said amino acid sequence.

6. A nucleic acid molecule encoding the antibody or fragment thereof of any one of claims 1 to 5 or encoding a heavy chain CDR, a light chain variable region, a heavy chain or a light chain comprised in said antibody or fragment thereof.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising or transformed or transfected with the nucleic acid molecule of claim 6 and/or the vector of claim 7.

9. A pharmaceutical composition comprising the antibody or fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, or the host cell of claim 8, and optionally a pharmaceutically acceptable excipient.

10. The pharmaceutical composition of claim 9, further comprising an additional antibody-based drug;

preferably, the antibody-based drug is a macrophage-based immune checkpoint antibody, more preferably an anti-CD47 antibody.

11. Use of the antibody or fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, or the pharmaceutical composition of claim 9 or 10 in the manufacture of a medicament for treating a Trop-2 high expressing cancer;

preferably, the disease is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, urothelial cancer, triple negative breast cancer or cervical cancer.

12. A kit comprising the antibody molecule or fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, or the pharmaceutical composition of claim 9 or 10.

13. A fusion protein comprising the antibody or fragment thereof of any one of claims 1 to 5.

14. A conjugate comprising the antibody or fragment thereof of any one of claims 1 to 5 and a drug conjugated thereto, wherein the drug is a cytotoxic agent.

15. The conjugate of claim 14, which is an Antibody Drug Conjugate (ADC) represented by the formula: (the antibody or fragment thereof of any one of claims 1 to 5) - (linker) - (cytotoxic agent);

preferably, the cytotoxic agent is a tubulin inhibitor (such as paclitaxel, docetaxel, etc.) or a DNA replication inhibitor (such as irinotecan or its metabolic active SN-38, etc.).

16. Use of the antibody or fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, or the host cell of claim 8 in the preparation of an Antibody Drug Conjugate (ADC) for the treatment of Trop-2 high expressing cancers;

preferably, the Trop-2 high-expression cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, urothelial cancer, triple negative breast cancer or cervical cancer.

Technical Field

The invention belongs to the field of biomedicine, and relates to a novel anti-human Trop-2 antibody or a functional fragment thereof. The invention also relates to the use of said antibody or functional fragment thereof.

Background

Trop-2(Tumor-associated calcium signal transducer 2), also known as Tumor-associated calcium signal sensor (tactd 2), epithelial glycoprotein-1 antigen (EGP-1), gastrointestinal Tumor-associated antigen 1 (733 GA-1), is a cell surface glycoprotein encoded by the tastd 2 gene, 323 amino acids in length, with the extracellular region consisting of 3 domains, and was shown to exist as a dimer.

Trop-2 is a transmembrane glycoprotein and, unlike other proto-oncogenes, Trop2 has no mutations, meaning that it has no genetic makeup changes that lead to overexpression. Trop-2 stimulates cell growth through ERK/MAPK and cyclin D1 pathways, thereby promoting mechanisms such as tumor invasion, angiogenesis, tumor progression and drug resistance. Trop-2 has been found to be highly expressed in a variety of tumors, particularly triple negative breast cancer, non-small cell lung cancer, and the like, and is associated with prognosis. In contrast, Trop-2 has extremely low expression in normal tissues and is an ideal target point of ADC medicines.

The antibody drug targeting trp-2 is mainly developed by ADC drug at present, and is not completely counted, and the clinical research varieties are more than 3, wherein the small molecule conjugate mainly comprises irinotecan derivatives and tubulin inhibitor. It is presently believed that it is desirable to select a novel, low toxicity, topoisomerase inhibitor metabolite, SN-38. The SN-38 has difference with the tumor inhibition of the existing microtubule inhibitor and DNA alkylating agent, and is particularly suitable for tumors with higher heterogeneity and multi-drug resistance mechanism, such as three-negative breast cancer, pancreatic cancer, gastric cancer and the like. The most advanced item in clinical practice is IMMU-132 item of immunology, and three-phase clinical trials for recurrent and metastatic three-negative breast cancer (ASCENT-Study), two-phase clinical trials for treating three-negative breast cancer (NCT02161679) alone or in combination with carboplatin, two-phase clinical trials for treating urothelial cancer (NCT03547973), one/two-phase clinical trials for treating solid tumors including gastric cancer, cervical cancer, and small cell lung cancer (NCT01631552), and the like have been performed. Antibody-conjugated drug items with similar technologies include the first three, the fevery company, and the drug companies each with a large ADC base.

At present, the anti-Trop-2 antibody is less in variety in clinical research. Therefore, there is still a need in the art to find new anti-Trop-2 antibodies that are particularly suited for the development of ADC drugs.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an anti-Trop-2 antibody through hybridoma screening and humanization technologies, wherein the antibody has high affinity to human Trop-2 and has the specific killing effect on cancer cells; meanwhile, the antibody has high internalization capacity and is particularly suitable for developing ADC drugs.

In view of the above technical problems, the present invention provides the following technical solutions.

In one aspect, the invention provides an 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 light chain variable region (VL) comprise a CDR combination (HCDR1, HCDR2, HCDR 3; LCDR1, LCDR2, LCDR3) selected from:

the antibody or fragment thereof binds to human Trop-2.

Preferably, the heavy chain variable region comprises a sequence selected from:

1 to 17 or an amino acid sequence having at least 75% identity to the amino acid sequence shown; and/or

The light chain variable region comprises a sequence selected from:

18 to 36 or an amino acid sequence having at least 75% identity to the amino acid sequence shown.

According to a particular embodiment of the invention, the heavy chain variable region and the light chain variable region comprised by the antibody or fragment thereof of the invention may be selected from the following combinations:

Typically, the antibody or fragment thereof is in any form, e.g., a monoclonal antibody, a single chain antibody, a diabody, a single domain antibody, a nanobody, a fully or partially humanized antibody, or a chimeric antibody, or the antibody or fragment thereof is a half-antibody or antigen-binding fragment of a half-antibody, e.g., scFv, BsFv, dsFv, (dsFv)₂、Fab、Fab'、F(ab')₂Or Fv; the antibody or fragment thereof may be of mouse, rat, human or any other origin;

more preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and/or a light chain constant region of the kappa or lambda type.

According to a particular embodiment of the invention, 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 of the IgG1 or IgG4 subtype and the light chain constant region is of the kappa type;

according to a particular embodiment of the invention, the heavy chain constant region of said monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO 37 or an amino acid sequence having at least 75% identity to said amino acid sequence;

The above-described at least 75% identity of the present invention is any percentage identity of at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity ≧ 75%.

Based on the antibody or fragment thereof of the present invention, in another aspect, the present invention also provides a nucleic acid molecule encoding the heavy chain CDR, the light chain CDR, the heavy chain variable region, the light chain variable region, the heavy chain or the light chain in any of the antibodies or fragments thereof of the present invention.

In yet another aspect, the invention provides a vector comprising a nucleic acid molecule of the invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector and the like.

The vectors or nucleic acid molecules of the invention may be used to transform or transfect host cells or in any way into host cells for the purpose of preserving or expressing antibodies, etc.

Thus, in a further aspect, the present invention provides a host cell comprising a nucleic acid molecule and/or vector of the invention, or transformed or transfected with a nucleic acid molecule and/or vector of the invention. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.

Based on the present disclosure, the antibodies or fragments thereof, nucleic acid molecules, vectors, and/or host cells provided herein can be obtained using any conventional techniques known in the art. The antibody or fragment thereof, nucleic acid molecule, vector and/or host cell may be comprised in a pharmaceutical composition, more particularly in a pharmaceutical preparation, for various purposes according to the actual needs.

Thus, in a further aspect, the invention also provides a pharmaceutical composition comprising an antibody or fragment thereof, a nucleic acid molecule, a vector and/or a host cell according to the invention, and optionally a pharmaceutically acceptable excipient.

The antibody or fragment thereof of the present invention can be used in combination with other antibody-based drugs having phagocytosis by macrophages. Therefore, preferably, the antibody drug promotes phagocytosis of the cell by macrophages through binding to a cell surface-expressed protein. Therefore, the pharmaceutical composition provided by the present invention may further comprise said other antibody-based drug, preferably a macrophage-based immune checkpoint antibody; according to a particular embodiment of the invention, the antibody is an anti-CD47 antibody.

The invention also provides related uses of the subject matter described above as antibodies that bind to human Trop-2 or any portion thereof.

In particular, in a further aspect, the invention provides the use of the antibody or fragment thereof, the nucleic acid molecule, the vector, the host cell and/or the pharmaceutical composition for the preparation of a medicament for the treatment of Trop-2 high expressing cancers; preferably, the Trop-2 high-expression cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, urothelial cancer, triple negative breast cancer or cervical cancer.

In this respect, the use encompasses the use of an antibody or fragment thereof of the invention in combination with other antibody-based drugs as described above for the preparation of said drugs.

The antibodies or fragments thereof provided by the present invention may also be fused or conjugated to other moieties. For example, the invention provides a fusion protein or conjugate comprising an antibody or fragment thereof of the invention.

With respect to fusion proteins, the fusion protein may comprise any other moiety, such as an amino acid, polypeptide, or protein, that modifies an antibody or fragment thereof of the invention.

With respect to conjugates, the conjugates can comprise an antibody or fragment thereof of the invention and a drug conjugated thereto, wherein the drug is, for example, a cytotoxic agent.

Preferably, the conjugate is an Antibody Drug Conjugate (ADC) represented by the formula: (antibody or fragment thereof of the invention) - (linker) - (cytotoxic agent);

preferably, the cytotoxic agent is a tubulin inhibitor (such as paclitaxel, docetaxel, etc.) or a DNA replication inhibitor (such as irinotecan or its metabolic active SN-38, etc.).

According to a particular embodiment of the invention, the conjugate is an "anti-TROP-2 antibody-linker-SN-38 antibody drug conjugate".

The invention also provides the use of the antibody or fragment thereof, nucleic acid molecule, vector, host cell and/or pharmaceutical composition in the preparation of an Antibody Drug Conjugate (ADC) for the treatment of Trop-2 high expressing cancers; preferably, the Trop-2 high-expression cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, urothelial cancer, triple negative breast cancer or cervical cancer.

The invention provides a novel anti-human Trop-2 antibody, which has good biological activity: the antibodies provided by the invention (including chimeric antibodies and humanized antibodies) can be effectively combined no matter the Trop-2 recombinant protein or the Trop-2 antigen expressed on the surface of cells, and are similar to a control antibody, namely Sacituzumab. Meanwhile, the antibody provided by the invention has high affinity to human Trop-2: compared with the control antibody, the humanized antibody of the invention has even higher specific binding capacity of the human Trop-2 protein and higher affinity than the Sacituzumab. Therefore, the antibody of the present invention has a good drug effect.

Experiments prove that the antibody disclosed by the invention also has good internalization capacity: the internalization rate of the humanized antibody is similar to that of a control antibody, namely, Sacituzumab; internalization capacity was significantly enhanced after labeling into ADCs. Thus, the antibodies of the invention have potential for the development of ADC drugs. The Trop-2 antibody of the invention can also have synergistic effects with other antibodies, for example, the antibody of the invention can be combined with CD47 to further promote the phagocytosis of tumor cells by macrophages.

In addition, the antibodies of the invention also demonstrate good in vivo efficacy. The antibody of the invention is used for preparing ADC, and the anti-Trop 2-ADC antibody is found to have a dose-dependent inhibition effect on tumor growth, under a high dose (10mg/kg), the drug effect of each ADC antibody is equivalent to that of a control antibody, namely, Sacituzumab, the obvious toxic effect of ADC micromolecule SN38 is not observed, the weight of animals in each experimental group is steadily increased, and the difference between the animal weight and the control is not obvious.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the screening results of positive hybridoma supernatants in combination with CHO cell surface Trop-2.

FIG. 2 shows the screening results of positive hybridoma supernatants in combination with CHO cell surface Trop-2.

FIG. 3 shows the results of ELISA detection of cross-reaction of supernatants from positive hybridoma clones with recombinant Trop-2 of different species.

Fig. 4 shows the results of ELISA detection of the binding activity of the anti-human Trop-2 chimeric antibody to the Trop-2 recombinant protein, wherein fig. 4A: ch 3-11; FIG. 4B: ch 4-3; FIG. 4C: ch 23-12; FIG. 4D: ch 11-4; FIG. 4E: ch 17-1.

Fig. 5 shows the results of FACS detection of the binding activity of the anti-human Trop-2 chimeric antibody to cell surface Trop-2 recombinant protein, in which fig. 5A: ch 3-11; FIG. 5B: ch 23-12; FIG. 5C: ch 11-4; FIG. 5D: ch 4-3; FIG. 5E: ch 17-1.

Fig. 6 shows the species-specific results of ELISA detection of anti-human Trop-2 antibodies binding to Trop-2, where fig. 6A: h 23-12; FIG. 6B: h 4-3; FIG. 6C: sacituzumab.

FIG. 7 shows the result of affinity analysis of anti-human Trop-2 antibody for human Trop-2 extracellular domain recombinant protein, in which FIG. 7A: sacituzumab; FIG. 7B: h 23-12; FIG. 7C: h 4-3.

FIG. 8 shows the observation of internalization of anti-Trop-2 humanized antibodies after binding to cell surface Trop-2 of N87.

Fig. 9 shows the drug-time curve (Trop-2 detected) of a single dose of anti-Trop-2 humanized antibody in nude mice, where fig. 9A: h 23-12; FIG. 9B: h 4-3.

FIG. 10 shows the inhibition of cell growth by anti-Trop-2-ADC antibodies.

FIG. 11 shows the body weight change curves of Balb/C nu tumor-bearing gastric cancer N87 mouse model mice, in which FIG. 11A: after the administration of ch4-3-SN 38; FIG. 11B: h23-12-SN38 after administration; FIG. 11C: after administration of isotype control antibody; FIG. 11D: high dose administration of ch4-3-SN38 and h23-12-SN 38.

Fig. 12 shows the tumor volume change curve of Balb/C nu tumor-bearing gastric cancer N87 mouse model, in which fig. 12A: after the administration of ch4-3-SN 38; FIG. 12B: h23-12-SN38 after administration; FIG. 12C: after administration of isotype control antibody; FIG. 12D: high dose administration of ch4-3-SN38 and h23-12-SN 38.

Fig. 13 shows the body weight change curves of SKOV3 subcutaneous graft tumor mouse model mice using anti-Trop 2 antibody in combination with anti-CD47 antibody.

Fig. 14 shows the tumor volume change curves of SKOV3 subcutaneous graft tumor mouse model using anti-Trop 2 antibody in combination with anti-CD47 antibody.

FIG. 15 shows the tumor volume change curve of Balb/C nu tumor-bearing gastric cancer NCI-N87 subcutaneous graft tumor mouse model.

FIG. 16 shows the body weight change curves of Balb/C nu tumor-bearing gastric cancer NCI-N87 subcutaneous graft tumor mouse model mice.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.

Example 1Preparation of anti-human Trop-2 antibody hybridoma

Immunization: adopting human Trop-2 recombinant protein (SEQ ID NO: NP-002344.2, 1aa-274aa) to immunize a Balb/c mouse, and using a 96-hole enzyme label plate coated with human Trop-2-his recombinant protein (SEQ ID NO: NP-002344.2, 1aa-274aa) to detect the serum titer by an ELISA method; mice with serum titers meeting the fusion requirements were used for the next cell fusion.

Cell fusion and hybridoma preparation: selecting mice with titer meeting the requirement, performing impact immunization, taking spleen of mice aseptically after 3 days, preparing B lymphocyte suspension, and mixing with SThe P2/0 myeloma cells were mixed at a ratio of 4:1 and the two cells were fused by PEG. The fused cells were resuspended in HAT medium and then plated in 96-well cell culture plates. Standing at 37 deg.C for 5% CO₂Culturing in an incubator.

Example 2Screening of anti-human Trop-2 antibody positive hybridoma cell strain

1. Positive hybridoma binding screen

10-14 days after fusion, coating the ELISA plate with human Trop-2-his recombinant protein (SEQ ID NO: NP-002344.2, 1aa-274aa) (20ng/ml), and standing overnight at 4 ℃; washing with PBS for three times, sealing with 4% skimmed milk powder-PBS, and standing at room temperature for 1 hr; washing with PBS for three times, adding hybridoma clone culture supernatant, and standing at room temperature for 1 hr. Let the following controls: (1) positive Control (PC): post-immunization mouse serum (diluted with PBS 1: 1000); (2) negative Control (NC): fusion wells without cell growth. Washing with PBST (0.05% Tween-PBS) for three times, washing with PBS for two times, adding HRP-goat anti-mouse IgG (Fc γ), and heating at 37 deg.C for 0.5 hr; washing with PBST (0.05% Tween20-PBS) for 3 times, adding TMB color development solution, developing for 15-30min in dark place, adding ELISA stop solution, and stopping reaction; the microplate reader reads the A450 value.

The first 95 clones with high reading values are selected according to the principle of high to low for secondary ELISA confirmation, 25 antibody secretion positive cells pool are selected for subcloning by a limiting dilution method, after plating for 10 days, monoclonal cell supernatants are selected for further screening of positive clones by an ELISA method, the ELISA method is the same as above, the first 21 clones with high reading values are selected according to the principle of high to low, 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 and m24-3 are subjected to next FACS binding screening.

2. Positive hybridoma and CHO cell surface Trop-2 binding screening

Cloning the reading frame of the Trop-2 gene from a carrier (Cat.: HG10428-M, Beijing Yiqiao Shenzhou) containing Trop-2cDNA by PCR, cloning the reading frame into a stable expression carrier containing a Glutamine Synthetase (GS) screening gene by an enzyme digestion method, electrotransfecting (Nuclear selector IIb, Lonza) suspension cultured CHO-K1 cells, and placing the transfected cells into a carrier containing the Trop-2cDNA50 μ M MSX (Cat.: M5379, Sigma) in CD CHO AGTTM medium (Cat.:12490-025, Gibco) were seeded in 96-well cell culture plates at 37 ℃ with 5% CO₂The cells were cultured for 2-3 weeks at rest, screened by MSX pressure, prescreened for 22 cell-growth wells and expanded to 24-well cell culture plates, and clones No. 1-T-21 (CHO/Trop-2 cells) were finally selected by flow cytometry (FACS), expanded and frozen for FACS detection.

According to the ELISA results, the hybridoma supernatants of the 21 clones were selected, diluted 100-fold, and incubated with the constructed CHO cell (CHO/Trop-2 cell) suspension at 37 ℃ for 30min, with the following controls: (1) positive Control (PC): murine IgG constant region form of Sacituzumab, 1 ug/ml; (2) negative Control (NC): irrelevant murine antibody, 1 ug/ml. After washing the cells 3 times with PBS, 1:200 dilution of goat anti-mouse IgG-FITC (Cat.: F9006, Sigma) was added and incubated for 30 min. After washing the cells 3 times with PBS, the Mean Fluorescence Intensity (MFI) of the cells was examined by flow cytometry (model B49007AD, SNAW31211, BECKMAN COULTER) to verify whether the antibody secreted by the hybridoma could bind to CHO cell surface Trop-2, and the results are shown in fig. 1.

Based on FIG. 1 and the cell state, clones 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, and m23-12 were selected, hybridoma supernatants were purified by ProA affinity chromatography, and binding of the purified murine antibodies was confirmed again. The antibodies were diluted to 13nM and 0.66nM, respectively, and then incubated with a suspension of CHO cells recombinantly expressing human Trop-2 (CHO/Trop-2 cells) for 30min at 37 deg.C, with the following controls: (1) positive Control (PC): murine IgG constant region form of Sacituzumab, 1 ug/ml; (2) negative Control (NC): irrelevant murine antibody, 1 ug/ml. After washing the cells 3 times with PBS, 1:200 dilution of goat anti-mouse IgG-FITC (Cat.: F9006, Sigma) was added and incubated for 30 min. After washing the cells 3 times with PBS, the Mean Fluorescence Intensity (MFI) of the cells was examined by flow cytometry (model B49007AD, SNAW31211, BECKMAN COULTER) to verify whether the antibodies secreted by the hybridomas bind to CHO cell surface Trop-2, as shown in fig. 2, the antibodies from the 15 clone supernatants all bind well to CHO cell surface Trop-2.

M3-11, m4-3, m11-4, m17-1 and m23-12 are selected as candidate clones for further screening.

3. Species-crossed ELISA screening of positive hybridoma clones

Human Trop-2-His recombinant protein (sequence number: NP-002344.2, 1aa-274aa), cynomolgus monkey Trop-2-His recombinant protein (sequence number: UniProtKB-A0A2K5UE71, 1aa-272aa), mouse Trop-2-His recombinant protein (Cat.:50922-M08H, Beijing Yigao Hooka) are coated overnight at 4 ℃, and the coating concentration is 0.2, 1 and 1 mu g/mL respectively; after washing the plate for 3 times with PBS, adding 5% BSA PBS, blocking for 60min at 37 ℃, and washing the plate for 3 times with PBST; the 15 purified murine antibodies were diluted to 1 μ g/mL with PBS, with the following controls: (1) positive Control (PC): 1ug/ml of murine IgG constant region form of Sacituzumab (WHO Drug Information (Vol.31, No.1,2017), SEQ ID NO:39 and SEQ ID NO: 40); (2) negative Control (NC): irrelevant hybridoma antibody, 1. mu.g/mL; (3) blank control: PBS. Incubating at 37 ℃ for 60min, and washing the plate for 4 times by PBST; adding HRP-goat anti-mouse IgG (Fcr) (Cat: 115-; adding a TMB substrate for color development, incubating at 37 ℃ for 10min, and adding 2M HCl to stop the reaction; the absorbance A450nm-630nm of the well plate at a wavelength of 450nm was read and recorded using 630nm as the reference wavelength. Except that the antibodies of clones m12-4, m17-1, m19-5 and m21-1 crossed with mouse Trop-2, the remaining antibodies did not cross with mouse, but all hybridoma antibodies could specifically bind to recombinant human and cynomolgus monkey Trop-2 (FIG. 3).

Example 3Sequence determination of murine anti-human Trop-2 antibody

After expanded culture of hybridoma cells m3-11, m4-3, m11-4, m17-1 and m23-12 secreting anti-human Trop-2 Antibody, subtype detection was performed using Mouse Monoclonal Antibody IgG Subclass Test Card (Cat.: A12403, VicNovo) and Mouse Monoclonal Antibody Light/Heavy Chain chair Test Card (Cat.: A12401, VicNovo) according to reagent protocol, and subtypes were identified as: the heavy chain is IgG1, the light chain is Kappa chain, and the cloning of antibody genes of m3-11, m4-3, m11-4, m17-1 and m23-12 provides basis.

Subjecting m3-11, m4-3, m11-4, m17-1, m23-12 hybridoma cells to TRIzolTotal cellular RNA was extracted using the kit (Cat.:15596026, Invitrogen) protocol; reverse transcribing hybridoma cell total RNA to cDNA using M-MuLV reverse transcriptase (Cat.: M0253S, NEB); degenerate primers (see book (DongShi, Wang Yan. antibody engineering (second edition); Beijing university of medicine publisher, 2001, 313-]) And Phusion kit (Cat.: E0553L, NEB) for the amplification of antibody light chain variable region IgVL (kappa) and heavy chain variable region V_HA sequence; purifying PCR amplification products by using a gel recovery kit (Cat.: AP-GX-250, Axygen); connecting the amplified PCR product to a T vector according to the specification of a T vector cloning kit (Cat.: ZC205, Jiang Jiu organism), transforming escherichia coli competent cells, amplifying strains, extracting plasmids, and then performing DNA sequencing to obtain the variable region sequence of the monoclonal antibody.

The sequencing result shows that:

the nucleotide sequence of the mouse antibody heavy chain variable region DNA of clone m3-11 is shown in SEQ ID NO:41, and the amino acid sequence of the mouse antibody heavy chain variable region of clone m3-11 is deduced from the DNA sequence and is shown in SEQ ID NO: 1; the nucleotide sequence of mouse antibody light chain variable region DNA of clone m3-11 is shown in SEQ ID NO:42, and the amino acid sequence of clone m3-11 mouse antibody light chain variable region is shown in SEQ ID NO: 18.

SEQ ID NO:1：

QVQLQQPGAELVKPGSSVKLSCKASGYTFTSYWMYWVKQRPGQGLEWIGEINPSNGRTNYNEKFKSKATLTVDKSSSTAYMQFSSLTSEDSAVYYCTREGHNYDGSLGAMDHWGQGTSVTVSS

SEQ ID NO:18：

DVVVTQTPLSLPVSFGDQVSISCRSSQSLTNSYGNTFLSWYLHKPGQSPQLLLYGISNRFSGVPDRFSGSGSGTDFTLKINTIKPEDLGMYYCFQSTHQPYTFGGGTKLEIK

The nucleotide sequence of the mouse antibody heavy chain variable region DNA of clone m4-3 is shown in SEQ ID NO:43, and the amino acid sequence of the mouse antibody heavy chain variable region of clone m4-3 is deduced from the DNA sequence and is shown in SEQ ID NO: 2; the nucleotide sequence of the mouse antibody light chain variable region DNA of clone m4-3 is shown in SEQ ID NO. 44, and the amino acid sequence of the mouse antibody light chain variable region of clone m4-3 is deduced from the DNA sequence and is shown in SEQ ID NO. 19.

SEQ ID NO:2：

QVQLQQSGPELVKPGASVKMSCKASGFTFTDYVIGWVKQRTGQGLEWIGEIYLGSGTIYYTEKFKGKATLTADTSSNTAYMQLSSLTSEDSAVYFCARGSIFPFDYWGQGTTLTVSS

SEQ ID NO:19：

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSTLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPYTFGGGTKLEIK

The nucleotide sequence of the mouse antibody heavy chain variable region DNA of clone m11-4 is shown in SEQ ID NO:47, and the amino acid sequence of the mouse antibody heavy chain variable region of clone m11-4 is shown in SEQ ID NO:5 by inference from the DNA sequence; the nucleotide sequence of mouse antibody light chain variable region DNA of clone m11-4 is shown in SEQ ID NO. 48, and the amino acid sequence of clone m11-4 mouse antibody light chain variable region is shown in SEQ ID NO. 24.

SEQ ID NO:5：

QVQLQQPGAELVRPGASVNLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSNSYTNYNQKFKDTATLTVDKSSSTAYMQLSSPTSEDSAVYFCSSYRSDGFAYWGQGTLVTVSA

SEQ ID NO:24：

DILLTQSPAILSVSPGEKVSFSCRASQNIGTSIHWYQQRTNGSPRLLIEFASESISGIPSRFSGSGSGTDFTLTINSVESEDIADYYCQQSNSWPFTFGGGTKLEIK

The nucleotide sequence of the mouse antibody heavy chain variable region DNA of clone m17-1 is shown in SEQ ID NO:51, and the amino acid sequence of the mouse antibody heavy chain variable region of clone m17-1 is deduced from the DNA sequence and is shown in SEQ ID NO: 11; the nucleotide sequence of the clone m17-1 mouse antibody light chain variable region DNA is shown in SEQ ID NO:52, and the amino acid sequence of the clone m17-1 mouse antibody light chain variable region is shown in SEQ ID NO:30 through the inference of the DNA sequence.

SEQ ID NO:11：

EVKLVESGGVLVKPGGSLKLSCAASGFTFSDSAMSWVRQTPEKRLEWVASISRGDDTYYPDSVKGRITISRDFARNILYLQMTSLRSEDTAMYYCTRDRFGFAYWGQGTLVTVSA

SEQ ID NO:30：

DIVMTQSPLTLSVTIGQPASISCKSGQSLLDSDGKTYFNWLLQRPGQSPKRLIYLVSMLDSGVPDRFTGSGSGTDFTLKISRVETEDLGVYYCWQGTHFPFTFGSGTKLEIK

The nucleotide sequence of the mouse antibody heavy chain variable region DNA of clone m23-12 is shown in SEQ ID NO:53, and the amino acid sequence of the mouse antibody heavy chain variable region of clone m23-12 is deduced from the DNA sequence and is shown in SEQ ID NO: 12; the nucleotide sequence of the mouse antibody light chain variable region DNA of clone m23-12 is shown in SEQ ID NO. 54, and the amino acid sequence of the mouse antibody light chain variable region of clone m23-12 is deduced from the DNA sequence and is shown in SEQ ID NO. 31.

SEQ ID NO:12：

QVQLQQPGAELVKPGASVKLSCKADGYIFTSYWMHWVKQRPGQGLEWIGEITPSDNYTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTRGHGNYVSFDYWGQGTTLTVSS

SEQ ID NO:31：

DIQMTQITSSLSASLGDRVTITCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGYTLPPYTFGGGTKLEIK

Example 4Preparation of anti-human Trop-2 chimeric antibody and control antibody

The light and heavy chain sequences of a control antibody (Sacituzumab) are completely synthesized, the light and heavy chain sequences are respectively cloned into a eukaryotic transient expression vector to obtain light chain and heavy chain expression plasmids of the control antibody, the light chain and heavy chain expression plasmids are transferred into escherichia coli for amplification, a large number of plasmids containing the light chain and heavy chain of the control antibody are obtained through separation, the light chain and heavy chain plasmids of the control antibody are respectively transferred into HEK293 cells for recombinant expression by utilizing the plasmids and according to the operation instruction of a transfection reagent 293fectin (Cat.:12347019, Gibco). 5-6 days after cell transfection, culture supernatant is taken and purified by a ProA affinity chromatography column to obtain a control antibody. Wherein, the amino acid sequence of the contrast antibody Sacituzumab is derived from WHO Drug Information (Vol.31, No.1,2017), the amino acid sequence of the heavy chain is shown in SEQ ID NO. 39, and the amino acid sequence of the light chain is shown in SEQ ID NO. 40.

The enzyme cutting sites are introduced into the light chain variable region and heavy chain variable region genes of corresponding murine antibodies 3-11, 4-3, 11-4, 17-1, 23-12 obtained from each clone through PCR, and the genes are respectively cloned into a eukaryotic transient expression vector containing a human-kappa light chain constant region and the upstream of a human IgG1 heavy chain constant region coding gene to obtain a human-murine chimeric light chain (pKN019-ch3-11L, pKN019-ch4-3L, pKN019-ch11-4L, pKN 019-17-1L, pKN019-ch23-12L) and a human-murine chimeric heavy chain (pKN041-ch3-11H, pKN019-ch4-3H, pKN019-ch11-4H, pKN019 ch-17-1H, pKN019-ch23-12H) expression plasmid, and the expression plasmid is transferred into escherichia coli for amplification, a large amount of plasmids containing the light chain and the heavy chain of the human-mouse chimeric antibody are obtained by separation, and the light chain and the heavy chain plasmids of the chimeric antibodies ch3-11, ch4-3, ch11-4, ch17-1 and ch23-12 are respectively transferred into HEK293 cells for recombinant expression by using the plasmids according to the operation instruction of a transfection reagent 293fectin (Cat.:12347019, Gibco). 5-6 days after cell transfection, culture supernatant was taken and purified by a ProA affinity column to obtain chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4 and ch 23-12.

Example 5ELISA (enzyme-Linked immuno sorbent assay) for detecting the binding activity of the anti-human Trop-2 chimeric antibody and the Trop-2 recombinant protein

Human Trop-2-his recombinant protein (SEQ ID NO: NP-002344.2, 1aa-274aa) at a concentration of 0.2. mu.g/mL, was coated overnight at 4 ℃ and blocked with 5% BSA in a 37 ℃ incubator for 60 min. Adding ch3-11, ch4-3, ch17-1, ch11-4, ch23-12 and a control antibody Sacituzumab (the initial concentration is 2 mu g/mL, 3 times of serial dilution and 8 gradients), and reacting in a constant temperature incubator at 37 ℃ for 60 min. PBST plate washing 4 times; then HRP-anti-human Fc (Cat.: 109-. The absorbance values A450nm-630nm were read and recorded for a well plate at a wavelength of 450nm, using 630nm as the reference wavelength.

The binding capacity of ch3-11, ch4-3, ch17-1, ch11-4, ch23-12 and the control antibody Saituzuzumab to human Trop-2 recombinant protein was determined by ELISA, and the half-effective binding concentration (EC50) values were 0.3147nM, 0.3195nM, 0.3278nM, 0.2366nM, 0.4581nM and 0.271nM (FIG. 4), which were substantially equivalent. The results show that the ch3-11, ch4-3, ch17-1, ch11-4 and ch23-12 chimeric antibodies have high affinity with human Trop-2 recombinant protein, and the sequence cloning of the murine antibodies 3-11, 4-3, 11-4, 17-1 and 23-12 is correct.

Example 6FACS detection of the binding activity of the anti-human Trop-2 chimeric antibody and human Trop-2 recombinant protein on the surface of CHO cells

A suspension of CHO cells (CHO/Trop-2 cells) recombinantly expressing human Trop-2 was incubated with chimeric antibodies (ch3-11, ch4-3, ch17-1, ch11-4, ch23-12) (concentration 30. mu.g/mL, 10. mu.g/mL, 5. mu.g/mL starting with 3-fold serial dilutions of 9 gradients, 11 gradients) for 30min at 37 ℃ with the following controls: (1) positive Control (PC): control antibody Sacituzumab; (2) negative Control (NC): IgG1 isotype control antibody NC-IgG 1. After washing the cells 3 times with PBS, goat anti-human IgG-FITC (Cat.: F9512, Sigma) diluted 1:100 was added and incubated for 30 min. After washing the cells 3 times with PBS, the Mean Fluorescence Intensity (MFI) of the cells was measured by flow cytometry (model B49007AD, SNAW31211, BECKMAN COULTER) to examine the binding ability of the chimeric antibody to human Trop-2 on the CHO cell surface.

The binding capacity of ch3-11, ch4-3, ch17-1, ch11-4, ch23-12 and the control antibody Saituzuzumab to human Trop-2 recombinant protein on the CHO cell surface was determined by FACS with half-maximal effective binding concentration (EC50) values of 0.993nM, 3.326nM, 2.918nM, 1.154nM, 2.748nM and 2.316nM, respectively (FIG. 5). Compared with the control antibody Sacituzumab, the binding activities of ch3-11 and ch11-4 are better, and the binding activities 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 the human Trop-2 recombinant protein on the surface of CHO cells.

Example 7Internalization activity of anti-human Trop-2 chimeric antibody combined with cell surface Trop-2

Taking BxPC-3, 5 × 10 human pancreatic cancer cells⁵Chimeric antibodies ch3-11, ch4-3, ch11-4, ch23-12 and a positive control antibody Sacituzumab diluted to 10. mu.g/ml are added to each cell/tube, and each antibody is divided into four groups (an experimental group and a control group which are incubated for 1h, 3h and 5 h), and each group has 2 tubes. Placing the experimental group into a 37 ℃ electric heating constant temperature incubator, respectively incubating for 1h, 3h and 5h, then placing on ice, and constantly incubating the control group as a negative control; after all the samples were incubated, the cells were centrifuged at 1,500rpm at 4 ℃ for 3min, the supernatant was discarded, the cells were washed with ice-cold PBS 1 time, a secondary antibody, i.e., anti-human IgG (Fc specific) -FITC antibody (Cat.: F9512, Sigma) was added, the cells were incubated for 30 minutes, centrifuged at 1,500rpm for 3min, the supernatant was discarded, the cells were washed with ice-cold PBS, 200. mu.l of ice-cold resuspended PBS cells were taken, and FACS detection was performed to detect mean fluorescenceIntensity MFI, and internalization efficiency was calculated by the formula: the% MFI at tx time point-MFI of 37 ℃ incubated sample x 100%/MFI of 4 ℃ incubated control sample; percent internalization at time tx ═ 100% to% MFI at time tx.

As shown in Table 1, the internalization ratios of ch4-3 and ch23-12 and the control antibody, Sacituzumab, were similar, while no significant internalization occurred in ch3-11 and ch 11-4.

TABLE 1 percent internalization of cell surface Trop-2 mediated anti-human Trop-2 chimeric antibodies

Example 8Stability against disruption of tolerance of anti-human Trop-2 chimeric antibodies

Chimeric antibodies ch3-11, ch4-3, ch11-4 and ch23-12 were placed in PBS, PBS containing 10% N, N-Dimethylacetamide (DMA) (Cat.: ARK2190, manufactured by Shanghai Selaginella chemical) and PBS containing 20% DMA at a concentration of 5mg/mL, respectively, and after standing at 37 ℃ for 2 hours, the samples were freed from DMA using an ultrafiltration centrifuge tube, the buffer was replaced with PBS, and the samples were analyzed for purity by high performance size exclusion chromatography (SEC-HPLC) using a G3000Wxl liquid chromatography column (Cat.: SEC-0046, Tosoh).

The results show that all 4 antibodies can well tolerate DMA, the purity is not obviously reduced at 10%, and the purity is slightly reduced at 20%, which suggests that the antibodies may have better tolerance to the subsequent ADC process.

TABLE 2 analysis of antibody HPLC purity before and after DMA treatment

Example 9Humanization and recombinant expression of anti-human Trop-2 monoclonal antibody

1. Humanization of murine monoclonal antibody 23-12

(1) CDR grafting

First, the heavy chain sequence of the murine antibody is comprehensively analyzed to determine the antigen Complementarity Determining (CDR) region where the antibody binds to the antigen and the framework region (framework) that supports the conserved three-dimensional conformation of the antibody. Then, based on the results of homology alignment, the most similar human antibody template was searched for in the human antibody germline library (http:// www2.mrc-lmb. cam. ac. uk/vbase/alignment 2.php # VHEX), and CDR grafting was performed by combining the full sequence blast results and the sequence characteristics of heavy chain CDR3, thereby realizing full humanization of the 23-12 heavy chain variable region (VH) in the framework region. According to the homology comparison result, the most similar human antibody template is searched in a human antibody germline library (http:// www2.mrc-lmb. cam. ac. uk/vbase/alignment 2.php # VHEX), and CDR grafting is carried out by combining the whole sequence blast result and the sequence characteristics of light chain CDR3, so that the high humanization of the light chain framework region is realized.

The nucleotide sequence of the humanized heavy chain variable region h23-12_ VH1 of the CDR grafted 23-12 antibody is shown in SEQ ID NO. 55, and the amino acid sequence is shown in SEQ ID NO. 13; the variable region h23-12_ VL1 of the humanized light chain has the nucleotide sequence shown in SEQ ID NO. 56 and the amino acid sequence shown in SEQ ID NO. 32.

SEQ ID NO:13：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGEITPSDNYTSYNQKFKGRVTITRDTSTSTAYMELSSLRSEDTAVYYCARGHGNYVSFDYWGQGTLVTVSS

SEQ ID NO:32：

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGYTLPPYTFGQGTKLEIKRTVAAP

(2) CDR region mutation design

According to the sequence characteristics of the murine antibody 23-12, the CDR sequences of the CDR grafted humanized light and heavy chain variable regions are subjected to mutation design, and the mutation sites are shown in the following table 3.

TABLE 3.23-12 humanized sequence design

Note: amino acid residue position numbering is according to the Kabat numbering system.

2. Recombinant expression of humanized monoclonal antibody 23-12

The humanized h23-12 antibody light chain variable region (h23-12_ VL1, h23-12_ VH1) sequence is synthesized completely, and humanized h23-12_ VH1 is cloned into the eukaryotic transient expression vector pKN041 at the upstream of the heavy chain constant region coding gene of human IgG1 by enzyme digestion, wherein the nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO:59, and the amino acid sequence is shown in SEQ ID NO: 37; humanized H23-12_ VL1 is cloned into the upstream of a coding gene of a human light chain C kappa of a eukaryotic transient expression vector pKN019 by enzyme digestion, the nucleotide sequence of a light chain constant region is shown in SEQ ID NO:60, the nucleotide sequence of an amino acid sequence is shown in SEQ ID NO:38, a humanized 23-12 light and heavy chain expression vector is constructed, expression plasmids of a light chain (pKN019-H23-12L1) and a heavy chain (pKN041-H23-12H1) are obtained, and the expression plasmids are transferred into escherichia coli for amplification and separated to obtain plasmids H23-12 antibody light chain and heavy chain H23-12L1 and H23-12H 1.

According to the mutation design shown in Table 3, using a StarMut gene site-directed mutagenesis kit (GenStar, Cat.: T111-01), respectively carrying out site-directed mutagenesis on expression plasmids of a light chain (pKN019-H23-12L1) and a heavy chain (pKN041-H23-12H1) and transferring the expression plasmids into escherichia coli for amplification to obtain light and heavy chain CDR region mutation expression plasmids (H23-12H 2-H23-12H 7, H23-12L 2-H23-12L 7) of the H23-12 antibody, wherein the light and heavy chain CDR region mutation expression plasmids respectively correspond to 23-12 humanized sequences in Table 3; the light and heavy chain plasmids of the 23-12 humanized antibody were combined according to the protocol of the transfection reagent 293fectin (Cat.:12347019, Gibco), and combined as shown in Table 4, and transferred into HEK293 cells for recombinant expression.

TABLE 4 humanized 23-12 light and heavy chain sequence combinations

Note: the table shows the sequences obtained for various 23-12 light heavy chain combinations, as H23-12-1 indicates that the antibody consists of 23-12 humanized antibody light chain H23-12L1 and humanized heavy chain H23-12H1, and so on.

5-6 days after cell transfection, culture supernatant is taken, expression supernatant is purified by a ProA affinity chromatography column, and the antibody affinity is measured by a method of capturing an Fc segment of an antibody by an Octet QKe system instrument of Fortebio company and a capture Antibody (AHC) biological probe of an anti-human antibody Fc segment, wherein the obtained 23-12 different humanized antibodies are different. The 23-12 antibody and the control antibody, Sacituzumab, were diluted to 4. mu.g/mL in PBS buffer and passed over the surface of an AHC probe (Cat.:18-0015, PALL) for 120 s. Human Trop-2-His recombinant protein (sequence number: NP-002344.2, 1aa-274aa) is used as a mobile phase, and the concentration of the Trop-2-His recombinant protein is 60 nM. The binding time was 100s and the dissociation time was 300 s. After the experiment, blank control response values were subtracted, 1:1Langmuir binding pattern fitting was performed with software, and kinetic constants for antigen-antibody binding were calculated.

The affinity of the h23-12 mutant combination antibody, the chimeric antibody ch23-12 and the control antibody, Sacituzumab, was determined by ForteBio to human Trop-2-His recombinant protein (Table 5).

TABLE 5.23-12 results of affinity determination of different antibodies with human Trop-2 extracellular region recombinant protein

Selecting h23-12-25, wherein the combined antibody has affinity (KD) of 5.02E-10M and is named as h23-12, and performing further functional verification, wherein the nucleotide sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:57, and the amino acid sequence is shown in SEQ ID NO: 14; the variable region of the light chain has the nucleotide sequence shown in SEQ ID NO. 58 and the amino acid sequence shown in SEQ ID NO. 33.

SEQ ID NO:14：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGEITPSDNYGSYNQKFKGRVTITRDTSTSTAYMELSSLRSEDTAVYYCARGHGNYVSFDYWGQGTLVTVSS

SEQ ID NO:33：

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGYTLPPYTFGQGTKLEIK

3. Humanization of murine monoclonal antibody 4-3

(1) CDR grafting

First, the heavy chain sequence of the murine antibody is comprehensively analyzed to determine the antigen Complementarity Determining (CDR) region where the antibody binds to the antigen and the framework region (framework) that supports the conserved three-dimensional conformation of the antibody. Then, according to the homology comparison result, the most similar humanized antibody template is searched in a human antibody germline library (http:// www2.mrc-lmb. cam. ac. uk/vbase/alignment 2.php # VHEX), and the CDR grafting is carried out by combining the whole sequence blast result and the sequence characteristics of the heavy chain CDR3, so that the full humanization of the 4-3 heavy chain variable region (VH) in the framework region is realized. According to the homology comparison result, the most similar human antibody template is searched in a human antibody germline library (http:// www2.mrc-lmb. cam. ac. uk/vbase/alignment 2.php # VHEX), and CDR grafting is carried out by combining the whole sequence blast result and the light chain CDR3 sequence characteristics, so that the whole humanization of the light chain framework region is realized.

The nucleotide sequence of the humanized heavy chain variable region h4-3_ VH1 grafted by the CDR of the 4-3 antibody is shown in SEQ ID NO:45, and the amino acid sequence is shown in SEQ ID NO: 3; the variable region h4-3_ VL1 of the humanized light chain has the nucleotide sequence shown in SEQ ID NO. 46 and the amino acid sequence shown in SEQ ID NO. 20.

SEQ ID NO:3：

EVQLVQSGPEVKKPGASVKVSCKASGFTFTDYVIGWVRQAPGQGLEWIGEIYLGSGTIYYTEKFKGRVTMTADTSTSTAYMELSSLRSEDTAVYYCARGSIFPFDYWGQGTLVTVSS

SEQ ID NO:20：

DIQLTQSPSSLSASVGDRVTITCSASSSVSYMYWYQQKPGKAPKLLIYDTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSYPYTFGQGTKLEIK

(2) CDR region mutation design

According to the sequence characteristics of the murine antibody 4-3, the CDR sequences of the CDR grafted humanized light and heavy chain variable regions are subjected to mutation design, and the mutation sites are shown in the following table 6.

TABLE 6.4-3 humanized sequence design

Note: amino acid residue position numbering is according to the Kabat numbering system.

4. Recombinant expression of humanized monoclonal antibody 4-3

The humanized h4-3 antibody light chain variable region (h4-3_ VL1, h4-3_ VH1) sequence is synthesized completely, and humanized h4-3_ VH1 is cloned into the eukaryotic transient expression vector pKN041 at the upstream of the heavy chain constant region coding gene of human IgG1 by enzyme digestion, wherein the nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO:59, and the amino acid sequence is shown in SEQ ID NO: 37; humanized H4-3_ VL1 is cloned to the upstream of the coding gene of the human light chain C kappa of a eukaryotic transient expression vector pKN019 by enzyme digestion, the nucleotide sequence of a light chain constant region is shown in SEQ ID NO:60, the nucleotide sequence of an amino acid sequence is shown in SEQ ID NO:38, a humanized 4-3 light and heavy chain expression vector is constructed, expression plasmids of a light chain (pKN019-H4-3L1) and a heavy chain (pKN041-H4-3H1) are obtained, and the expression plasmids are transferred into escherichia coli for amplification and separated to obtain plasmids H4-3 antibody light chain and heavy chain H4-3L1 and H4-3H 1.

According to the mutation design shown in Table 6, a StarMut gene site-directed mutagenesis kit (Cat.: T111-01, GenStar) is utilized to carry out site-directed mutagenesis on expression plasmids of a light chain (pKN019-H4-3L1) and a heavy chain (pKN041-H4-3H1) respectively, and the expression plasmids are transferred into escherichia coli for amplification to obtain light and heavy chain CDR region mutation expression plasmids (H4-3H 2-H4-3H 4, H4-3L 2-H4-3L 5) of the H4-3 antibody, wherein the light and heavy chain CDR region mutation expression plasmids respectively correspond to 4-3 humanized sequences in the Table 6; the light and heavy chain plasmids of the 4-3 humanized antibody were combined according to the protocol of the transfection reagent 293fectin (Cat.:12347019, Gibco), combined as shown in Table 7, and transferred into HEK293 cells for recombinant expression.

TABLE 7 humanized 4-3 light and heavy chain sequence combinations

	h4-3 H1	h4-3 H2	h4-3 H3	h4-3 H4
					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

Note: the table shows the sequences obtained for various 4-3 light heavy chain combinations, as H4-3-1 indicates that the antibody consists of a 4-3 humanized antibody light chain H4-3L1 and a humanized heavy chain H4-3H1, and so on.

5-6 days after cell transfection, culture supernatant is taken, expression supernatant is purified by a ProA affinity chromatography column, and the antibody affinity is measured by a method of capturing an Fc segment of an anti-human antibody by an Octet QKe system instrument of Fortebio company and a capture Antibody (AHC) biological probe of the Fc segment of the anti-human antibody, wherein the obtained 4-3 different humanized antibodies are obtained. The 4-3 antibody and the control antibody, Sacituzumab, were diluted to 4. mu.g/mL in PBS buffer and passed over the surface of an AHC probe (Cat.:18-0015, PALL) for 120 s. Human Trop-2-His recombinant protein (sequence number: NP-002344.2, 1aa-274aa) is used as a mobile phase, and the concentration of the Trop-2-His recombinant protein is 60 nM. The binding time was 100s and the dissociation time was 300 s. After the experiment, blank control response values were subtracted, 1:1Langmuir binding pattern fitting was performed with software, and kinetic constants for antigen-antibody binding were calculated.

The affinity of the h4-3 mutant combination antibody, the chimeric antibody ch4-3 and the control antibody, Sacituzumab, was determined by ForteBio to human Trop-2-His recombinant protein (Table 8).

TABLE 8.4-3 results of affinity determination of different humanized antibodies with the recombinant protein of the extracellular region of human Trop-2

Antibody combinations	KD value (M)	Antibody combinations	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

Selecting h4-3-1, wherein the combined antibody has affinity (KD) of 3.04E-10M and is named as h4-3, and performing further functional verification, wherein the nucleotide sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 45, and the amino acid sequence is shown in SEQ ID NO. 3; the variable region of the light chain has the nucleotide sequence shown in SEQ ID NO. 46 and the amino acid sequence shown in SEQ ID NO. 20.

SEQ ID NO:3：

EVQLVQSGPEVKKPGASVKVSCKASGFTFTDYVIGWVRQAPGQGLEWIGEIYLGSGTIYYTEKFKGRVTMTADTSTSTAYMELSSLRSEDTAVYYCARGSIFPFDYWGQGTLVTVSS

SEQ ID NO:20：

DIQLTQSPSSLSASVGDRVTITCSASSSVSYMYWYQQKPGKAPKLLIYDTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSYPYTFGQGTKLEIK

5. Humanization of murine monoclonal antibody 11-4

(1) CDR grafting

First, the heavy chain sequence of the murine antibody is comprehensively analyzed to determine the antigen Complementarity Determining (CDR) region where the antibody binds to the antigen and the framework region (framework) that supports the conserved three-dimensional conformation of the antibody. Then, based on the results of homology alignment, the most similar human antibody template was searched for in the human antibody germline library (http:// www2.mrc-lmb. cam. ac. uk/vbase/alignment 2.php # VHEX), and CDR grafting was performed by combining the results of full sequence blast and the sequence characteristics of heavy chain CDR3, thereby realizing full humanization of the 11-4 heavy chain variable region (VH) in the framework region. According to the homology comparison result, the most similar human antibody template is searched in a human antibody germline library (http:// www2.mrc-lmb. cam. ac. uk/vbase/alignment 2.php # VHEX), and CDR grafting is carried out by combining the whole sequence blast result and the light chain CDR3 sequence characteristics, so that the whole humanization of the light chain framework region is realized.

The nucleotide sequence of the humanized heavy chain variable region h11-4_ VH1 of the 11-4 antibody CDR grafted is shown in SEQ ID NO:49, and the amino acid sequence is shown in SEQ ID NO: 6; the variable region of the light chain h11-4_ VL1 has the nucleotide sequence shown in SEQ ID NO. 50 and the amino acid sequence shown in SEQ ID NO. 25.

SEQ ID NO:6

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPSNSYTNYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYRSDGFAYWGQGTLVTVSS

SEQ ID NO:25

EIVLTQSPATLSLSPGERATLSCRASQNIGTSIHWYQQKPGQAPRLLIYFASESISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNSWPFTFGGGTKVEIK

(2) CDR region mutation design

According to the sequence characteristics of the murine antibody 11-4, the CDR grafted humanized light and heavy chain variable region sequence is subjected to mutation design, and the mutation sites are shown in the following table 9.

TABLE 9.11-4 humanized sequence design

Note: amino acid residue position numbering is according to the Kabat numbering system.

6. Recombinant expression of humanized monoclonal antibody 11-4

The humanized h11-4 antibody light chain variable region (h11-4_ VL1, h11-4_ VH1) sequences are synthesized completely, and humanized h11-4_ VH1 is cloned into the eukaryotic transient expression vector pKN041 at the upstream of the coding gene of the heavy chain constant region of human IgG1 by enzyme digestion, wherein the nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO:59, and the amino acid sequence is shown in SEQ ID NO: 37; humanized H11-4_ VL1 is cloned into the upstream of a coding gene of a human light chain C kappa of a eukaryotic transient expression vector pKN019 by enzyme digestion, the nucleotide sequence of a light chain constant region is shown in SEQ ID NO:60, the nucleotide sequence of an amino acid sequence is shown in SEQ ID NO:38, a humanized 11-4 light and heavy chain expression vector is constructed, expression plasmids of a light chain (pKN019-H11-4L1) and a heavy chain (pKN041-H11-4H1) are obtained, and the expression plasmids are transferred into escherichia coli for amplification and separated to obtain plasmids H11-4 antibody light chain and heavy chain H11-4L1 and H11-4H 1.

According to the mutation design shown in Table 9, using a StarMut gene site-directed mutagenesis kit (Cat.: T111-01, GenStar), performing site-directed mutagenesis on expression plasmids of a light chain (pKN019-H11-4L1) and a heavy chain (pKN041-H11-4H1) respectively, transferring the plasmids into escherichia coli for amplification, and obtaining light and heavy chain mutation expression plasmids (H11-4H 2-H11-4H 7, H11-4L 2-H11-4L 5) of the H11-4 antibody, wherein the light and heavy chain mutation expression plasmids respectively correspond to 11-4 humanized sequences in Table 9; the light and heavy chain plasmids of the 11-4 humanized antibody were combined according to the protocol of the transfection reagent 293fectin (Cat.:12347019, Gibco), and combined as shown in Table 10, and transferred into HEK293 cells for recombinant expression.

TABLE 10 humanized 11-4 light and heavy chain sequence combinations

Note: the table shows the sequences obtained by combining various 11-4 light and heavy chains, e.g., H11-4-1 indicates that the antibody consists of 11-4 humanized antibody light chain H11-4L1 and humanized heavy chain H11-4H1, and so on.

5-6 days after cell transfection, culture supernatant is taken, expression supernatant is purified by a ProA affinity chromatography column, and the antibody affinity of the obtained 11-4 different humanized antibodies is measured by a method of capturing an Fc segment of an anti-human antibody Fc segment by an Octet QKe system instrument of Fortebio company and adopting a capture Antibody (AHC) biological probe of the anti-human antibody Fc segment. For the assay, the 11-4 antibody and the control antibody, Sacituzumab, were diluted to 4. mu.g/mL in PBS buffer and passed over the surface of an AHC probe (Cat.:18-0015, PALL) for 120 s. Human Trop-2-His recombinant protein (sequence number: NP-002344.2, 1aa-274aa) is used as a mobile phase, and the concentration of the Trop-2-His recombinant protein is 60 nM. The binding time was 100s and the dissociation time was 300 s. After the experiment, blank control response values were subtracted, 1:1Langmuir binding pattern fitting was performed with software, and kinetic constants for antigen-antibody binding were calculated.

The affinity of the h11-4 mutant combination antibody, the chimeric antibody ch11-4 and the control antibody, Sacituzumab, was determined by ForteBio to human Trop-2-His recombinant protein (Table 11).

TABLE 11.11-4 results of affinity determination of different humanized antibodies with the recombinant protein of the extracellular region of human Trop-2

Antibody combinations	KD value (M)	Antibody combinations	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 10ELISA detection of species specificity of anti-Trop-2 humanized antibody and Trop-2 combination

Human Trop-2-His recombinant protein (sequence number: NP-002344.2, 1aa-274aa), cynomolgus monkey Trop-2-His recombinant protein (sequence number: UniProtKB-A0A2K5UE71, 1aa-272aa), mouse Trop-2-His recombinant protein (Cat.:50922-M08H, Beijing Yigao) is coated overnight at 4 ℃ with the coating concentration of 1 mug/mL; after washing the plate for 3 times with PBS, adding 5% BSA PBS, blocking for 60min at 37 ℃, and washing the plate for 3 times with PBST; adding h23-12 (initial concentration is 10 mug/mL, 3-fold gradient is used for sequentially diluting 14 concentrations), h4-3 (initial concentration is 3 mug/mL, 3-fold gradient is used for sequentially diluting 12 concentrations), Sacituzumab (initial concentration is 3 mug/mL, 3-fold gradient is used for sequentially diluting 12 concentrations), each concentration is provided with a parallel hole, incubating is carried out at 37 ℃ for 60min, and PBST is washed for 4 times; HRP-anti-human Fc (Cat.: 109. 035. 098, Jackson Immuno Research) was added at a dilution of 1:5000, incubated at 37 ℃ for 30min, and the plates were washed 4 times with PBST; adding a TMB substrate for color development, incubating at 37 ℃ for 10min, and adding 2M HCl to stop the reaction; the absorbance A450nm-630nm of the well plate at a wavelength of 450nm was read and recorded using 630nm as the reference wavelength.

The experimental results show that the h23-12, the h4-3 and the control antibody Sacituzumab can be specifically combined with the recombinant human and cynomolgus monkey Trop-2, but have no combination activity with the recombinant mouse Trop-2 (figure 6, table 12), and provide a basis for performing pharmacological toxicological experiments on humanized antibodies.

TABLE 12 EC50 binding of anti-Trop-2 humanized antibodies to different species of Trop-2

Example 11anti-Trop-2 humanized antibody affinity assay

The antibody affinity was determined by capturing the Fc fragment of the antibody with an Ocet QKe system instrument from Fortebio using an anti-human antibody Fc fragment capture Antibody (AHC) biological probe.

For the assay, the antibodies (h23-12, h4-3, and the control antibody, Sacituzumab) were diluted to 4ug/mL in PBS buffer and passed over the surface of an AHC probe (Cat.:18-0015, PALL) for 120 s. Human Trop-2-his recombinant protein (SEQ ID NO: NP-002344.2, 1aa-274aa) is used as a mobile phase, and the concentration of the Trop-2-his corresponding to each antibody is h 23-12: 23. 30, 45, 75 nM; h 4-3: 23. 30, 45, 60 nM; sacituzumab: 23. 30, 45, 75 nM. The binding time was 100s and the dissociation time was 300 s. After the experiment, blank control response values were subtracted, 1:1Langmuir binding pattern fitting was performed with software, and kinetic constants for antigen-antibody binding were calculated.

The reaction curves of h23-12, h4-3 and the control antibody, Sacituzumab, with the human Trop-2 recombinant protein are shown in FIG. 7, and the affinity is calculated by fitting the curves, with h23-12 affinity (KD) of 6.40E-10M, h4-3 affinity (KD) of 5.45E-10M, and Sacituzumab affinity (KD) of 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, are equivalent to the control antibody Sacituzumab, and the dissociation value of h23-12 is superior to that of the Sacituzumab.

TABLE 13 determination of affinity between anti-Trop-2 humanized antibody and human Trop-2 extracellular domain recombinant protein

	KD value (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

Example 12Internalization activity of anti-human Trop-2 humanized antibody combined with cell surface Trop-2

Naturally expressing human Trop-2 human gastric cancer cell NCI-N87 at 2X 10³The density of individual cells/well was plated on 96-well cell culture plates and cultured for 24 hours. Cells were washed 1 time with PBS and the supernatant was discarded. Mix-n-Stain will be used^TM CF^TM488A (Cat.: MX488AS100, Sigma) labeled h23-12 and control antibody Sacituzumab were diluted to 15. mu.g/mL with RPMI 1640 (containing 10% FBS), added to NCI-N87 cells, one set was placed in a 37 ℃ electrothermal incubator, and one set was placed in a4 ℃ refrigerator AS a negative control; the negative control was incubated for 30 minutes and then washed 3 times with PBS, observed with a fluorescence microscope and photographed, and the experimental group was incubated at 37 ℃ for 5 hours and then observed with a fluorescence microscope and photographed.

The results of the experiment (FIG. 8) show that both the humanized h23-12 and the control antibody, Sacituzumab, were able to be internalized by Trop-2 mediated at 37 ℃ with a punctate distribution within the cytoplasm. Suggesting that the internalization activity of the antibody can be still maintained after the antibody is humanized.

The internalization rate on BxPC cells at 3h was measured by FACS using the method shown in example 7, and the results are shown in table 14, suggesting that the internalization rate after humanization is comparable to Sac.

TABLE 14 percentage internalization of BXPC-3 cell surface Trop-2 mediated anti-human Trop-2 antibodies

Example 13Pharmacokinetics research of Balb/C single administration nude miceIs especially suitable for the treatment of diabetes

Healthy female 5-week-old Balb/C nude mice are divided into 2 groups, after a single dose (15mg/kg) is injected into the abdominal cavity with h23-12, serum is collected at 5h, 25h, 48h, 96h, 168h and 240h respectively, is stored at the temperature of minus 20 ℃, a control group is arranged, and the serum is compared with a control product Sacituzumab which is injected into the abdominal cavity with the same dose of h23-12, and the pharmacokinetic characteristics of the control product are observed.

Healthy female Balb/C nude mice of 5 weeks old are divided into 4 groups, and after a single-dose (20mg/kg) intraperitoneal injection of h4-3, sera are collected at 4h, 8h, 24h, 48h, 96h, 144h, 192h and 240h respectively and stored at-20 ℃ to observe the pharmacokinetic characteristics of the sera.

The drug concentration in serum was measured by using an ELISA method coated with human Trop-2-his (SEQ ID NO: NP-002344.2, 1aa-274aa) and a standard curve was prepared. Fitting a linear curve with the concentration of the standard antibody as the Y axis and the OD value as the X axis, substituting the OD value of the detected serum into a formula to obtain the antibody content in the serum, and calculating according to the formula T_1/2Calculating the half-life T of the drug_1/2。

The results of the drug-time curves show that h23-12, h4-3 and the control antibody Sacituzumab have longer half-life in mice and drug metabolism half-life T_1/2The performance is equivalent (fig. 9A, fig. 9B, table 15), suggesting that the antibody has no obvious inactivation phenomenon in vivo and has better structural stability. Its metabolism corresponds to the basic characteristics of monoclonal antibody drugs, T_1/2About 170 h.

TABLE 15 pharmacokinetic parameters of a single administration of anti-Trop-2 antibodies in nude mice

	T_1/2(Trop-2 detection)
		Sacituzumab(n＝2)	168±14
h23-12(n＝2)	174±12
		h4-3(n＝4)	180±57

Example 14anti-Trop-2 naked antibody and affinity analysis of ADC antibody thereof

First, an antibody labeled with ADC drug SN38 was prepared. The antibody was reduced in sodium phosphate buffer at pH 7.0. + -. 0.5 for 2h using 20 equivalents of Dithiothreitol (DTT), and the reduced antibody was subsequently purified using ultrafiltration centrifuge tubes to remove excess DTT and replaced in a sodium phosphate wash at pH 7.0. + -. 0.5. The reduced antibody was incubated with CL2A-SN-38 for 30min at ambient temperature using 7-15% v/v DMSO as a co-solvent. Finally, the excess small molecules are removed by ultrafiltration centrifuge tubes. Analyzing the molecular weight of the antibody coupling drug by using mass spectrometry, and calculating to obtain an antibody coupling ratio (DAR) value of the antibody. Eventually each antibody carries an average of 7.5 molecules of SN 38.

SN 38-labeled antibody prepared as described above was assayed: h23-12-SN38, ch4-3-SN38, ch11-4-SN38, positive control antibody Sacituzumab-SN38, and naked anti-h 23-12, ch4-3, ch11-4, positive control antibody Sacituzumab were diluted to 4. mu.g/mL with PBS buffer and flowed over the surface of AHC probe (Cat.:18-0015, PALL) for 120 s. Human Trop-2-his recombinant protein (SEQ ID NO: NP-002344.2, 1aa-274aa) was used as a mobile phase at a concentration of 60 nM. The binding time was 300s and the dissociation time was 300 s. After the experiment, blank control response values were subtracted, 1:1Langmuir binding pattern fitting was performed with software, and kinetic constants for antigen-antibody binding were calculated.

As shown in Table 16, the affinity of the ADC antibody labeled with SN38 to human Trop-2 recombinant protein was not significantly changed compared with that of naked antibody.

TABLE 16 determination of the affinity of anti-Trop-2 naked antibody and its ADC antibody with human Trop-2 recombinant protein

Example 15FACS detection of BXPC-3 mediated anti-Trop-2 naked antibody and internalization of ADC antibody thereof

The internalization rates of BXPC-3, a human pancreatic cancer cell, and NCI-N87, a human gastric cancer cell, were determined as described in example 7, respectively. The antibodies to be tested include the ADC drug SN 38-labeled antibody prepared as described above: h23-12-SN38, ch4-3-SN38, ch11-4-SN38, positive control antibody Sacituzumab-SN38, and naked anti-h 232-12, ch4-3, ch11-4, positive control antibody Sacituzumab and negative isotype control antibody NC-IgG1, 10 ug/ml.

As shown in table 17 and table 18, the internalization ratios of h23-12 naked antibody and SN38 labeled ADC antibody were similar, and the degree of internalization was similar to that of the control antibody; and the internalization rate of the ADC antibody of ch4-3 and ch11-4 labeled SN38 is higher than that of naked antibody.

TABLE 17 percent internalization of anti-human Trop-2 antibodies mediated by NCI-N87 cell surface Trop-2

TABLE 18 percentage internalization of BXPC-3 cell surface Trop-2 mediated anti-human Trop-2 antibodies

Example 16Detection of cell killing activity of anti-Trop-2-ADC antibody

Taking human pancreatic cancer cells (BxPC-3), according to the ratio of 2X 10³One cell/well was inoculated into 96-well cell culture plates and placed at 37 ℃ in 5% CO₂After overnight incubation in an incubator, samples of SN 38-labeled anti-Trop-2 antibodies (two parallel wells per concentration and blank wells (no treatment)) were added at different concentrations according to Table 19 and placed at 37 ℃ in 5% CO₂After 3h incubation, the incubator was replaced with fresh complete medium. The treatment pattern of the previous day was repeated the next day, and the Cell killing activity of the SN 38-labeled anti-Trop-2 antibody against cells was examined with the Cell Counting Kit-8(CCK-8) Kit after 4 consecutive days of treatment.

TABLE 19 anti-Trop-2-ADC antibodies and action concentrations

The results show (FIG. 10, Table 20) that each anti-Trop-2 ADC antibody specifically kills target cells with no significant difference in killing activity from the control antibody, Sacituzumab-SN 38.

TABLE 20 inhibitory Activity of anti-Trop-2 ADC antibodies on cell growth

Name (R)	IC₅₀(ug/ml)
		Blank cell	/
h23-12-SN38	1.409
		Sacituzumab-SN38	4.311
ch4-3-SN38	3.299
		ch11-4-SN38	2.879

Example 17Pharmacodynamic evaluation of anti-Trop 2-ADC antibodies in N87 subcutaneous transplantation tumor model

Taking five-week-old female BALB/c nude mice, and inoculating 3X 10 subcutaneous tissue⁶Human gastric cancer cell (NCI-N87) to allow tumor growth to 150mm³The random grouping is carried out on the left and the right, 6 mice are divided into groups, the dose and the frequency of the administration are shown in the table 21, each group is administrated by intravenous injection twice every week, the tumor volume and the mouse weight are measured when the mouse weight is reduced by more than 15%, or the tumor volume of a single animal is more than 3000mm³Or the average tumor volume of a group of animals exceeds 2000mm³The experiment was stopped and mice were euthanized.

TABLE 21 grouping of nude mice and dose, frequency of administration

As shown in FIG. 11 and FIG. 12, the anti-Trop 2-ADC antibody has a dose-dependent inhibition effect on tumor growth, no difference of good and bad drug effects is observed among the ADC antibodies at a high dose (10mg/kg), no obvious toxic effect of the ADC small molecule SN38 is observed, and the weight of animals in each experimental group is steadily increased and has no obvious difference with a control group.

Example 18Pharmacodynamic evaluation of anti-Trop 2 antibody in combination with anti-CD47 antibody in SKOV3 subcutaneous graft tumor modelPrice of

Taking five-week-old female BALB/c nude mice, and subcutaneously inoculating 3X 10 hypochondriac parts of each mouse⁶Individual ovarian cancer cell (SKOV3), when tumor grows to 150mm³The random grouping is carried out on the left and the right, 6 mice are grouped per group, the grouping and the administration dosage and the frequency are shown in a table 22, each group is administrated by intraperitoneal injection twice every week, the administration is 5 times totally, the tumor volume and the mouse weight are measured while the administration is carried out, the state of the mouse is observed, and the mouse is euthanized after the last administration of the experiment. anti-CD47 antibody is described in patent application publication US20150183874a1, humanized 5F9 version 2.

TABLE 22 grouping of nude mice and dose, frequency of administration

Group of	Medicine	Dosage to be administered	Frequency of administration
				1	Anti-CD47	10mg/kg	Biw
2	h23-12	2mg/kg	Biw
				3	h23-12+Anti-CD47	2mg/kg+10mg/kg	Biw
4	Negative control hIgG4	2mg/kg	Biw

As shown in FIG. 13 and FIG. 14, compared with the negative control hIgG4, the h23-12+ Anti-CD47 combination group showed a certain tumor suppression activity, and the Anti-CD47 and the h23-12 single drug group had no obvious tumor suppression effect. Therefore, the Trop2 antibody and the anti-CD47 antibody can synergistically promote phagocytosis of tumor cells by macrophages, and therefore have a synergistic tumor inhibition effect.

Example 19Pharmacodynamic evaluation of anti-Trop 2-ADC antibodies in N87 subcutaneous transplantation tumor model

Taking five-week-old female BALB/c nude mice, and inoculating 3X 10 subcutaneous tissue⁶Human gastric cancer cell (NCI-N87) to allow tumor growth to 100mm³The random grouping is carried out on the left and the right, 6 animals are grouped, the grouping and the administration dosage and the frequency are shown in a table 23, each group is administrated by intravenous injection twice weekly for 6 weeks, the administration is carried out, the tumor volume and the mouse weight are measured simultaneously, when the mouse weight is reduced by more than 15%, or the tumor volume of a single animal is more than 3000mm³Or the average tumor volume of a group of animals exceeds 2000mm³The experiment was stopped and mice were euthanized.

TABLE 23 grouping of nude mice and dose, frequency of administration

Group of	Medicine	Dosage to be administered	Frequency of administration
				1	ch3-11-SN38	5mg/kg	Biw×6
2	ch11-4-SN38	5mg/kg	Biw×6
				3	Sacituzumab-SN38	5mg/kg	Biw×6
4	Negative control hIgG1	5mg/kg	Biw×6
				5	ADC control hIgG1-SN38	5mg/kg	Biw×6

As shown in figure 15 and figure 16, the anti-Trop 2-ADC antibody has a dose-dependent inhibition effect on tumor growth, and the ADC antibodies ch3-11-SN38 and ch11-4-SN38 have slightly better drug effects than Sacituzumab-SN38 at a dose of 5mg/kg, and no obvious toxic effect of ADC small molecule SN38 is observed.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.

Sequence listing

<110> Mewey (Shanghai) Biotech Co., Ltd

Beijing konuoxincheng Technology Co.,Ltd.

<120> anti-human Trop-2 antibody and application thereof

<130> LC19110043

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Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe

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Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

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Met Gln Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Thr Arg Glu Gly His Asn Tyr Asp Gly Ser Leu Gly Ala Met Asp His

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Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

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Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Val Ile Gly Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Leu Gly Ser Gly Thr Ile Tyr Tyr Thr Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Ser Ile Phe Pro Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

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Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Val Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Leu Gly Ser Gly Thr Ile Tyr Tyr Thr Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Ile Phe Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 4

<211> 117

<212> PRT

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<223> heavy chain variable region

<400> 4

Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Val Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Leu Gly Ser Gly Thr Ile Tyr Tyr Ala Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Ile Phe Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 5

<211> 117

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 5

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Asn Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Ser Asn Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Thr Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ser Ser Tyr Arg Ser Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115

<210> 6

<211> 117

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 6

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Ser Asn Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Arg Ser Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 7

<211> 117

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 7

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Ser Asn Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Ser Tyr Arg Ser Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 8

<211> 117

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 8

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Ser Asn Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Ser Tyr Arg Ser Glu Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 9

<211> 117

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 9

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Ser Asn Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Ser Tyr Arg Ser Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 10

<211> 117

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 10

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Ser Asn Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Ser Tyr Arg Ser Asp Ala Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 11

<211> 115

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 11

Glu Val Lys Leu Val Glu Ser Gly Gly Val Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Arg Gly Asp Asp Thr Tyr Tyr Pro Asp Ser Val Lys

50 55 60

Gly Arg Ile Thr Ile Ser Arg Asp Phe Ala Arg Asn Ile Leu Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Thr

85 90 95

Arg Asp Arg Phe Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 12

<211> 119

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 12

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Asp Gly Tyr Ile Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Thr Pro Ser Asp Asn Tyr Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly His Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 13

<211> 119

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 13

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Thr Pro Ser Asp Asn Tyr Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly His Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 14

<211> 119

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 14

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Thr Pro Ser Asp Asn Tyr Gly Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly His Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 15

<211> 119

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Thr Pro Gly Asp Asn Tyr Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly His Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 16

<211> 119

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Thr Pro Ser Asp Asn Tyr Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Glu Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 17

<211> 119

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain variable region

<400> 17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Thr Pro Ser Asp Asn Tyr Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gln Gly Asn Tyr Val Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 18

<211> 112

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 18

Asp Val Val Val Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Phe Gly

1 5 10 15

Asp Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Thr Asn Ser

20 25 30

Tyr Gly Asn Thr Phe Leu Ser Trp Tyr Leu His Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Leu Tyr Gly Ile Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Asn Thr Ile Lys Pro Glu Asp Leu Gly Met Tyr Tyr Cys Phe Gln Ser

85 90 95

Thr His Gln Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 19

<211> 106

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 19

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Thr Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 20

<211> 106

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 20

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 21

<211> 106

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 21

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 22

<211> 106

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 22

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 106

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 23

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 24

<211> 107

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 24

Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Lys Val Ser Phe Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Glu Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 25

<211> 107

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 25

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 26

<211> 107

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 26

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Glu Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 27

<211> 107

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 27

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser

20 25 30

Ile Glu Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Glu Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 28

<211> 107

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 28

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser

20 25 30

Ile Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Glu Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 29

<211> 107

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 29

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Glu Phe Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 30

<211> 112

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 30

Asp Ile Val Met Thr Gln Ser Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Gly Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Phe Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Met Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 31

<211> 108

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 31

Asp Ile Gln Met Thr Gln Ile Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 32

<211> 114

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 32

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro

<210> 33

<211> 108

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 33

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 34

<211> 114

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro

<210> 35

<211> 114

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 35

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Thr Leu Pro Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro

<210> 36

<211> 114

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain variable region

<400> 36

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Ser Leu Pro Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro

<210> 37

<211> 330

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> heavy chain constant region

<400> 37

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 38

<211> 107

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> light chain constant region

<400> 38

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 39

<211> 451

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> Sacituzumab, heavy chain

<400> 39

Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 40

<211> 214

<212> PRT

<213> Artificial (Artificial)

<220>

<221> PEPTIDE

<222> ()..()

<223> Sacituzumab, light chain

<400> 40

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 41

<211> 369

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> heavy chain variable region

<400> 41

caggtccaac tgcagcagcc tggggctgaa ctggtgaagc ctgggtcttc agtgaagctg 60

tcctgcaagg cttctggcta caccttcact agttactgga tgtactgggt gaagcagagg 120

cctggacagg gccttgagtg gattggagag attaatccta gtaacggtcg tactaattac 180

aatgagaagt tcaagagcaa ggccacactg actgtagaca aatcgtccag cacagcctac 240

atgcaattca gcagcctgac atctgaggac tctgcggtct attactgtac aagagaaggc 300

cataattacg atggttccct cggggctatg gaccactggg gtcaaggaac ctcagtcacc 360

gtctcctca 369

<210> 42

<211> 336

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> light chain variable region

<400> 42

gatgttgtgg tgactcaaac tccactctcc ctgcctgtca gctttggaga tcaggtttct 60

atctcttgca ggtctagtca gagtcttaca aacagttatg ggaacacctt tttgtcttgg 120

tacctgcaca agcctggcca gtctccacag ctcctcctct atgggatttc caacagattt 180

tctggggtgc cagacaggtt cagtggcagt ggttcaggga cagatttcac actcaagatc 240

aacacaataa agcctgagga cctgggaatg tattactgct ttcaaagtac acatcagccg 300

tacacgttcg gaggggggac caagctggaa ataaaa 336

<210> 43

<211> 351

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> heavy chain variable region

<400> 43

caggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggatt cacattcact gactatgtta taggctgggt gaagcagaga 120

actggacagg gccttgagtg gattggagag atttatcttg gaagtggtac tatttactac 180

actgagaagt tcaagggcaa ggccacactg actgcagaca catcctccaa cacagcctac 240

atgcagctca gcagcctgac gtctgaagac tctgcggtct atttctgtgc aaggggatct 300

attttcccct ttgactactg gggccaaggc accactctca cagtctcctc a 351

<210> 44

<211> 318

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> light chain variable region

<400> 44

caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60

atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccagca gaagccagga 120

tcctccccca gactcctgat ttatgacaca tccaccctgg cttctggagt ccctgttcgc 180

ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 240

gatgctgcca cttactactg ccagcagtgg agtagttacc cttacacgtt cggagggggg 300

accaagctgg aaataaaa 318

<210> 45

<211> 351

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> heavy chain variable region

<400> 45

gaggtgcagc tggtgcagtc tggacccgag gtgaagaagc ctggagcctc cgtgaaggtg 60

tcctgcaagg cctccggctt caccttcacc gactacgtga tcggctgggt gcgacaggct 120

cctggccagg gactggagtg gatcggcgag atctacctgg gctccggcac catctactac 180

accgagaagt tcaagggacg ggtgaccatg acagccgaca cctccacctc caccgcctac 240

atggagctgt cctccctgcg gtccgaggac accgccgtgt actactgcgc tcgaggctcc 300

atcttcccct tcgactactg gggccagggc accctggtga ccgtgtcctc t 351

<210> 46

<211> 318

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> light chain variable region

<400> 46

gacatccagc tgacccagtc tccctcctcc ctgtctgcct ccgtgggcga cagggtgacc 60

atcacctgct ctgcctcctc ctccgtgtcc tacatgtact ggtaccagca gaagcctggc 120

aaggctccca agctgctgat ctacgacacc tccaccctgg cctctggcgt gccctccagg 180

ttctctggct ccggatctgg caccgacttc accctgacca tctcctccct gcagcccgag 240

gacttcgcca cctactactg ccagcagtgg tcctcctacc cctacacctt cggacagggc 300

accaagctgg agatcaag 318

<210> 47

<211> 351

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> heavy chain variable region

<400> 47

caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgaacctg 60

tcctgcaagg cttctggcta caccttcacc agctactgga taaactgggt gaagcagagg 120

cctggacaag gccttgagtg gatcggaaat atttatcctt ctaatagtta tactaactac 180

aatcaaaagt tcaaggacac ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca gcagcccgac atctgaggac tctgcggtct atttctgttc aagttatagg 300

tccgacgggt ttgcttactg gggccaaggg actcttgtca ctgtctctgc a 351

<210> 48

<211> 321

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> light chain variable region

<400> 48

gacatcttgc tgactcagtc tccagccatc ctgtctgtga gtccaggaga aaaagtcagt 60

ttctcctgca gggccagtca gaacattggc acaagcatac actggtatca gcaaagaaca 120

aatggttctc caaggcttct catagaattt gcttctgagt ctatctctgg gatcccttcc 180

aggtttagtg gcagtggatc agggacagat tttactctta ccatcaacag tgtggagtct 240

gaagatattg cagattatta ctgtcaacaa agtaatagct ggccgttcac gttcggaggg 300

gggaccaagc tggaaataaa a 321

<210> 49

<211> 351

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> heavy chain variable region

<400> 49

caggtgcagc tggtgcagtc tggagccgag gtgaagaagc ctggagcctc cgtgaaggtg 60

tcctgcaagg cctccggcta caccttcacc tcctactgga tcaactgggt gcggcaggct 120

cctggccagg gactggagtg gatgggcaac atctacccat ccaactccta caccaactac 180

aaccagaagt tcaaggacag ggtgaccatg accagagaca cctccacctc caccgtgtac 240

atggagctgt cctccctgcg gtccgaggac acagccgtgt actactgcgc tcggtaccgg 300

tctgacggct tcgcctactg gggacagggc accctggtga ccgtgtcctc c 351

<210> 50

<211> 321

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> light chain variable region

<400> 50

gagatcgtgc tgacccagtc tcctgccacc ctgtccctgt ctcctggcga gagagccacc 60

ctgtcctgca gagcctccca gaacatcggc acctccatcc actggtacca gcagaagcct 120

ggccaggctc ctcggctgct gatctacttc gcctccgagt ccatctctgg catccctgct 180

cggttctctg gctccggatc tggcaccgac ttcaccctga ccatctcctc cctggagcct 240

gaggacttcg ccgtgtacta ctgccagcag tccaactcct ggcccttcac cttcggaggt 300

ggcaccaagg tggagatcaa g 321

<210> 51

<211> 345

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> heavy chain variable region

<400> 51

gaggtgaagc tggtggagtc tgggggagtc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcagt gactctgcca tgtcttgggt tcgccagact 120

ccagagaaga ggctggagtg ggtcgcatcc attagtcgtg gtgatgacac atattatcca 180

gacagtgtga agggccgaat caccatttcc agagattttg ccagaaacat cctgtatttg 240

caaatgacca gtctgaggtc tgaggacacg gccatgtatt actgtacaag agatcggttc 300

gggtttgctt actggggcca agggactctg gtcactgtct ctgca 345

<210> 52

<211> 336

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> light chain variable region

<400> 52

gacattgtga tgacccagtc tccactcact ttgtcggtta ccattggaca acctgcctcc 60

atctcttgca agtcaggtca gagcctctta gatagtgatg gaaagacata ttttaattgg 120

ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc tatgctggac 180

tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240

agcagagtgg agactgagga tttgggagtt tattattgct ggcaaggtac acattttcca 300

ttcacgttcg gctcggggac aaagttggaa ataaag 336

<210> 53

<211> 357

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> heavy chain variable region

<400> 53

caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagctg 60

tcctgtaagg ctgatggcta catcttcacc agttactgga tgcactgggt gaaacagagg 120

cctggacaag gccttgagtg gatcggagag attactcctt ctgataatta tacttcctac 180

aatcaaaagt tcaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca gcagcctgac gtctgaggac tctgcggtct attactgtac aagaggccac 300

ggtaactacg tcagctttga ctactggggc caaggcacca ctctcacagt ctcctca 357

<210> 54

<211> 324

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> light chain variable region

<400> 54

gacatccaga tgacacagat tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcacttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccctca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggttatacgc ttcctccgta cacgttcgga 300

ggggggacca agctggaaat aaaa 324

<210> 55

<211> 357

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> heavy chain variable region

<400> 55

caggtgcagc tggtgcagtc cggagccgag gtgaagaagc ctggagcctc cgtgaaggtg 60

tcctgcaagg cctccggcta caccttcacc tcctactgga tgcactgggt gcggcaggct 120

cctggccagg gactggagtg gatgggcgag atcacaccct ccgacaacta cacctcctac 180

aaccagaagt tcaagggacg ggtgaccatc accagggaca cctccacctc caccgcctac 240

atggagctgt cctccctgcg gtccgaggac accgccgtgt actactgcgc tcgaggccac 300

ggcaactacg tgtccttcga ctactgggga cagggcaccc tggtgaccgt gtcctcc 357

<210> 56

<211> 324

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> light chain variable region

<400> 56

gacatccaga tgacccagtc tccctcctcc ctgtctgcct ccgtgggaga ccgggtgacc 60

atcacctgca gagcctccca ggacatctcc aactacctga actggtacca gcagaagcct 120

ggcaaggctc ccaagctgct gatctactac acctccaggc tgcactccgg agtgccctcc 180

cggttctccg gctctggctc cggaaccgac ttcaccctga ccatctcctc cctgcagccc 240

gaggacttcg ccacctactt ctgccagcag ggctacaccc tgcctcccta caccttcggc 300

cagggcacca agctggagat caag 324

<210> 57

<211> 357

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> heavy chain variable region

<400> 57

caggtgcagc tggtgcagtc cggagccgag gtgaagaagc ctggagcctc cgtgaaggtg 60

tcctgcaagg cctccggcta caccttcacc tcctactgga tgcactgggt gcggcaggct 120

cctggccagg gactggagtg gatgggcgag atcacaccct ccgacaacta cggctcctac 180

aaccagaagt tcaagggacg ggtgaccatc accagggaca cctccacctc caccgcctac 240

atggagctgt cctccctgcg gtccgaggac accgccgtgt actactgcgc tcgaggccac 300

ggcaactacg tgtccttcga ctactgggga cagggcaccc tggtgaccgt gtcctcc 357

<210> 58

<211> 324

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> light chain variable region

<400> 58

gacatccaga tgacccagtc tccctcctcc ctgtctgcct ccgtgggaga ccgggtgacc 60

atcacctgca gagcctccca ggacatctcc aactacctga actggtacca gcagaagcct 120

ggcaaggctc ccaagctgct gatctactac acctccaggc tggagtccgg agtgccctcc 180

cggttctccg gctctggctc cggaaccgac ttcaccctga ccatctcctc cctgcagccc 240

gaggacttcg ccacctactt ctgccagcag ggctacaccc tgcctcccta caccttcggc 300

cagggcacca agctggagat caag 324

<210> 59

<211> 990

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> heavy chain constant region

<400> 59

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

cagaagagcc tctccctgtc cccgggtaaa 990

<210> 60

<211> 321

<212> DNA

<213> Artificial (Artificial)

<220>

<221> gene

<222> ()..()

<223> light chain constant region

<400> 60

agaaccgtgg cggcgccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggtaccgcta gcgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtg t 321

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Anti-human Trop-2 antibody and application thereof

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