CN112243443B

CN112243443B - anti-TROP-2 antibodies, antigen-binding fragments thereof, and medical uses thereof

Info

Publication number: CN112243443B
Application number: CN202080003175.1A
Authority: CN
Inventors: 花海清; 包如迪
Original assignee: Changzhou Hengbang Pharmaceutical Co ltd; Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Current assignee: Changzhou Hengbang Pharmaceutical Co ltd; Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Priority date: 2019-05-10
Filing date: 2020-05-08
Publication date: 2023-03-17
Anticipated expiration: 2040-05-08
Also published as: TW202108623A; CN112243443A; WO2020228604A1

Abstract

Relates to anti-TROP-2 antibodies, antigen-binding fragments thereof, and pharmaceutical uses thereof. Further relates to chimeric antibodies, humanized antibodies comprising the CDR regions of the anti-TROP-2 antibodies, and pharmaceutical compositions comprising the anti-TROP-2 antibodies or antigen-binding fragments thereof, and their use as anti-cancer drugs. In particular to a humanized anti-TROP-2 antibody and application thereof in preparing a medicament for treating TROP-2 mediated diseases or symptoms and tumor detection and diagnosis.

Description

anti-TROP-2 antibodies, antigen-binding fragments thereof, and medical uses thereof

Technical Field

The present application relates to an anti-TROP-2 antibody that is specifically immunoreactive with a human TROP-2 receptor, and antigen-binding fragments thereof, chimeric antibodies comprising the CDR regions of said anti-TROP-2 antibody, humanized antibodies, and pharmaceutical compositions comprising the human anti-TROP-2 antibody and antigen-binding fragments thereof, as well as their use as anti-cancer drugs and their use for detecting or diagnosing tumors.

Background

With the continuous and deep research on tumor genomics, proteomics and signal transduction pathways, the interaction of cancer genes and cancer suppressor genes of tumor cells and the influence of the interaction on the tumor microenvironment become clearer, which also makes it possible to design new anti-tumor treatment schemes aiming at specific molecular targets of tumors.

The molecular target therapy of tumor is a new therapeutic mode different from traditional operation, radiotherapy and chemotherapy, and has the advantages that the medicine is usually combined with corresponding target site only, and the function of target site molecule is directly influenced or the carried physical or chemical effector molecule is used to kill or inhibit target cell. Because the target position is clear, the medicine usually has high selectivity, can effectively kill or inhibit target cells, and does not generate or only generates less toxic and side effects on normal tissue cells. Therefore, the development of molecular targeted drugs becomes a hot spot for clinical research of tumors.

Human trophoblast cell surface antigen 2 (human trophoblast cell surface antigen 2, trop-2) is a cell surface glycoprotein encoded by the tactd 2 gene. TROP-2 is composed of 323 amino acids, wherein the signal peptide is 26 amino acids, the extracellular region is 248 amino acids, the transmembrane region is 23 amino acids, and the cytoplasmic region is 26 amino acids. There are 4 heterogeneous N-binding glycosylation sites in the TROP-2 extracellular domain, and the apparent molecular weight increases by 11 to 13kD upon addition of sugar chains. The extracellular domain of the tactd gene family has a characteristic Thyroglobulin (TY) sequence, which is generally thought to be involved in the proliferation, infiltration, and metastasis of cancer cells.

To date, no physiological ligand for TROP-2 has been identified, the molecular function has not been elucidated, but since intracellular serine 303 residue is phosphorylated by Protein Kinase C (PKC), which is a Ca2+ -dependent protein kinase, and the intracellular domain has PIP2 binding sequence, it is suggested that it has a signaling function in tumor cells.

A large number of clinical studies and literature reports indicate that TROP-2 is over-expressed in various epithelia cancers such as gastric cancer, lung cancer, large intestine cancer, ovarian cancer, breast cancer, prostatic cancer, pancreatic cancer, liver cancer, esophageal cancer and the like. In contrast, TROP-2 was expressed only in a small amount in cells in the epithelial region, and was not expressed in normal tissues of adults, and the expression level was lower than that in cancer, indicating that TROP-2 is involved in tumor formation. The overexpression of TROP-2 in tumor tissues is closely related to poor prognosis of patients and metastasis of cancer cells, and affects the overall survival rate of patients. Thus, TROP-2 has become an attractive target for molecular targeted therapy of tumors.

Several studies of the anti-tumor effect of anti-hTROP-2 antibodies have been reported.

U.S. Pat. No. 5840854 reports cytotoxicity of anti-hTROP-2 monoclonal antibody (BR 110) binding to cytotoxin to human cancer cell lines H3619, H2987, MCF-7, H3396 and H2981.

U.S. Pat. No. 6653104 discloses an antibody (RS 7) which exhibits antitumor activity in a nude mouse xenograft model when tested in an in vivo model using an antibody labeled with a radioactive substance, but the antitumor effect of the naked antibody alone is not reported.

U.S. Pat. No. 7420040 also reports: an isolated monoclonal antibody produced from hybridoma cell strain ar47a6.4.2 or ar52a301.5 obtained by immunizing a mouse with human ovarian cancer tissue binds to hTROP-2 and shows antitumor activity in a nude mouse xenograft model.

CN102827282A discloses a humanized anti-TROP-2 genetic engineering antibody IgG and application thereof, and in vitro test results show that the anti-TROP-2 antibody IgG has a significant inhibitory effect on the proliferation of pancreatic cancer cells.

CN104114580a discloses an antibody (particularly a humanized antibody) specifically reactive with hTROP-2 and having anti-tumor activity in vivo; and a hybridoma producing the antibody, a complex of the antibody and a drug, a pharmaceutical composition for diagnosis or treatment of a tumor, a method for detecting a tumor, and a kit for detection or diagnosis of a tumor.

Disclosure of Invention

The present invention provides novel anti-TROP-2 antibodies or antigen-binding fragments thereof having enhanced tumor cell killing.

According to some embodiments of the present application, there is provided an anti-TROP-2 antibody or antigen-binding fragment thereof, comprising an antibody heavy chain variable region and an antibody light chain variable region, wherein said antibody heavy chain variable region comprises at least 1 HCDR selected from the group consisting of seq id nos: the amino acid sequence of SEQ ID NO: 3. SEQ ID NO:4 and SEQ ID NO:5; and the antibody light chain variable region comprises at least 1 LCDR selected from the group consisting of seq id nos: SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8.

in some embodiments, the heavy chain variable region of an anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application comprises:

SEQ ID NO:3, HCDR1,

The amino acid sequence of SEQ ID NO:4 HCDR2 and

SEQ ID NO:5 HCDR3 as shown.

In some embodiments, the light chain variable region of the anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application comprises:

SEQ ID NO: LCDR1 shown in 6,

SEQ ID NO: LCDR2 and LCDR 7

SEQ ID NO: LCDR3 as shown at 8.

In some embodiments, an anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application comprises:

the amino acid sequence of SEQ ID NO:3, HCDR1,

SEQ ID NO:4 HCDR2 and

SEQ ID NO:5 HCDR3; and

SEQ ID NO: LCDR1 shown in 6,

SEQ ID NO: LCDR2 and LCDR 7

The amino acid sequence of SEQ ID NO: LCDR3 as shown at 8.

In some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application is selected from the group consisting of: murine antibodies, chimeric antibodies, human antibodies, humanized antibodies.

In some embodiments, an anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application further comprises a heavy chain constant region derived from human IgG1, igG2, igG3, or IgG4, or a variant thereof.

In particular embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application further comprises an IgG1 heavy chain constant region derived from an IgG1 heavy chain having enhanced ADCC toxicity following amino acid mutation, in some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application further comprises an IgG1 heavy chain constant region incorporating E239D and M241L mutations, in some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 or SEQ ID NO:49, in particular embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application further comprises a constant region derived from a human kappa chain, lambda chain, or variant thereof, and in some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application further comprises a heavy chain constant region selected from the group consisting of SEQ ID NOs: 50.

In some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application, whose heavy chain variable region is a heavy chain variable region selected from the group consisting of those represented by the following sequences: SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO: 15. SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO:25, or a heavy chain variable region sequence having at least 70%,75%,80%,85%,90%,95%, or 99% homology thereto.

In some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application, whose light chain variable region is a light chain variable region selected from the group consisting of those represented by the following sequences: SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. the amino acid sequence of SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO:26, or a light chain variable region sequence having at least 70%,75%,80%,85%,90%,95%, or 99% homology thereto.

In some embodiments, the heavy chain of an anti-TROP-2 antibody or antigen-binding fragment thereof according to the application is selected from the group consisting of heavy chains comprising: SEQ ID NO: 27. the amino acid sequence of SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO: 33. SEQ ID NO: 35. SEQ ID NO: 37. SEQ ID NO: 39. SEQ ID NO: 41. SEQ ID NO: 43. SEQ ID NO: 45. SEQ ID NO:47, or a full-length heavy chain sequence having at least 80%,85%,90%,95% or 99% homology thereto.

In some embodiments, the light chain of an anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application is selected from the group consisting of light chains comprising: SEQ ID NO:28. SEQ ID NO: 30. SEQ ID NO: 32. SEQ ID NO: 34. SEQ ID NO: 36. SEQ ID NO: 38. SEQ ID NO: 40. SEQ ID NO: 42. SEQ ID NO:44, or a full-length light chain sequence having at least 80%,85%,90%,95%, or 99% homology thereto.

In a specific embodiment, an anti-TROP-2 antibody or antigen-binding fragment thereof according to the application, wherein:

the heavy chain variable region of the anti-TROP-2 antibody is SEQ ID NO:9 is shown in the figure;

the variable region of the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 10.

In another specific embodiment, an anti-TROP-2 antibody or antigen-binding fragment thereof according to the application, wherein:

the heavy chain variable region of the anti-TROP-2 antibody is SEQ ID NO:11 is shown in the figure;

the variable region of the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 12.

In another specific embodiment, an anti-TROP-2 antibody or antigen-binding fragment thereof, according to the application, wherein:

the heavy chain variable region of the anti-TROP-2 antibody is SEQ ID NO:13 is shown in the figure;

the variable region of the light chain of the anti-TROP-2 antibody is SEQ ID NO: as shown at 14.

the heavy chain variable region of the anti-TROP-2 antibody is SEQ ID NO:15, respectively;

the variable region of the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 16.

the heavy chain variable region of the anti-TROP-2 antibody is SEQ ID NO:17 is shown;

the variable region of the light chain of the anti-TROP-2 antibody is SEQ ID NO:18, respectively.

the heavy chain variable region of the anti-TROP-2 antibody is SEQ ID NO:19 is shown in the figure;

the variable region of the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 20.

the heavy chain variable region of the anti-TROP-2 antibody is SEQ ID NO:21 is shown in the figure;

the variable region of the light chain of the anti-TROP-2 antibody is SEQ ID NO:22, respectively.

the heavy chain variable region of the anti-TROP-2 antibody is SEQ ID NO:23 is shown;

the variable region of the light chain of the anti-TROP-2 antibody is SEQ ID NO: as shown at 24.

the heavy chain variable region of the anti-TROP-2 antibody is SEQ ID NO:25 is shown;

the variable region of the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 26.

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:27 is shown;

the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 28.

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:29 is shown;

the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 30.

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:31, shown in the figure;

the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 32.

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO: 33;

the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 34.

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:35 is shown in the figure;

the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 36.

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:37 is shown in the figure;

the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 38.

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO: 39;

the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 40.

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:41 is shown;

the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 42.

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:43 is shown;

the light chain of the anti-TROP-2 antibody is SEQ ID NO: as shown at 44.

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:45 is shown;

the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 38.

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:47 is shown;

the light chain of the anti-TROP-2 antibody is SEQ ID NO: shown at 28.

According to some embodiments of the present application, there is provided a polynucleotide encoding an anti-TROP-2 antibody or antigen-binding fragment thereof of the present application.

According to some embodiments of the present application, there is provided an expression vector comprising a polynucleotide of the present application.

According to some embodiments of the present application, there is provided a host cell introduced or containing the expression vector of the present application.

In a particular embodiment, the host cell is a bacterium, preferably E.coli.

In another specific embodiment, the host cell is a yeast, preferably pichia pastoris.

In another specific embodiment, the host cell is a mammalian cell, preferably a CHO cell or a HEK293 cell.

According to some embodiments of the present application, there is provided a method of producing an anti-TROP-2 antibody, comprising the steps of: culturing a host cell according to the present application, isolating the antibody from the culture, and purifying the antibody.

According to some embodiments of the present application, there is provided a pharmaceutical composition comprising an anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application and a pharmaceutically acceptable excipient, diluent or carrier.

According to some embodiments of the present application, there is provided a detection or diagnostic kit comprising an anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application.

According to some embodiments of the present application, there is provided a use of an anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application in the manufacture of a medicament for treating or preventing a TROP-2 mediated disease or condition.

According to some embodiments of the present application, there is provided use of an anti-TROP-2 antibody or antigen-binding fragment thereof according to the present application in the preparation of an agent for detecting or diagnosing a TROP-2 mediated disease or disorder.

In some embodiments, the disease or disorder is cancer.

In some embodiments, the disease or disorder is a cancer that expresses TROP-2.

In particular embodiments, the disease or condition is selected from the group consisting of: breast cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, and melanoma.

According to some embodiments of the present application, there is provided a method of treating or preventing a TROP-2 mediated disease, comprising the steps of: providing a therapeutically effective amount, or a prophylactically effective amount, of an anti-TROP-2 antibody, or antigen-binding fragment thereof, according to the present application to a subject.

According to some embodiments of the present application, there is provided a method of treating or preventing a TROP-2 mediated disease, comprising the steps of: providing a therapeutically effective amount or a prophylactically effective amount of a pharmaceutical composition according to the present application to a subject.

In some embodiments, the subject is suspected of having, has, or is susceptible to a TROP-2 mediated disease selected from the group consisting of breast cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gall bladder cancer, glioblastoma, and melanoma.

Drawings

FIG. 1: ELISA in vitro binding experiments of the antibodies showed the binding activity of 11 humanized anti-TROP-2 antibodies to human TROP-2 antigen.

Detailed Description

1. Term(s) for

In order that the present application may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Amino acid three letter codes and one letter codes as used herein are as described in j.biol.chem,243, p3558 (1968).

The term "antibody" as used herein refers to an immunoglobulin, which is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., igM, igD, igG, igA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. The second of the five classes of Ig may have either a kappa chain or a lambda chain.

In the present application, the antibody light chain variable region described herein may further comprise a light chain constant region comprising a human-or murine-derived kappa, lambda chain or variant thereof.

In the present application, the antibody heavy chain variable region described herein may further comprise a heavy chain constant region comprising IgG1, igG2, igG3, igG4 of human or murine origin, or a variant thereof.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence-conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and the heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues in the VL and VH regions of the antibodies or antigen-binding fragments described herein conform in number and position to the known Kabat numbering convention and the Kabat or ABM definition convention (http:// bio in.

The term "antigen presenting cell" or "APC" is a cell that displays foreign antigens complexed with MHC on its surface. T cells recognize this complex using the T Cell Receptor (TCR). Examples of APCs include, but are not limited to, dendritic Cells (DCs), peripheral Blood Mononuclear Cells (PBMCs), monocytes, B lymphoblastoid cells, and monocyte-derived dendritic cells.

The term "antigen presentation" refers to the process by which APCs capture antigens and enable them to be recognized by T cells, for example as a component of an MHC-I/MHC-II conjugate.

The term "TROP-2" includes any variant or isoform of TROP-2 that is naturally expressed by a cell. The antibodies of the present application can cross-react with TROP-2 from non-human species. Alternatively, the antibody may also be specific for human TROP-2 and may not exhibit cross-reactivity with other species. TROP-2, or any variant or isoform thereof, may be isolated from cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques common in the art and those described herein. Preferably, the anti-TROP-2 antibody targets human-derived TROP-2 with a normal glycosylation pattern.

The term "recombinant human antibody" includes human antibodies made, expressed, created or isolated by recombinant methods, involving techniques and methods well known in the art, such as:

1. antibodies isolated from transgenes for human immunoglobulin genes, transchromosomal animals (e.g., mice), or hybridomas made therefrom;

2. antibodies isolated from host cells transformed to express the antibodies, such as transfectomas;

3. antibodies isolated from a library of recombinant combinatorial human antibodies; and

4. antibodies produced, expressed, created or isolated by methods such as splicing of human immunoglobulin gene sequences to other DNA sequences.

Such recombinant human antibodies comprise variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as occur during antibody maturation.

The term "murine antibody" is used herein to refer to a monoclonal antibody to human TROP-2 prepared according to the knowledge and skill in the art. Preparation is performed by injecting a subject with the TROP-2 antigen and then isolating hybridomas that express antibodies having the desired sequence or functional properties. In a preferred embodiment of the present application, the murine TROP-2 antibody or antigen binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, igG2, igG3 or IgG4 or variant thereof.

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the present application can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., "humanized antibodies").

The term "humanized antibody", also known as CDR-grafted antibody (CDR), refers to an antibody produced by grafting a mouse CDR sequence into a human antibody variable region framework. The humanized antibody can overcome the disadvantage of strong immune response induced by the chimeric antibody due to carrying a large amount of mouse protein components. To avoid a decrease in activity associated with a decrease in immunogenicity, the human antibody variable regions may be subjected to minimal back-mutation to maintain activity.

The term "chimeric antibody" refers to an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. Establishing a chimeric antibody, selecting and establishing a hybridoma secreting a mouse-derived specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to needs, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into a human vector, and finally expressing a chimeric antibody molecule in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of the human antibody may be selected from a heavy chain constant region of human IgG1, igG2, igG3 or IgG4 or a variant thereof, preferably comprising a human IgG1, igG2 or IgG4 heavy chain constant region, or an IgG1 heavy chain constant region that enhances ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which typically include at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of a parent antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of the parent binding activity when expressed as activity on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%,95%, or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen-binding fragments include, but are not limited to: fab, fab ', F (ab') 2, fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson,2005, nat. Biotechnol.23: 1126-1136.

The "Fab fragment" consists of the CH1 and variable regions of one light and one heavy chain. The heavy chain of a Fab molecule is unable to form a disulfide bond with another heavy chain molecule.

The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains.

A "Fab ' fragment" contains a portion of one light chain and one heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of the two Fab ' fragments to form a F (ab ') 2 molecule.

An "F (ab') 2 fragment" contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks the constant region.

The term "multispecific antibody" is used in its broadest sense to encompass antibodies having polyepitopic specificity. These multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has polyepitopic specificity; an antibody having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; an antibody having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full length antibodies, antibody fragments, diabodies (diabodies), bispecific diabodies and triabodies (triabodies), antibody fragments that have been covalently or non-covalently linked together, and the like.

The term "single-chain antibody" is a single-chain recombinant protein formed by connecting a heavy chain variable region VH and a light chain variable region VL of an antibody via a linker peptide, and is the smallest antibody fragment having a complete antigen-binding site.

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment that contains only heavy chain variable regions or light chain variable regions. In certain instances, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment may target the same or different antigens.

The term "in conjunction with TROP-2" in the present application means capable of interacting with human TROP-2.

The term "antigen binding site" in the present application refers to a three-dimensional spatial site recognized by an antibody or antigen binding fragment of the present application.

The term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after denaturing solvent treatment. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art and include immunoblot and immunoprecipitation detection assays, and the like. Methods of determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.

The terms "specific binding", "selectively binding" and "selective binding" as used herein refer to binding of an antibody to an epitope on a predetermined antigen. In general, when usedHuman TROP-2 as an analyte and using an antibody as a ligand, the antibody being present at a level of about less than 10 when measured in an instrument by Surface Plasmon Resonance (SPR) techniques ^-7 M or even smaller equilibrium dissociation constant (K) _D ) Binds to a predetermined antigen with at least twice the affinity as it binds to a non-specific antigen other than the predetermined antigen or closely related antigens (e.g., BSA, etc.). The term "antibody recognizing an antigen" is used interchangeably herein with the term "specifically binding antibody".

The term "cross-reactive" refers to the ability of an antibody of the present application to bind to TROP-2 from a different species. For example, an antibody of the present application that binds human TROP-2 may also bind to TROP-2 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens, or binding or functional interactions with cells that physiologically express TROP-2, in binding assays (e.g., SPR and ELISA). Methods of determining cross-reactivity include standard binding assays as described herein, such as Surface Plasmon Resonance (SPR) analysis, or flow cytometry.

The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of a ligand preferably reduces or alters the normal level or type of activity that occurs in the absence of inhibition or blocking when ligand binding occurs. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with an anti-TROP-2 antibody compared to a ligand not contacted with an anti-TROP-2 antibody.

The term "inhibit growth" (e.g., in relation to a cell) is intended to include any measurable decrease in cell growth.

The terms "induce an immune response" and "enhance an immune response" are used interchangeably and refer to stimulation (i.e., passive or adaptive) of an immune response to a particular antigen. The term "induction" with respect to induction of CDC or ADCC refers to stimulation of a specific direct cell killing mechanism.

"ADCC", i.e., antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity), as used herein, refers to the direct killing of antibody-coated target cells by Fc fragments that recognize antibodies by Fc receptors. The ADCC effector function of an antibody may be enhanced or reduced or eliminated by modification of the Fc portion of the IgG. The modification refers to mutation in the heavy chain constant region of the antibody.

Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the antibody Experimental technical guidance of Cold spring harbor, chapters 5-8 and 15. For example, mice can be immunized with human TROP-2 or a fragment thereof, and the resulting antibodies can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to incorporate one or more human FR regions in a CDR region of non-human origin. The human FR germline sequence can be derived from the website http of Imminogenetics (IMGT): i/imgt. Cines. Fr or from the immunoglobulin journal, 2001ISBN 012441351.

The engineered antibodies or antigen binding fragments of the present application can be prepared and purified using conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into the GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the FC region. Stable clones were obtained by expression of antibodies that specifically bind to the human antigen. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified and collected by conventional techniques. The antibody can be concentrated by filtration by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

The antibody of the present application refers to a monoclonal antibody. A monoclonal antibody (mAb), as used herein, refers to an antibody obtained from a single clonal cell line, not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombinant technology, phage display technology, synthetic techniques (e.g., CDR-grafting), or other known techniques.

"administration," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering", "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by an agent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering a therapeutic agent, such as comprising any of the antibodies of the present application, either internally or externally to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically useful degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the present application (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of the target disease in every patient, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, kruskal-Wallis test (H-test), jonckhere-Terpstra test, and Wilcoxon test.

The term "consisting essentially of … …" or variations thereof as used throughout the specification and claims is meant to encompass all such elements or groups of elements, and optionally includes other elements of similar or different nature than the elements, which other elements do not materially alter the basic or novel properties of a given dosing regimen, method or composition.

The term "naturally occurring" as applied to an object as described herein refers to the fact that the object may be found in nature. For example, a polypeptide sequence or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is naturally occurring.

An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

"exogenous" refers to a substance that is to be produced outside an organism, cell, or human body by context.

"endogenous" refers to a substance produced in a cell, organism, or human body by background.

"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny thereof. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clearly visible from the context.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may, but need not, be present.

"pharmaceutical composition" means a composition containing one or more of the antibodies or antigen-binding fragments thereof described herein, as well as other components such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

The present application is further described below with reference to examples, but these examples do not limit the scope of the present application. The experimental methods without specifying specific conditions in the examples of the present application are generally performed under conventional conditions such as the antibody technique laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1: antigen preparation

The protein encoding His-tagged human TROP-2 (TROP-2-His) was synthesized by SinoBiologics corporation (10428-H08H).

TROP-2-His sequence:

example 2: mouse hybridoma and obtaining of antibody sequence

Immunizing animals with human antigen TROP-2-His, wherein 5 Balb/c mice and 5A/J mice, female, are aged for 10 weeks, and are fully mixed and emulsified by Sigma Complete Freund's Adjuvant (CFA) and Sigma Incomplete Freund's Adjuvant (IFA) in a ratio of 1: 1 to prepare stable water-in-oil liquid; the injection dose was 25. Mu.g/200. Mu.L/mouse.

TABLE 1 immunization protocol

Day 1	First immunization, complete Freund's adjuvant.
		Day 21	Second immunization, incomplete Freund's adjuvant.
Day 35	And the third immunization, incomplete Freund's adjuvant.
		Day 42	Blood sampling and serum titer test (3-blood-free)
Day 49	Fourth immunization, incomplete Freund's adjuvant.
		Day 56	Blood sampling and serum titer test (4 blood-free)

Serum titers and the ability to bind cell surface antigens were assessed using an indirect ELISA method as described in example 3 on immune mouse sera, with control titer measurements (greater than 10 ten thousand dilutions) determining the initiation of cell fusion. Selecting an immune mouse with strong serum titer, affinity and FACS combination, carrying out primary final immunization, killing the mouse, taking splenocytes, fusing the splenocytes with SP2/0 myeloma cells, paving a plate to obtain hybridomas, screening target hybridomas by indirect ELISA, and establishing a strain as a monoclonal cell strain by a limiting dilution method. The resulting positive antibody strains were further screened using indirect ELISA to select hybridomas that bind the recombinant protein. Hybridoma cells in the logarithmic growth phase were harvested, RNA extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript) ^TM Reverse Transcriptase, takara # 2680A). And (3) carrying out PCR amplification on cDNA obtained by reverse transcription by adopting a mouse Ig-primer group (Novagen, TB326 Rev.B 0503) and then sequencing to finally obtain the sequence of the murine antibody.

The variable regions of the mouse monoclonal antibody M1 in the heavy and light chains have the following sequences:

M1 HCVR

M1 LCVR

TABLE 2 CDR sequences of the heavy and light chain variable regions of murine mAb M1

Name(s)	Sequence of	Numbering
			HCDR1	NYWMN	SEQ ID NO：3
HCDR2	RIDPNDSETHYNQKFKD	SEQ ID NO：4
			HCDR3	SGFGSTYWFFDV	SEQ ID NO：5
LCDR1	KASQDVSTAVA	SEQ ID NO：6
			LCDR2	SASYRYT	SEQ ID NO：7
LCDR3	QQHYSTPLT	SEQ ID NO：8

Example 3: method for detecting in vitro binding activity of antibody

(1) In vitro indirect ELISA binding experiments:

TROP-2His protein (Nano Biological Inc., cat # 10428-H08H) was diluted to a concentration of 1. Mu.g/ml with PBS pH7.4, added to a 96-well high affinity microplate at a volume of 100. Mu.l/well, and incubated overnight (16-20 hours) at 4 ℃ in a refrigerator. After washing the plate 4 times with PBST (0.05% Tween-20 in PBS, pH 7.4), 150. Mu.l/well of a blocking solution of 3% Bovine Serum Albumin (BSA) diluted with PBST was added, and the plate was incubated at room temperature for 1 hour for blocking. After blocking was complete, the blocking solution was discarded and the plate was washed 4 times with PBST buffer.

The test antibody was diluted with 3% BSA in PBST, 10. Mu.M starting, 5-fold gradient, 9 doses, 100. Mu.l/well in an ELISA plate, and incubated at room temperature for 1 hour. After completion of incubation, the plate was washed 4 times with PBST, 100. Mu.l/well of HRP-labeled secondary goat-anti-human antibody (Abcam, cat # ab 97225) diluted with 3% BSA-containing PBST was added, and incubated at room temperature for 1 hour. After washing the plate 4 times with PBST, 100. Mu.l/well of TMB chromogenic substrate (Cell Signaling Technology, cat # 7004S) was added, the plate was incubated at room temperature for 1 minute in the absence of light, the reaction was stopped by adding 100. Mu.l/Kong Zhongzhi solution (Cell Signaling Technology, cat # 7002S), and the absorbance was read at 450nm with a microplate reader (BioTek, model Synergy H1) and the data was analyzed. The results of the concentration signal value curve analysis are shown in Table 3 below:

TABLE 3 affinity (EC) of murine antibodies to human TROP-2 antigen ₅₀ Value)

Murine antibodies	EC binding to human TROP-2His antigen ₅₀ (nM)
		M1	0.56

(2) In vitro cell binding experiments:

collecting cultured TROP-2 high expression cells (CHO or 293 cells over-expressing TROP-2 and TROP-2-expressing tumor cells such as HCC-827, MDA-MB-468, etc.), adjusting cell density, and spreading on 96-well U-bottom plate with 1 × 10 cells per well ⁵ To 2X 10 ⁵ And (4) cells. 1200g,5min centrifugation, supernatant removal, addition of 100ul of antibody solution or mouse immune serum which is diluted in a gradient manner, and incubation for 60min at the temperature of 4 ℃;1200g,5min separationThe supernatant was removed from the heart, cells were washed with PBS 2 times, and then 100ul of a fluorescently labeled secondary antibody (PE-GAM or PE-GAH) was added to each well, followed by incubation at 4 ℃ for 60min.1200g, and 5min to remove the supernatant by centrifugation. After washing the cells 2 times with PBS, the cells were resuspended in PBS, and the signal was detected using a flow cytometer and analyzed for concentration curves.

Example 4: mouse antibody humanization experiments

Humanization of murine anti-human TROP-2 monoclonal antibodies was performed as described in many publications in the art. Briefly, murine antibody M1 was humanized using human constant domains instead of the parent (murine antibody) constant domains, and human antibody sequences were selected based on the homology of the murine and human antibodies.

Based on the obtained typical structure of VH/VL CDR of the murine antibody, the variable region sequences of the heavy and light chains are compared with the germline database of the human antibody to obtain a human germline template with high homology.

The CDR regions of murine antibody M1 were grafted onto the corresponding humanized templates that were selected. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, residues directly interacting with the CDR region, and residues having important influence on the conformation of VL and VH are subjected to back mutation, and through expression test and comparison of the number of back mutations, an antibody is selected, which is formed by designing the combination of humanized heavy chain variable region HCVR and light chain variable region LCVR sequences, and the sequence is as follows:

HU1 HCVR

HU1 LCVR

HU2 HCVR

HU2 LCVR

HU3 HCVR

HU3 LCVR

HU4 HCVR

HU4 LCVR

HU5 HCVR

HU5 LCVR

HU6 HCVR

HU6 LCVR

HU7 HCVR

HU7 LCVR

HU8 HCVR

HU8 LCVR

HU9 HCVR

HU9 LCVR

the designed heavy chain and light chain variable region sequences are respectively connected with IgG1 heavy chain constant region and light chain constant region sequences, and the connected human IgG1 heavy chain constant region sequences are as follows:

IgG1 C1

IgG1 C2

the designed heavy and light chain variable region sequences were linked to IgG1 heavy and light chain constant region sequences, respectively, and the human kappa chain constant region sequences were as follows:

Ig kappa C

after ligation, the resulting exemplary heavy and light chain sequences were as follows (where HU1-HU9 heavy chains were derived from joining sequences SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 to sequences SEQ ID NO:49, respectively, and HU10 heavy chains were derived from joining sequences SEQ ID NO:19, SEQ ID NO:9 to sequences SEQ ID NO:48, respectively):

HU1 HC

HU1 LC

HU2 HC

HU2 LC

HU3 HC

HU3 LC

HU4 HC

HU4 LC

HU5 HC

HU5 LC

HU6 HC

HU6 LC

HU7 HC

HU7 LC

HU8 HC

HU8 LC

HU9 HC

HU9 LC

HU6DL HC

HU6DL LC

HU10 HC

HU10 LC

TABLE 4 sequence numbering of antibodies and their heavy, light, variable regions

cDNA fragments were synthesized based on the amino acid sequences of the light and heavy chains of each of the humanized antibodies above and inserted into pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and transfection reagent PEI (Polysciences, inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1: 2 and placed in CO ₂ Incubate in incubator for 4-5 days. Collecting cell culture solution, centrifuging, filtering, loading to an antibody purification affinity column, washing the column with phosphate buffer, eluting with glycine-HCl buffer (pH2.7.0.1M Gly-HCl), neutralizing with 1M Tris-HCl pH 9.0, and dialyzing with phosphate buffer to obtain the humanized antibody protein of the present application, wherein the concentration and purity of the humanized antibody protein are shown in Table 5 below.

TABLE 5 concentration and purity of each humanized antibody

Humanized antibody numbering	Concentration (mg/ml)	Purity (%)
			HU1	0.72	98.2％
HU2	0.62	98.4％
			HU3	0.75	96.2％
HU4	0.96	96.4％
			HU5	1.17	97.1％
HU6	1.35	96.8％
			HU7	1.26	98.5％
HU8	1.36	98.3％
			HU6DL	1.25	97.4％
HU10	1.21	98.2％

Example 5: in vitro binding affinity and kinetics experiments:

affinity (EC) of each humanized antibody for human TROP-2 antigen determined using the in vitro indirect ELISA binding assay described in example 3 (1) ₅₀ ) As shown in table 6 below:

TABLE 6 affinity (EC) of each humanized antibody for human TROP-2 antigen ₅₀ )

To examine the binding ability of each humanized antibody to the target protein TROP-2 on tumor cells, the affinity (EC) of each humanized antibody to HCC827 tumor cells (non-small cell lung cancer) was determined using the in vitro cell binding assay described in example 3 (2) ₅₀ ) As shown in table 7 below:

TABLE 7 affinity (EC) of each humanized antibody for HCC827 tumor cells ₅₀ )

Affinity (EC) of each humanized antibody for MAB-MB-468 tumor cells determined using the in vitro cell binding assay described in example 3 (2) ₅₀ ) (breast cancer, invasive ductal carcinoma) as shown in table 8 below:

TABLE 8 affinity (EC) of each humanized antibody for MAB-MB-468 tumor cells ₅₀ )

Example 6: humanized antibody mediated killing of tumor cells

The humanized antibody can exert the killing effect on tumor cells from various aspects, and one of the effects is to mediate the killing effect of immune cells on the tumor cells. To test the killing effect of the humanized antibody-mediated immunocytes on tumor cells, human Peripheral Blood Mononuclear Cells (PBMC) were co-cultured with HCC827 tumor cells (non-small cell lung cancer) for evaluation. HCC827 cells were collected, and cell density was adjusted to 0.44X 10 with complete medium after centrifugal counting ⁶ one/mL, spread in 60 wells in the middle of a white 96-well plate, 90. Mu.L per well, and the number of cells was 40000. Commercial human PBMC cells were collected and after centrifugation, cell density was adjusted to 2.2X 10 with complete medium ⁶ one/mL, spread in the middle 60 wells of a white 96-well plate with HCC827 cells, 90. Mu.L per well, and the number of cells is 200000. The remaining wells were filled with 200. Mu.L of PBS, and the cell plates were incubated at 37 ℃ in a 5% CO2 incubator overnight. The following day of the experiment, humanized antibody solutions were prepared in 96-well V-bottom plates with PBS at a concentration of 1000nM starting, 3-fold dilutions, 9 concentrations, added to white 96-well plates at 20 μ L per well, duplicate wells, and the plates were placed in 37 ℃ c, 5% co2 incubator for a further 72 hours. Fifth day of experiment, assay reading: the cell culture plate was removed, allowed to equilibrate to room temperature, 50. Mu.L of CTG solution (Promega G7573) was added to each well, shaken, mixed, allowed to stand in the dark for 10 minutes, and then detected using the luminescence protocol of the microplate reader. The results of the experiment are shown in table 9 below:

TABLE 9 humanized antibody mediated killing of tumor cells

The same method is adopted to measure the killing effect of the HU6 antibody on HCC827 tumor cells, and the result shows that the highest dose killing effect is 52.3%.

Example 7: humanized antibody-mediated TROP2 endocytosis

To study humanized antibody-mediated endocytosis of TROP-2 protein in tumor cells, SW780 cells were trypsinized, harvested and resuspended in precooled PBS to a cell concentration of 1X 10 ⁶ one/mL. Taking an EP tube, adding 1mL of cell suspension, centrifuging at 1500rpm for 5 minutes, removing supernatant, adding 1mL of prepared antibody to be detected for heavy suspension of cells, wherein the final concentration of the antibody is 20 mu g/mL, incubating for 1h by a shaking table at 4 ℃, centrifuging, removing supernatant (4 ℃,1500rpm multiplied by 5 min), washing twice by PBS, and removing supernatant. Add 100. Mu.L of fluorescent secondary antibody working solution to each tube to resuspend the cells, incubate them in a shaker at 4 ℃ for 30min, centrifuge and discard the supernatant (4 ℃,1500 rpm. Times.5 min), wash them twice with PBS, and remove the supernatant. Adding 1.0mL of preheated SW780 cells into each tube, suspending the cells by a complete culture medium, uniformly mixing, subpackaging into 4 tubes, wherein each tube is 200 mu L, and the volume of each tube is respectively 0min group, blank group, 30min group and 2h group, taking out 0min and blank and placing on ice, placing the rest in an incubator at 37 ℃, carrying out endocytosis for 30min and 2h respectively, taking out 1 tube at different corresponding time points, placing on ice for precooling for 5min, centrifuging all treatment groups, discarding supernatant (4 ℃,1500rpm multiplied by 5 min), washing once by PBS (phosphate buffer solution), and removing the supernatant. mu.L strip buffer was added to the tubes of all treatment groups except the 0min group, incubated at room temperature for 8min, centrifuged to discard the supernatant (4 ℃,1500 rpm. Times.5 min), washed twice with PBS, and the supernatant was removed. All treatment groups were added with 100. Mu.L of immunostaining fixative, left at 4 ℃ for more than 30min, and tested on the machine with a flow cytometer DxFlex. From the 0min group, 200. Mu.l of the tube was directly added with the immunostaining fixative. From the blank group, 200. Mu.l were taken, and strip buffer and immunostaining fixative were added directly. Detecting the DNA by a flow cytometer DxFlex on a computer. Data statistics and analysis: 30min average percent endocytosis (%) = (3)0min group MIF-blank group MFI)/(0 min group MFI-blank group MFI) × 100%,2h mean endocytosis percentage (%) = (2 h group MIF-blank group MFI)/(0 min group MFI-blank group MFI) = 100%. The percent endocytosis of the humanized antibody detected using the above method is shown in table 10 below:

TABLE 10 humanized antibody mediated TROP-2 protein endocytosis

Example 8: competitive binding of humanized antibodies to antigens

The binding pattern and binding site of different antibodies to antigens are studied, usually by competitive binding experiments. The hRS7 antibody protein was diluted to a concentration of 1. Mu.g/ml with PBS, pH7.4, added to a 96-well high affinity plate at a volume of 100. Mu.l/well, and incubated overnight (16-20 hours) at 4 ℃ in a refrigerator. After washing the plate 4 times with PBST (0.05% Tween-20 in PBS, pH 7.4), 150. Mu.l/well of a 2% Bovine Serum Albumin (BSA) blocking solution diluted with PBST was added, and the plate was incubated at room temperature for 1 hour for blocking. After blocking was complete, the blocking solution was discarded and the plate was washed 4 times with PBST buffer.

The test antibody was diluted to 100. Mu.g/ml with 2% BSA in PBST and added to the microplate at 50. Mu.l/well. TROP-2His protein (Nano Biological Inc., cat # 10428-H08H) was diluted with 2% BSA-containing PBST and added to the microplate at 50. Mu.l/well. The microplate was incubated at room temperature for 1 hour. After the end of incubation, the plate was washed 4 times with PBST, 100. Mu.l/well of anti-His HRP-labeled secondary antibody (Abcam, cat # ab 197049) diluted with PBST containing 2% BSA was added and incubated at room temperature for 1 hour. After washing the plate 4 times with PBST, 100. Mu.l/well of TMB chromogenic substrate (Cell Signaling Technology, cat # 7004S) were added, the reaction was stopped by adding 100. Mu.l/well of Stop Solution (Cell Signaling Technology, cat # 7002S) after incubation for 1 minute at room temperature in the dark, and the absorbance was read at 450nm with a microplate reader (BioTek, model. Synergy H1) and the data was analyzed as shown in the following table. The humanized antibody of the present invention has a very low inhibition rate of binding of hRS7 antibody to TROP2 protein, suggesting that the humanized antibody of the present invention does not compete with hRS7 antibody for binding to the same epitope.

TABLE 11 humanized antibodies compete for binding to hRS7 antigen

Humanized antibodies	Inhibition rate
		hRS7	94.7％
HU1	13.5％
		HU6	6.7％
HU6DL	5.9％
		HU10	10.2％

Sequence listing

<110> Jiangsu Haofen pharmaceutical group Co., ltd

SHANGHAI HANSOH BIOMEDICAL Co.,Ltd.

Changzhou Hengbang Pharmaceutical Co.,Ltd.

<120> anti-TROP-2 antibody, antigen-binding fragment thereof, and medical use thereof

<140> CN 202080003175.1

<141> 2022-08-15

<150> CN201910389587.2

<151> 2019-05-10

<150> CN201911073081.7

<151> 2019-11-05

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35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 11

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(121)

<223> HU2 HCVR

<400> 11

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 12

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> HU2 LCVR

<400> 12

Glu Thr Thr Leu Thr Gln Ser Pro Ala Phe Met Ser Ala Thr Pro Gly

1 5 10 15

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

20 25 30

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

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 13

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(121)

<223> HU3 HCVR

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 14

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> HU3 LCVR

<400> 14

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 15

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(121)

<223> HU4 HCVR

<400> 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His 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 Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 16

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> HU4 LCVR

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 17

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(121)

<223> HU5 HCVR

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His 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 Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 18

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> HU5 LCVR

<400> 18

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 Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Phe 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 Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 19

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(121)

<223> HU6 HCVR

<400> 19

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 20

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> HU6 LCVR

<400> 20

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr 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 Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 21

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(121)

<223> HU7 HCVR

<400> 21

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His 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 Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 22

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> HU7 LCVR

<400> 22

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

1 5 10 15

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

20 25 30

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

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 23

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(121)

<223> HU8 HCVR

<400> 23

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 Asn Tyr

20 25 30

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

35 40 45

Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His 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 Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 24

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> HU8 LCVR

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 25

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(121)

<223> HU9 HCVR

<400> 25

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

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

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 26

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> HU9 LCVR

<400> 26

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

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

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 27

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(451)

<223> HU1 HC

<400> 27

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Thr 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 Lys 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> 28

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(214)

<223> HU1 LC or HU10 LC

<400> 28

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu 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> 29

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(451)

<223> HU2 HC

<400> 29

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Thr 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 Lys 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> 30

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(214)

<223> HU2 LC

<400> 30

Glu Thr Thr Leu Thr Gln Ser Pro Ala Phe Met Ser Ala Thr Pro Gly

1 5 10 15

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

20 25 30

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

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu 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> 31

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(451)

<223> HU3 HC

<400> 31

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Met 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 Lys 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> 32

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(214)

<223> HU3 LC

<400> 32

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu 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> 33

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(451)

<223> HU4 HC

<400> 33

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His 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 Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Met 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 Lys 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> 34

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(214)

<223> HU4 LC

<400> 34

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu 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> 35

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(451)

<223> HU5 HC

<400> 35

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His 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 Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr 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 Lys 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> 36

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(214)

<223> HU5 LC

<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 Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Phe 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 Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu 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> 37

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(415)

<223> HU6 HC

<400> 37

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Thr 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 Lys 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> 38

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(214)

<223> HU6 LC or HU6DL LC

<400> 38

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr 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 Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu 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> 39

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(415)

<223> HU7 HC

<400> 39

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His 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 Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr 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 Lys 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 Sequence

<220>

<221> CHAIN

<222> (1)..(214)

<223> HU7 LC

<400> 40

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

1 5 10 15

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

20 25 30

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

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu 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> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(415)

<223> HU8 HC

<400> 41

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 Asn Tyr

20 25 30

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

35 40 45

Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His 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 Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr 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 Lys 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> 42

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(214)

<223> HU8 LC

<400> 42

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu 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> 43

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(413)

<223> HU9 HC

<400> 43

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

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

100 105 110

Gln Gly Thr Thr 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 Lys 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> 44

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(214)

<223> HU9 LC

<400> 44

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

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

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu 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> 45

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(451)

<223> HU6DL HC

<400> 45

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Thr 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 Lys 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 Asp Glu Leu 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> 46

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<222> (1)..(451)

<223> HU10 HC

<400> 46

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Thr 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 Lys 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 Asp Glu Leu 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> 47

<211> 330

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(330)

<223> IgG1 C1

<400> 47

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

Lys 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 Asp Glu

225 230 235 240

Leu 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> 48

<211> 330

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(330)

<223> IgG1 C2

<400> 48

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

Lys 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> 49

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> Ig kappa C

<400> 49

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

Claims

1. An anti-TROP-2 antibody or antigen-binding fragment thereof, comprising: an antibody heavy chain variable region comprising: HCDR1 shown in SEQ ID NO. 3, HCDR2 shown in SEQ ID NO. 4 and HCDR3 shown in SEQ ID NO. 5; and an antibody light chain variable region comprising: LCDR1 shown in SEQ ID NO. 6, LCDR2 shown in SEQ ID NO. 7 and LCDR3 shown in SEQ ID NO. 8.

2. The anti-TROP-2 antibody or antigen-binding fragment thereof of claim 1, wherein said antibody is a murine antibody, a chimeric antibody or a humanized antibody.

3. The anti-TROP-2 antibody or antigen-binding fragment thereof of claim 2, wherein said anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, igG2, igG3, or IgG4.

4. The anti-TROP-2 antibody or antigen-binding fragment thereof of claim 3, wherein said anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, igG2, or IgG4.

5. The anti-TROP-2 antibody or antigen-binding fragment thereof of claim 3, wherein said anti-TROP-2 antibody or antigen-binding fragment thereof further comprises an IgG1 heavy chain constant region incorporating the E239D and M241L mutations.

6. The anti-TROP-2 antibody or antigen-binding fragment thereof of claim 3, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region selected from SEQ ID NO 48 or SEQ ID NO 49.

7. The anti-TROP-2 antibody or antigen-binding fragment thereof of claim 3, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain, lambda chain.

8. The anti-TROP-2 antibody or antigen-binding fragment thereof of claim 3, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region selected from SEQ ID NO 50.

9. The anti-TROP-2 antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein:

the anti-TROP-2 antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:9 and SEQ ID NO: 10; or the like, or, alternatively,

the anti-TROP-2 antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:11 and SEQ ID NO: 12; or the like, or, alternatively,

the anti-TROP-2 antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:13 and SEQ ID NO:14, a light chain variable region as shown; or the like, or, alternatively,

the anti-TROP-2 antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:15 and SEQ ID NO:16, a light chain variable region shown in seq id no; or the like, or, alternatively,

the anti-TROP-2 antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:17 and SEQ ID NO:18, the light chain variable region shown in; or the like, or, alternatively,

the anti-TROP-2 antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:19 and the heavy chain variable region of SEQ ID NO:20, a light chain variable region; or

The anti-TROP-2 antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:21 and the heavy chain variable region of SEQ ID NO: 22; or the like, or, alternatively,

the anti-TROP-2 antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:23 and SEQ ID NO:24, a light chain variable region; or the like, or, alternatively,

the anti-TROP-2 antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:25 and the heavy chain variable region of SEQ ID NO:26, or a light chain variable region as shown.

10. The anti-TROP-2 antibody or antigen-binding fragment thereof of claim 9, wherein:

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:27, light chain SEQ ID NO:28; or the like, or, alternatively,

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:29, light chain of SEQ ID NO:30, of a nitrogen-containing gas; or the like, or, alternatively,

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:31, light chain of SEQ ID NO:32, a first step of removing the first layer; or the like, or, alternatively,

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:33, light chain SEQ ID NO:34; or the like, or, alternatively,

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:35, light chain of SEQ ID NO:36; or the like, or, alternatively,

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:37, light chain SEQ ID NO:38; or the like, or, alternatively,

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:39, light chain of SEQ ID NO:40; or the like, or, alternatively,

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:41 and the light chain is SEQ ID NO:42; or the like, or, alternatively,

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:43, light chain of SEQ ID NO:44; or the like, or, alternatively,

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:45, light chain of SEQ ID NO:38; or the like, or, alternatively,

the heavy chain of the anti-TROP-2 antibody is SEQ ID NO:47, light chain SEQ ID NO:28.

11. a polynucleotide encoding the anti-TROP-2 antibody or antigen-binding fragment thereof of any one of claims 1 to 10.

12. An expression vector comprising the polynucleotide of claim 11.

13. A host cell transformed with the expression vector of claim 12.

14. The host cell of claim 13, wherein said host cell is selected from the group consisting of a bacterial, yeast, or mammalian cell.

15. The host cell of claim 14, wherein the host cell is selected from the group consisting of E.coli, pichia or CHO cells or HEK293 cells.

16. A method of producing an anti-TROP-2 antibody comprising the steps of:

culturing the host cell of any one of claims 14-15;

isolating the antibody from the culture; and

purifying the antibody.

17. A pharmaceutical composition comprising:

the anti-TROP-2 antibody or antigen-binding fragment thereof of any one of claims 1 to 10 and a pharmaceutically acceptable excipient, diluent or carrier.

18. A detection or diagnostic kit comprising:

the anti-TROP-2 antibody or antigen-binding fragment thereof of any one of claims 1 to 10.

19. Use of the anti-TROP-2 antibody or antigen-binding fragment thereof of any one of claims 1 to 10 in the preparation of a medicament for treating or preventing a TROP-2 expressing cancer;

the cancer is selected from: breast cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, and melanoma.

20. Use of the anti-TROP-2 antibody or antigen-binding fragment thereof of any one of claims 1 to 10 in the preparation of an agent for detecting, diagnosing, prognosing a TROP-2 expressing cancer;

21. The anti-TROP-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 for use in the treatment or prevention of a TROP-2 expressing cancer;

22. The anti-TROP-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 for use in detecting, diagnosing, prognosing a TROP-2 expressing cancer;