CN116284408A - Antibody binding to human MUC17, preparation method and application thereof - Google Patents

Antibody binding to human MUC17, preparation method and application thereof Download PDF

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CN116284408A
CN116284408A CN202111500224.5A CN202111500224A CN116284408A CN 116284408 A CN116284408 A CN 116284408A CN 202111500224 A CN202111500224 A CN 202111500224A CN 116284408 A CN116284408 A CN 116284408A
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朱祯平
顾昌玲
邓岚
黄浩旻
徐菲
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Sunshine Guojian Pharmaceutical Shanghai Co Ltd
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Sunshine Guojian Pharmaceutical Shanghai Co Ltd
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Abstract

The present invention relates to novel antibodies or antigen-binding fragments thereof that specifically bind to human MUC17 and compositions containing the antibodies or antigen-binding fragments thereof. The invention also relates to polynucleotides encoding the antibodies or antigen binding fragments thereof and host cells comprising the same, as well as related uses. The invention furthermore relates to therapeutic and diagnostic uses of these antibodies or antigen binding fragments thereof.

Description

Antibody binding to human MUC17, preparation method and application thereof
Technical Field
The invention belongs to the field of tumor treatment and biotechnology, and relates to an antibody combined with human MUC17, a preparation method and application thereof.
Background
Mucin17 (MUC 17) is a transmembrane protein expressed on the top membrane of normal gastrointestinal mucosal epithelial cells and belongs to a Mucin (Mucin) family member whose primary structure comprises a signal peptide, a large extracellular domain consisting of a central region of 61 tandem repeats, two Epidermal Growth Factor (EGF) domains, a collectin (SEA) and a cytoplasmic tail of 80 amino acids. MUC17 is aberrantly expressed in some cancers, for example, MUC17 mRNA is expressed in one pancreatic cell line and three colon cancer cell lines (Gun et al 2002); immunohistochemical studies confirmed the expression of MUC17 protein in pancreatic cancer (Moniaux et al 2006); over-expression of MUC17 on cancer cell membranes at gastric cancer and gastroesophageal junction; however, in colon cancer, the MUC17 protein expression level is low (Senapati et al, J.Clin. Pathol. 2010). Nonetheless, the expression pattern of MUC17 makes it a potential target for the treatment of different forms of malignancy.
Thus, there is an urgent need to develop novel drugs targeting human MUC17 and to provide more, more effective treatment regimens for patients suffering from diseases in which MUC17 expression is abnormal.
Disclosure of Invention
The present invention aims to provide an antibody or antigen-binding fragment thereof that binds to human MUC 17; providing a polynucleotide molecule encoding the antibody or antigen binding fragment thereof that binds human MUC 17; providing an expression vector comprising said polynucleotide molecule; providing a host cell comprising the expression vector; providing a method of preparing the antibody or antigen-binding fragment thereof that binds to human MUC 17; providing a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds human MUC 17; the use of the antibody or antigen binding fragment thereof that binds human MUC17 in the manufacture of a medicament is provided.
In a first aspect of the present invention there is provided an antibody or antigen-binding fragment thereof that binds to human MUC17, the antibody or antigen-binding fragment thereof that binds to human MUC17 comprising:
(a) Heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequence of said HCDR1 selected from the group consisting of SEQ ID NOs: 2. 10, 18, 26, 34, 42, 50, 58, 66, 74, 82 and 90, the amino acid sequence of said HCDR2 is selected from the group consisting of SEQ ID NO: 3. 11, 19, 27, 35, 43, 51, 59, 67, 75, 83 and 91, and the amino acid sequence of HCDR3 is selected from the group consisting of SEQ ID NO: 4. 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, and 92; and
(b) Light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, said LCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6. 14, 22, 30, 38, 46, 54, 62, 70, 78, 86 and 94, the amino acid sequence of LCDR2 is selected from the group consisting of SEQ ID NOs: 7. 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, and 95, and the amino acid sequence of LCDR3 is selected from the group consisting of SEQ ID NOs: 8. 16, 24, 32, 40, 48, 56, 64, 72, 80, 88 and 96.
In another preferred embodiment, the antibody or antigen-binding fragment thereof that binds to human MUC17 is characterized in that:
(1) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 2. 3 and 4, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively as set forth in SEQ ID NOs: 6. 7 and 8;
(2) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 10. 11 and 12, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 14. 15 and 16;
(3) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 18. 19 and 20, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 22. 23 and 24;
(4) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 26. 27 and 28, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 30. 31 and 32;
(5) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 34. 35 and 36, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 38. 39 and 40;
(6) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 42. 43 and 44, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively set forth in SEQ ID NOs: 46. 47 and 48;
(7) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 50. 51 and 52, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 54. 55 and 56;
(8) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 58. 59 and 60, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 62. 63 and 64;
(9) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 66. 67 and 68, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively set forth in SEQ ID NOs: 70. 71 and 72;
(10) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 74. 75 and 76, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 78. 79 and 80;
(11) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 82. 83 and 84, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 86. 87 and 88; or (b)
(12) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 90. 91 and 92, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 94. 95 and 96.
In another preferred embodiment, the antibody that binds to human MUC17 is a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.
In another preferred embodiment, the antibody that binds to human MUC17 is a single chain antibody or a double chain antibody.
In another preferred example, the antibody that binds to human MUC17 is a murine antibody, a chimeric antibody, or a humanized antibody.
In another preferred embodiment, the antibody that binds to human MUC17 is a humanized antibody.
In another preferred embodiment, the antigen binding fragment that binds to human MUC17 comprises a Fab fragment, a F (ab ') 2 fragment, a Fab' fragment, an Fv fragment, or a single chain Fv (scFv) fragment.
In another preferred embodiment, the antigen-binding fragment that binds human MUC17 comprises a heavy chain variable region (VH) and a light chain variable region (VL),
(1) The amino acid sequence of VH is selected from the group consisting of SEQ ID NO: 1. 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 99 and 103; and
(2) The amino acid sequence of VL is selected from the group consisting of SEQ ID NO: 5. 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 100 and 104.
In another preferred embodiment, the antibody or antigen-binding fragment thereof that binds to human MUC17 is characterized in that:
(1) The amino acid sequence of the VH is shown in SEQ ID NO:1, wherein the amino acid sequence of the VL is shown in SEQ ID NO:5 is shown in the figure;
(2) The amino acid sequence of the VH is shown in SEQ ID NO:9, the amino acid sequence of the VL is shown in SEQ ID NO: 13;
(3) The amino acid sequence of the VH is shown in SEQ ID NO:17, the amino acid sequence of the VL is shown in SEQ ID NO: 21;
(4) The amino acid sequence of the VH is shown in SEQ ID NO:25, said VL comprises the amino acid sequence set forth in SEQ ID NO:29, and a polypeptide comprising the amino acid sequence shown in seq id no;
(5) The amino acid sequence of the VH is shown in SEQ ID NO:33, the amino acid sequence of the VL is shown in SEQ ID NO: shown at 37;
(6) The amino acid sequence of the VH is shown in SEQ ID NO:41, wherein the amino acid sequence of the VL is shown in SEQ ID NO: 45;
(7) The amino acid sequence of the VH is shown in SEQ ID NO:49, the amino acid sequence of the VL is set forth in SEQ ID NO: 53;
(8) The amino acid sequence of the VH is shown in SEQ ID NO:57, the amino acid sequence of the VL is shown in SEQ ID NO: indicated at 61;
(9) The amino acid sequence of the VH is shown in SEQ ID NO:65, the amino acid sequence of the VL is shown in SEQ ID NO: 69;
(10) The amino acid sequence of the VH is shown in SEQ ID NO:73, the amino acid sequence of the VL is shown in SEQ ID NO: indicated at 77;
(11) The amino acid sequence of the VH is shown in SEQ ID NO:81, the amino acid sequence of the VL is shown in SEQ ID NO: indicated at 85;
(12) The amino acid sequence of the VH is shown in SEQ ID NO:89, the amino acid sequence of the VL is shown in SEQ ID NO:93, shown in the figure; .
(13) The amino acid sequence of the VH is shown in SEQ ID NO:99, the amino acid sequence of the VL is shown in SEQ ID NO: 100; or (b)
(14) The amino acid sequence of the VH is shown in SEQ ID NO:103, the amino acid sequence of the VL is shown in SEQ ID NO: 104.
In another preferred embodiment, the antibody or antigen-binding fragment thereof that binds human MUC17 comprises a heavy chain constant region selected from an IgG1, igG2, igG3 or IgG4 heavy chain constant region and a light chain constant region selected from a kappa or lambda light chain constant region; preferably, the heavy chain constant region is an IgG1 heavy chain constant region and the light chain constant region is a kappa light chain constant region; preferably, the amino acid sequence of the heavy chain constant region is as set forth in SEQ ID NO:97, the amino acid sequence of the light chain constant region is shown in SEQ ID NO: shown at 98.
In another preferred embodiment, the antibody or antigen-binding fragment thereof that binds human MUC17 comprises a heavy chain and a light chain selected from the group consisting of:
(1) The amino acid sequence of the heavy chain is shown in SEQ ID NO:101, wherein the amino acid sequence of the light chain is shown in SEQ ID NO: 102; and
(2) The amino acid sequence of the heavy chain is shown in SEQ ID NO:105, the amino acid sequence of the light chain is shown in SEQ ID NO: shown at 106.
In a second aspect of the invention there is provided an isolated polynucleotide molecule encoding an antibody or antigen-binding fragment thereof that binds human MUC17 as described in the first aspect of the invention.
In a third aspect of the invention there is provided an expression vector comprising a polynucleotide molecule according to the second aspect of the invention.
In another preferred embodiment, the expression vector is a virus or plasmid, preferably a phage or phagemid.
In another preferred embodiment, the expression vector is selected from the group consisting of: pcDNA3.4, pDR1, pcDNA3.1 (+), pcDNA3.1/ZEO (+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, preferably pTT5.
In a fourth aspect of the invention there is provided a host cell comprising an expression vector according to the third aspect of the invention.
In another preferred embodiment, the host cell is selected from the group consisting of: COS, CHO, 293F, 293E, NS0, sf9, sf21, DH 5. Alpha., BL21 (DE 3) or TG1, more preferably E.coli TG1, BL21 (DE 3) cells (expressing single chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full length IgG antibodies).
In another preferred embodiment, the host cell is a eukaryotic cell, preferably a CHO cell, 293F cell or 293E cell.
In a fifth aspect of the present invention there is provided a method of preparing an antibody or antigen binding fragment thereof that binds to human MUC17 according to the first aspect of the present invention, the method comprising the steps of:
(a) Culturing a host cell according to the fourth aspect of the invention under expression conditions so as to express an antibody or antigen-binding fragment thereof that binds human MUC 17;
(b) Isolating and purifying the antibody or antigen-binding fragment thereof that binds human MUC17 as described in step (a).
In a sixth aspect of the invention there is provided a pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof that binds human MUC17 according to the first aspect of the invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.
In another preferred embodiment, the pharmaceutical composition further comprises one or more additional therapeutic agents.
In a seventh aspect of the invention there is provided the use of an antibody or antigen-binding fragment thereof that binds human MUC17 as described in the first aspect of the invention, or a pharmaceutical composition as described in the sixth aspect of the invention, in the manufacture of a medicament for cancer or tumour.
In another preferred embodiment, the cancer or tumor is selected from: pancreatic cancer, colorectal cancer, colon cancer, colorectal cancer, small intestine cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, gastroesophageal cancer, or a combination thereof.
In an eighth aspect of the invention, there is provided an immunoconjugate comprising:
(a) An antibody or antigen-binding fragment thereof that binds to human MUC17 according to the first aspect of the invention; and
(b) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
In another preferred embodiment, the conjugate moiety is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.) capable of producing a detectable product.
In another preferred embodiment, the immunoconjugate comprises an antibody-drug conjugate (ADC).
In another preferred embodiment, the immunoconjugate is used for the preparation of a pharmaceutical composition for the treatment of cancer or tumor.
In a ninth aspect of the invention there is provided a method of treating cancer or tumour, the method comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof that binds human MUC17 as described in the first aspect of the invention, a pharmaceutical composition as described in the sixth aspect of the invention, or an immunoconjugate as described in the eighth aspect of the invention.
In another preferred embodiment, the method further comprises co-administration with an additional antineoplastic agent.
Drawings
Figure 1 shows ELISA detection of binding of hybridoma antibodies to MUC17 antigen.
FIG. 2 shows FACS detection of binding of hybridoma antibodies to cellular LS 174T.
Figure 3 shows ELISA to detect binding of chimeric antibodies to MUC17 antigen.
FIGS. 4A-4B show FACS detection of chimeric antibody binding to cellular LS 174T.
Figure 5 shows ELISA detection of binding of humanized antibodies to MUC17 antigen.
FIG. 6A shows FACS detection of binding of humanized antibody hu-27A3 to cell LS 174T; FIG. 6B shows FACS detection of binding of humanized antibody hu-19E11 to cell LS 174T.
Detailed Description
Through extensive and intensive research, the inventor obtains a brand-new murine monoclonal antibody capable of specifically combining human MUC17 through antigen immunization, hybridoma screening and antibody expression purification to biological activity identification, and further constructs and obtains chimeric antibodies and humanized antibodies of the murine monoclonal antibody. The antibody of the invention has high affinity and specificity to human MUC17, can be applied to preparing medicaments for treating cancers or tumors, and has good clinical application prospect.
Terminology
In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meanings given below, unless expressly specified otherwise herein. Other definitions are set forth throughout the application.
The term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured.
As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
In the present invention, the terms "Antibody (abbreviated Ab)" and "Immunoglobulin G (abbreviated IgG)" are hetero tetrameric proteins having identical structural characteristics, which are composed of two identical light chains (L) each of which is linked to a heavy chain by one covalent disulfide bond, and two identical heavy chains (H) each of which has a regularly spaced intrachain disulfide bond, while the number of disulfide bonds between the heavy chains of different Immunoglobulin isotypes (isotype) is different. One end of each heavy chain is composed of a variable region (VH), followed by a constant region, the heavy chain constant region is composed of three domains CH1, CH2, and CH3, one end of each light chain is composed of a variable region (VL), the other end has a constant region, and the light chain constant region includes one domain CL; the constant region of the light chain is paired with the CH1 domain of the constant region of the heavy chain, the variable region of the light chain is paired with the variable region of the heavy chain, the constant regions are not directly involved in binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC, anti-independent cell-mediated cytotoxicity) and the like, the heavy chain constant region comprising IgG1, igG2, igG3, igG4 subtypes; the light chain constant region includes Kappa (Kappa) or Lambda (Lambda). The heavy and light chains of an antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of an antibody are covalently linked together by inter-polypeptide disulfide bonds formed between the hinge regions.
In the present invention, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the culture of hybridoma cells and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.
In the present invention, the term "murine antibody" refers to an antibody derived from a rat or mouse. The murine antibody is obtained by immunizing a mouse with human MUC17 as an antigen and screening hybridoma cells. More preferably, the murine antibodies of the present invention include 8H2, 19E11, 23F6, 27A3, 42H4, 44H4, 11C8, 15D10, 45D1, 50A5, 51E12, and 56E8.
In the present invention, the term "chimeric antibody" refers to an antibody comprising heavy and light chain variable region sequences derived from one species and constant region sequences derived from another species, for example, an antibody having murine heavy and light chain variable regions linked to human constant regions. Preferably, the heavy chain of the chimeric antibody of the present invention is obtained by splicing the heavy chain variable region sequence of a murine antibody to the human IgG1 constant region, and the light chain is obtained by splicing the light chain variable region of a murine antibody to the human Kappa chain. More preferably, the chimeric antibody of the present invention includes ch-8H2, ch-19E11, ch-23F6, ch-27A3, ch-42H4, ch-44H4, ch-11C8, ch-15D10, ch-45D1, ch-50A5, ch-51E12 and ch-56E8.
In the present invention, the term "humanized antibody" means an antibody whose CDRs are derived from a non-human species (preferably murine), and the remainder of the antibody molecule (including the framework and constant regions) is derived from a human antibody. In addition, framework region residues may be altered to maintain binding affinity. Preferably, the humanized antibody of the present invention is obtained by recombining the CDR regions of the murine antibody 27A3 or 19E11 and the non-CDR regions derived from the human antibody, and back-mutating the embedded residues, residues having direct interactions with the CDR regions, and residues having an important influence on the conformation of VL and VH of 27A3 or 19E11. More preferably, the humanized antibodies of the invention include hu-27A3 and hu-19E11.
In the present invention, the term "antigen-binding fragment" refers to a fragment of an antibody capable of specifically binding to human MUC 17. Non-limiting examples of antigen binding fragments of the invention include Fab fragments, F (ab ') 2 fragments, fab' fragments, fv fragments, single chain antibodies (scFv), single domain antibodies (sdabs), and the like. The Fab fragment is cleaved by papain digestion of the antibody into two identical Fab fragments consisting of VH and CH1 of the heavy chain and VL and CL domains of the light chain and one Fc fragment. F (ab ') 2 fragments are fragments produced by pepsin digestion of antibodies, and F (ab ') 2 fragments have two antigen-binding Fab ' portions linked together by disulfide bonds. Fv fragments are composed of dimers of the antibody in which the heavy and light chain variable regions are closely non-covalently associated. A single chain antibody (scFv) is an antibody in which a heavy chain variable region and a light chain variable region of an antibody are linked by a short peptide linker (linker) of 15 to 20 amino acids. Single domain antibodies (sdabs), also known as nanobodies (nanobodies) or heavy chain antibodies, consist of only a heavy chain, with an antigen binding region that is only a single domain linked to an Fc region through a hinge region.
In the present invention, the term "variable" means that some portion of the variable region in an antibody differs in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC, anti-independent cell-mediated cytotoxicity), and the like.
As used herein, the term "Framework Region (FR)" refers to the amino acid sequence inserted between CDRs, i.e., those portions of the light and heavy chain variable regions of immunoglobulins that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of immunoglobulins each have four FRs, designated FR1-L, FR2-L, FR3-L, FR-L and FR1-H, FR2-H, FR3-H, FR-H, respectively. Accordingly, the light chain variable domain may thus be referred to as (FR 1-L) - (CDR 1-L) - (FR 2-L) - (CDR 2-L) - (FR 3-L) - (CDR 3-L) - (FR 4-L) and the heavy chain variable domain may thus be denoted as (FR 1-H) - (CDR 1-H) - (FR 2-H) - (CDR 2-H) - (FR 3-H) - (CDR 3-H) - (FR 4-H). Preferably, the FR of the invention is a human antibody FR or a derivative thereof which is substantially identical to a naturally occurring human antibody FR, i.e. has a sequence identity of up to 85%, 90%, 95%, 96%, 97%, 98% or 99%.
Knowing the amino acid sequence of the CDRs, one skilled in the art can readily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR-H.
As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.
In the present invention, the terms "anti", "binding", "specific binding" refer to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. Typically, the antibody is present at less than about 10 -7 M, e.g. less than about 10 -8 M、10 -9 M、10 -10 M、10 -11 An equilibrium dissociation constant (KD) of M or less binds to the antigen. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. For example, the binding affinity of an antibody to an antigen is determined in a BIACORE instrument using surface plasmon resonance (Surface Plasmon Resonance, abbreviated SPR) or the relative affinity of an antibody to antigen binding is determined using ELISA.
In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. An epitope of the invention is a region of an antigen to which an antibody binds.
Coding nucleic acids and expression vectors
In another aspect, the present invention provides a polynucleotide molecule encoding an antibody or antigen-binding fragment thereof that binds human MUC17 as described above. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
The preparation method of the polynucleotide molecule of the invention is a preparation method conventional in the art, and preferably comprises the following preparation methods: the polynucleotide molecule encoding the above antibody or antigen-binding fragment thereof is obtained by a gene cloning technique such as a PCR method or the like, or by a method of artificial total sequence synthesis.
It will be appreciated by those skilled in the art that nucleotide sequences encoding the amino acid sequences of the antibodies or antigen binding fragments thereof described above may be appropriately introduced into substitutions, deletions, alterations, insertions or additions to provide a homolog of the polynucleotide. Homologs of the polynucleotides of the invention may be prepared by substitution, deletion, or addition of one or more bases of the gene encoding the antibody or antigen binding fragment thereof within a range that retains antibody activity.
In another aspect, the invention provides an expression vector comprising a polynucleotide molecule as described above.
Wherein the expression vector is a conventional expression vector in the art, and refers to an expression vector comprising suitable regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other suitable sequences. The expression vector may be a virus or plasmid, such as a suitable phage or phagemid, see, e.g., sambrook et al Molecular Cloning for further technical details: a Laboratory Manual, second edition, cold Spring Harbor Laboratory Press,1989. A number of known techniques and protocols for nucleic acid manipulation are described in Current Protocols in Molecular Biology, second edition, ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.1 (+), pcDNA3.4, pcDNA3.1/ZEO (+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pTT5.
The present invention further provides a host cell comprising the expression vector described above.
The host cell of the present invention is a variety of host cells conventional in the art, as long as it can stably self-replicate the recombinant expression vector described above, and the nucleotide carried thereby can be expressed efficiently. Wherein the host cell comprises a prokaryotic expression cell and a eukaryotic expression cell, preferably the host cell comprises: COS, CHO (chinese hamster ovary ), 293E, 293F, NS0, sf9, sf21, DH5 a, BL21 (DE 3) or TG1, more preferably e.coli TG1, BL21 (DE 3) cells (expressing single chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full length IgG antibodies). The expression vector is transformed into a host cell to obtain the preferred recombinant expression transformant of the invention. Wherein the conversion process is conventional in the art, preferably chemical, heat shock or electrotransformation.
Preferably, the host cell is a eukaryotic cell. CHO cells, 293F cells or 293E cells are preferred.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
In another aspect, the present invention provides a method for preparing the above-described antibody or antigen-binding fragment thereof that binds to human MUC17, the method comprising the steps of:
(a) Culturing the host cell under expression conditions, thereby expressing an antibody or antigen-binding fragment thereof that binds human MUC 17;
(b) Isolating and purifying the antibody or antigen-binding fragment thereof that binds human MUC17 as described in step (a).
The method for culturing host cells and the method for separating and purifying the antibody are conventional methods in the field, and specific operation methods refer to corresponding cell culture technical manuals and antibody separation and purification technical manuals. The method for producing an antibody or an antigen-binding fragment thereof that binds to human MUC17 disclosed in the present invention comprises: culturing the host cell under expression conditions, thereby expressing an antibody or antigen-binding fragment thereof that binds human MUC 17; isolating and purifying the antibody or antigen binding fragment thereof that binds human MUC 17. Using the above method, the recombinant protein can be purified to a substantially homogeneous substance.
The antibody or antigen-binding fragment thereof binding to human MUC17 disclosed in the present invention may be isolated and purified by affinity chromatography, and the antibody or antigen-binding fragment thereof binding to human MUC17 bound to the affinity column may be eluted by conventional methods such as high salt buffer, pH change, etc., depending on the characteristics of the affinity column used. The inventor of the present invention performs a detection experiment on the obtained antibody or antigen binding fragment thereof binding to human MUC17, and the experimental result shows that the anti-MUC 17 antibody can well bind to target cells and has higher affinity.
Pharmaceutical composition
In another aspect of the invention there is provided a composition comprising an antibody or antigen-binding fragment thereof as described above that binds human MUC17, and one or more pharmaceutically acceptable carriers, diluents or excipients. Preferably, the composition is a pharmaceutical composition.
The anti-MUC 17 antibody or antigen binding fragment thereof provided by the invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert curative effect more stably, and the preparations can ensure the structural integrity of the amino acid core sequence of the antibody, and simultaneously protect the multifunctional group of the protein from degradation (including but not limited to aggregation, deamination or oxidation). Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 4 to 8, preferably about 5 to 8, and preferably about 5 to 7 or 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavity injection. In general, for liquid formulations, it is generally possible to preserve at least one year at 2-8 ℃ and for lyophilized formulations, it is stable for at least six months at 30 ℃. The antibody preparation can be a suspension, a water injection, a freeze-drying preparation and the like which are commonly used in the pharmaceutical field.
In the present invention, the term "pharmaceutical composition" refers to a pharmaceutical formulation composition comprising the antibody or antigen-binding fragment thereof that binds to human MUC17 of the present invention, together with a pharmaceutically acceptable carrier, to provide more stable therapeutic effects, which formulation can ensure the conformational integrity of the amino acid core sequence of the disclosed antibody, while also protecting the multifunctional groups of the protein from degradation (including, but not limited to, aggregation, deamination or oxidation).
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the antibodies of the invention may also be used with other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the antibody or immunoconjugate thereof is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Application of
In a further aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof as described above that binds human MUC17, or a pharmaceutical composition as described above, in the manufacture of a medicament for the prevention, diagnosis, or treatment of cancer or tumour.
The drug for treating cancer or tumor according to the present invention is a drug for inhibiting and/or treating tumor, and may include a delay in the development of symptoms associated with tumor and/or a reduction in the severity of these symptoms, further includes a reduction in the symptoms associated with existing tumor and prevention of occurrence of other symptoms, and further includes a reduction or prevention of metastasis of tumor, etc.
Tumors to which the medicaments of the present invention are directed preferably include, but are not limited to: pancreatic cancer, colorectal cancer, colon cancer, colorectal cancer, small intestine cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, gastroesophageal cancer, or a combination thereof.
When the anti-MUC 17 antibody and the composition thereof of the present invention are administered to animals including humans, the administration amount varies depending on the age and weight of the patient, the disease characteristics and severity, and the administration route, and the results and various conditions of animal experiments may be referred to, and the total administration amount may not exceed a certain range. The anti-MUC 17 antibodies and compositions thereof of the present invention may also be administered in combination with other anti-neoplastic agents to achieve a more effective treatment of neoplasms.
The anti-MUC 17 antibodies, or antigen-binding fragments thereof, compositions or conjugates thereof, of the invention may also be used to detect the presence or level of MUC17 in a sample.
Advantageous effects
The invention provides novel antibodies and antigen binding fragments thereof that bind human MUC17, which have high affinity and specificity for human MUC17, and are expected to be better potential therapeutic agents for patients with MUC17 expression abnormality.
The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which the detailed conditions are not noted in the following examples, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
The experimental materials and sources used in the following examples and the method of preparing the reagents are specifically described below.
Experimental reagents and materials:
mouse myeloma cells SP2/0: purchased from ATCC under accession number CRL-1581;
balb/c mice: purchased from Shanghai Ling Biotechnology Co., ltd;
LS174T cells: ATCC, cat# CL-188 TM ；
Goat anti-mouse secondary antibody: purchased from boolon immunotechnology limited under the trade designation BF03001-1ML;
donkey anti-mouse PE fluorescent secondary antibody: purchased from Jackson under the accession number 715-116-150;
sheep anti-human PE fluorescent secondary antibody: purchased from Jackson under the trade designation 109-115-098;
96-well elisa plate: purchased from costar, cat No. 9018;
HRP-labeled goat anti-human Fc antibody: purchased from sigma, cat No. a0170;
PBS buffer: shanghai source culture Biotechnology Co., ltd., product number B320KJ;
bovine Serum Albumin (BSA): purchased from the division of bioengineering (Shanghai) under the designation A600332-0100;
PBST：PBS+0.05％Tween 20；
TMB: purchased from BD company, cat number 555214;
RPMI 1640Medium: purchased from Gibco company under the accession number 61870127;
FBS: purchased from Gibco under the accession number 10099;
penicillin-streptomycin (Penicillin-streptomycin): purchased from Gibco company under the accession number 15140122;
fetal bovine serum: purchased from Gibco company under the accession number 10091-148;
polyethylene glycol solution (PEG): purchased from sigma company under the product number P7181;
hybrid-SFM: purchased from life technologies, cat No. 12045-076;
HAT: purchased from Gibco, cat No. 21060017;
the experimental apparatus used in the following examples is illustrated as follows:
PCR instrument: purchased from BioRad, cat# C1000 Touch Thermal Cycler;
beckman Coulter CytoFLEX flow cytometer: purchased from Beckman corporation;
SpectraMax M5 microplate reader: purchased from Molecular Devices company.
Example 1 preparation and selection of antigen immunized animals and hybridoma cells
1. Antigen immunized mice
A human MUC17-His protein expressed by a mammalian cell 293E (amino acid aa4131-4390 segment sequence is taken according to a sequence Q685J3 provided by UniProtKB, a signal peptide sequence is added at the N end of the sequence, a His tag is added at the C end of the sequence, the sequence is respectively constructed into an expression vector through two enzyme cutting sites of EcoRI and HindIII, HEK-293E cells are transfected for expression and purification, and the purity after expression and purification is more than 95%) is obtained by conventional immunization of Balb/C mice. On day 1, after emulsification of soluble human MUC17-His protein with Freund's complete adjuvant, balb/c mice were injected subcutaneously at multiple points (human MUC17-His 100. Mu.g/mouse/0.5 mL); on day 14, after emulsification of soluble human MUC17-His protein with Freund's incomplete adjuvant, balb/c mice were subcutaneously injected (human MUC17-His 50. Mu.g/mouse/0.5 mL); on day 28, after emulsification of soluble human MUC17-His protein with Freund's incomplete adjuvant, balb/c mice were subcutaneously injected (human MUC17-His 50. Mu.g/mouse/0.5 mL); three weeks later, the soluble human MUC17-His protein, 50. Mu.g/mouse/0.2 mL, was challenged by intraperitoneal injection, and 3-4 days later, the mouse spleen was taken for fusion experiments.
2. Preparation and selection of hybridoma cells
The spleen cells of the mice were PEG-fused with myeloma cells SP2/0 of the mice 3-4 days after the last immunization of the mice using conventional hybridoma protocols. The fused cells were suspended uniformly in a complete medium composed of RPMI1640-GLUMAX supplemented with 1% Penicillin-streptomycin,20% FBS (fetal bovine serum) and 1 XHAT. The fused cells were cultured at a rate of 3X 10 4 Cells/200. Mu.L/well were plated in 60 96 well plates. After 7-12 days, supernatants were harvested and hybridoma wells positive for human MUC17 binding activity were screened by indirect enzyme-linked immunosorbent assay (ELISA).
Wherein, the ELISA method is used for screening the hybridoma holes positive for the binding activity of the human MUC17, and the method is as follows: MUC17-Fc (amino acids aa4131-4390 were sequenced according to sequence Q685J3 provided by UniProtKB, the N-terminus was added with a signal peptide sequence, the C-terminus was added with an Fc tag, and the fragment was passed through EcoRI and HiThe ndIII two cleavage sites are respectively constructed into an expression vector, transfected HEK-293E cells are expressed and purified), diluted to 1 mug/mL with PBS buffer, 100 mug/well added into a plate and cultured overnight at 4 ℃. The supernatant was thrown off the next day, 5% skim milk powder was added and blocked at 37℃for 2 hours, and the plates were washed 3 times with PBST for use. The collected hybridoma supernatants were sequentially added to the blocked plates at 100. Mu.L/well and left at 37℃for 1 hour. PBST washing the plate for 3 times, adding HRP-marked goat anti-mouse IgG secondary antibody, and standing at 37 ℃ for 30 minutes; after PBST washing the plate for 3 times, the residual liquid drops are beaten as much as possible on the absorbent paper, 100 mu L of TMB is added into each hole, and the plate is placed for 5 minutes at room temperature (20+/-5 ℃) in a dark place; 50 mu L of 2M H are added to each well 2 SO 4 The stopping solution stops the substrate reaction, the OD value is read at 450nm of the enzyme label instrument, and the binding capacity of the antibody to be detected and the target antigen MUC17 is analyzed. A total of 12 hybridoma cell lines were obtained by screening. Amplifying and screening the obtained 12 Hybridoma cell strains in a serum-containing complete culture medium, centrifuging and changing the solution to a serum-free culture medium hybrid oma-SFM culture medium to ensure that the cell density is 1-2 multiplied by 10 7 Per mL, at 8% CO 2 Culturing at 37deg.C for 1 week, centrifuging to obtain culture supernatant, and purifying by Protein G affinity chromatography to obtain antihuman MUC17 monoclonal antibody proteins respectively named 8H2, 19E11, 23F6, 27A3, 42H4, 44H4, 11C8, 15D10, 45D1, 50A5, 51E12 and 56E8.
Example 2 binding ability of murine antibody to human MUC17-Fc protein
ELISA the binding capacity of murine antibodies to human MUC17-Fc protein was determined. The specific method comprises the following steps:
MUC17-Fc protein was diluted with coating solution (50 mM carbonate coating buffer, pH 9.6) to 1. Mu.g/mL coating plate 4℃overnight; sealing with 5% skimmed milk powder, and incubating at 37deg.C for 2 hr; after washing the plates 3 times with PBST, the anti-human MUC17 antibody protein prepared in example 1 was diluted 3-fold from 1. Mu.g/mL with 1% BSA buffer for 11 gradients, 100. Mu.L/well was added to the pre-coated MUC17-Fc plates and incubated at 37℃for 1 hour; PBST washing the plate for 3 times, adding HRP-marked goat anti-mouse IgG secondary antibody, and standing at 37 ℃ for 30 minutes; after washing the plate 3 times with PBST, the residual droplets were removed as much as possible on absorbent paper, 100. Mu.L TMB was added to each well, and the plate was left at room temperature (20.+ -. 5 ℃ C.) in the absence of light for 5 minutes, and 50. Mu.L 2M H was added to each well 2 SO 4 The stopping solution stops the substrate reaction, the OD value is read at 450nm of the enzyme label instrument, and the binding capacity of the antibody to be detected and the target antigen human MUC17-Fc is analyzed. GraphPad Prism9 data analysis, mapping and calculation of EC 50 . The results are shown in FIG. 1. The data are shown in Table 1. The strongest affinity was clone No. 27 A3; of these, 42H4, 11C8 have poor affinity.
TABLE 1 ELISA detection of binding of hybridoma antibodies to human MUC17 antigen
Sample 42H4 27A3 8H2 56E8 50A5 11C8
Top 2.605 2.419 2.737 1.97 2.644 2.253
HillSlope 0.9669 1.281 0.8767 1.143 0.9144 0.748
EC 50 (ng/mL) 237 20.2 55.56 27.57 81.34 108
Sample 23F6 44H4 15D10 51E12 45D1 19E11
Top 2.883 2.696 2.787 2.883 2.865 2.528
HillSlope 0.957 1.081 1.369 1.212 1.063 1.181
EC 50 (ng/mL) 62.33 67.69 31.71 37.15 35.96 37.55
Example 3 binding ability of murine antibodies to colorectal cancer cell LS174T
The binding activity of murine antibodies to colorectal cancer LS174T cells was examined by flow cytometry (FACS) as follows:
LS174T cells are collected, cell culture solution is removed through centrifugation, and the cells are washed with PBS buffer solution for 2 times; count and dilute to 2X 10 with 1% BSA FACS buffer 6 Spreading the cells to a 96-well round bottom plate for later use, wherein the cell is 100 mu L/well; the anti-human MUC17 antibody protein prepared in example 1 was added to the above cell circular bottom plate in a 3-fold dilution of 11 gradients of 1% BSA buffer from 1. Mu.g/mL, and incubated at 4℃for 1 hour; after centrifugation, the supernatant was discarded, washed 3 times with 1% BSA FACS buffer, 100. Mu.L of donkey anti-mouse PE or sheep anti-human PE fluorescent secondary antibody was added per well at a ratio of 1:500 (see fluorescent secondary antibody specification for details), and incubated at 4℃for 1 hour; washing 3 times with 1% BSA FACS buffer, re-suspending with 1% BSA FACS buffer, 200. Mu.L/well, and using BD FACSCelesta TM To determine the sample to be analyzed. The resulting data were analyzed by fit using GraphPad Prism9, plotted and EC calculated 50 . The results of binding of the antibodies to the cells are shown in FIG. 2 and the data are shown in Table 2.
As can be seen from Table 2, the hybridoma antibody 27A3 had the strongest affinity, and 19E11, 51E12, respectively.
TABLE 2 FACS detection of hybridoma antibody binding to cell LS174T
Sample 42H4 27A3 8H2 56E8 50A5 11C8
Top 725.8 734.9 444.6 296.2 500.4 468.9
HillSlope 1.335 1.422 1.276 1.448 1.166 0.9413
EC 50 (ng/mL) 468.3 6.562 164.3 110.3 541.6 869.6
Sample 23F6 44H4 15D10 51E12 45D1 19E11
Top 459.5 574 382.1 878.4 456.7 560.8
HillSlope 1.219 1.407 0.959 1.279 1.079 1.457
EC 50 (ng/mL) 395.4 48.8 3426 47.21 273.2 23.98
EXAMPLE 4 preparation and Activity characterization of chimeric antibodies against human MUC17
1. Chimeric antibody production
Antibody genes of 12 hybridoma cells prepared in example 1 were obtained.
The present example obtained the heavy chain variable region and the light chain variable region of the hybridoma by a related method of molecular biology, and further constructed a chimeric antibody.
RNA from hybridoma cells was extracted by Trizol and mRNA was reverse transcribed to obtain cDNA, which was then used as a template to carry out PCR with heavy and light chain degenerate primers of murine antibodies (Antibody Engineering, volume 1,Edited by Roland Kontermann and Stefan D ubel, combined primer sequences from page 323), the PCR products obtained were sequenced and analyzed by Kabat database to determine the variable region sequence of murine antibodies. The sequence information is shown in Table 6.
The obtained heavy chain variable region sequence is spliced with a human IgG1 constant region (the amino acid sequence is shown as SEQ ID NO: 97), the light chain variable region sequence is spliced with a human kappa chain constant region (the amino acid sequence is shown as SEQ ID NO: 98), a chimeric antibody heavy chain and light chain are respectively constructed to pTT5 expression vectors, HEK-293E cells are transfected, protein A is subjected to affinity purification to obtain the chimeric antibody, a 0.22 mu M filter membrane is subjected to sterile filtration and then placed at 4 ℃ for later use, and the chimeric antibody is respectively named as ch-8H2, ch-19E11, ch-23F6, ch-27A3, ch-42H4, ch-44H4, ch-11C8, ch-15D10, ch-45D1, ch-50A5, ch-51E12 and ch-56E8.
2. Determination of affinity of chimeric antibodies for the target antigen human MUC17
The affinity of the chimeric antibodies to human MUC17 was determined by ELISA (experimental procedure reference example 2, secondary antibody HRP-goat anti-human Fc). The resulting data were analyzed by fit using GraphPad Prism9, plotted and EC calculated 50 。
The results are shown in FIG. 3 and the data are shown in Table 3. The results indicate that the chimeric antibody has a comparable binding affinity to human MUC17 as the murine antibody. As shown by ELISA results, the chimeric antibody ch-56E8 has a low plateau, and the ch-42H4 does not detect the binding to the antigen MUC17 under the test conditions, but has a good clone affinity.
TABLE 3 ELISA detection of affinity of chimeric antibodies to MUC17
Sample ch-8H2 ch-19E11 ch-23F6 ch-27A3 ch-42H4 ch-44H4
Top 2.53 2.328 2.106 2.31 7.583 2.26
HillSlope 1.368 1.335 1.304 1.347 1.622 1.386
EC 50 (nM) 0.1164 0.1404 0.1203 0.07855 2646 0.1247
Sample ch-11C8 ch-15D10 ch-45D1 ch-50A5 ch-51E12 ch-56E8
Top 1.984 2.465 2.516 2.004 2.315 1.697
HillSlope 1.248 1.362 1.261 1.232 1.37 1.148
EC 50 (nM) 0.1553 0.1172 0.1233 0.1747 0.2178 0.1207
3. Determination of binding affinity of chimeric antibodies to colorectal cancer LS174T cells
The binding affinity of the chimeric antibodies to colorectal cancer LS174T cells was determined by FACS method (experimental method reference example 3). The resulting data were analyzed by fit using GraphPad Prism9, plotted and EC calculated 50 。
The results are shown in FIGS. 4A-4B. The data are shown in tables 4A-4B. As can be seen from the data, ch-27A3, ch-19E11 and ch-51E12 bind LS174T cells with a strong affinity.
TABLE 4A FACS detection of chimeric antibody binding to cell LS174T
Sample ch-8H2 ch-23F6 ch-27A3 ch-42H4
Top 7034 8046 7961 5975
HillSlope 0.9588 1.041 0.7482 0.8707
EC 50 (nM) 1.762 4.866 0.04347 10.32
TABLE 4B FACS detection of chimeric antibody binding to cell LS174T
Figure BDA0003402397930000181
Figure BDA0003402397930000191
Example 5 preparation of humanized antibodies against human MUC17
Murine antibody 27A3 and 19E11 with higher affinity were selected for antibody humanization.
The amino acid sequences of the light chain variable region and the heavy chain variable region were analyzed, and 3 Complementarity Determining Regions (CDRs) and 4 Framework Regions (FRs) of the murine antibodies 27A3 and 19E11, respectively, were determined according to the Kabat's rule. 27A3 has the heavy chain variable region sequence set forth in SEQ ID NO:25, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 26. 27 and 28; 27A3 has the light chain variable region sequence set forth in SEQ ID NO:29, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 30. 31 and 32. 19E11 is set forth in SEQ ID NO:9, HCDR1, HCDR2 and HCDR3 are each as set forth in SEQ ID NO: 10. 11 and 12; 19E11 is set forth in SEQ ID NO:13, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO: 14. 15 and 16.
The humanized template that best matches the 27A3 non-CDR regions was selected from the gemline database. The CDR region of 27A3 is then grafted onto a selected humanized template to replace the CDR region of the humanized template, and the embedded residues, residues that interact directly with the CDR region, and residues that have an important effect on the conformation of the VL and VH of 27A3 are back mutated based on the three-dimensional structure of the antibody to yield the heavy chain variable region (SEQ ID NO: 99) and the light chain variable region (SEQ ID NO: 100) of humanized 27A 3. The back mutated variable region sequence was recombined with human IgG1 (SEQ ID NO: 97) and Kappa chain constant region (SEQ ID NO: 98) to obtain the full length of the heavy chain (SEQ ID NO: 101) and the full length of the light chain (SEQ ID NO: 102) of the 27A3 humanized antibody (hu-27A 3). The heavy chain and light chain gene sequences of 27A3 are respectively linked to pTT5 expression vector, HEK-293E cells are transfected, and humanized antibodies are obtained by Protein A affinity purification. The 0.22 μm filter was sterile filtered and placed at 4℃for further use.
Similarly, the humanized template that best matches the 19E11 non-CDR regions was selected in the Germline database. The CDR region of 19E11 is then grafted onto the selected humanized template, replacing the CDR region of the humanized template, and back-mutating the embedded residues, residues that interact directly with the CDR region, and residues that have an important effect on the conformation of the VL and VH of 19E11 based on the three-dimensional structure of the antibody, to yield the heavy chain variable region (SEQ ID NO: 103) and the light chain variable region (SEQ ID NO: 104) of humanized 19E 11. The back mutated variable region sequence was recombined with human IgG1 (SEQ ID NO: 97) and Kappa chain constant region (SEQ ID NO: 98) to obtain the full length of the heavy chain (SEQ ID NO: 105) and the full length of the light chain (SEQ ID NO: 106) of the 19E11 humanized antibody (hu-19E 11). The heavy chain and light chain gene sequences of 19E11 are respectively linked to pTT5 expression vector, HEK-293E cells are transfected, and Protein A is subjected to affinity purification to obtain humanized antibodies. The 0.22 μm filter was sterile filtered and placed at 4℃for further use.
Example 6 affinity identification of humanized antibodies against human MUC17
ELISA detection of the affinity of humanized antibodies for the target antigen MUC17
The affinity of the humanized antibodies for MUC17 was determined by ELISA (experimental methods refer to example 2). The resulting data were analyzed by fit using GraphPad Prism9, plotted and EC calculated 50 。
The results are shown in FIG. 5 and Table 5. The results show that the humanized 27A3 and 19E11 well maintain the affinity of the original murine antibody.
TABLE 5 ELISA detection of affinity of hu-27A3 and hu-19E11 humanized antibodies to MUC17
Sample ch-27A3 hu-27A3 ch-19E11 hu-19E11
EC 50 (nM) 0.07855 0.07659 0.1404 0.1311
2. Determination of binding affinity of humanized antibodies to colorectal cancer cells LS174T
The binding affinity of the humanized antibodies to colorectal cancer cells LS174T was determined by FACS method (experimental method reference example 3). The resulting data were analyzed by fit using GraphPad Prism9, plotted and EC calculated 50 。
The results are shown in FIGS. 6A-6B. EC of hu-27A3 50 EC of 0.03nM, hu-19E11 50 0.085nM, and hu-27A3 has a higher affinity than hu-19E11. The results show that the humanized 27A3 and 19E11 well maintain the affinity of the original murine antibody.
In summary, the present invention fuses mouse spleen cells with myeloma cells by immunizing the mouse with MUC17 protein, and performs affinity screening of anti-MUC 17 hybridoma antibodies. The antibody gene sequence of 12 hybridomas is obtained by modulating and sequencing the genes of the hybridomas. Chimeric antibodies were constructed and affinity compared, and based on the affinity versus sequencing alignment, 27A3 and 19E11 were selected for humanization. The affinity detection result shows that the humanized 27A3 and 19E11 maintain the affinity of the original rat antibody well.
The high-affinity anti-MUC 17 humanized antibody obtained by the invention can be used for screening anti-tumor drugs.
TABLE 6 antibody sequence listing
Figure BDA0003402397930000201
Figure BDA0003402397930000211
Figure BDA0003402397930000221
Figure BDA0003402397930000231
Figure BDA0003402397930000241
Figure BDA0003402397930000251
Sequence listing
<110> Dansheng medicine technology (Shanghai) Co., ltd
<120> antibody binding to human MUC17, method for producing the same, and use thereof
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Glu Val Asn Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Asn Tyr
20 25 30
Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Asn Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ser Val Asn Trp Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ala
115
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<211> 10
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Gly Phe Ala Phe Ser Asn Tyr Asp Met Ser
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<210> 3
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Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Asn Val Lys
1 5 10 15
Gly
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<211> 7
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Val Asn Trp Gly Phe Pro Tyr
1 5
<210> 5
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Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ile Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Arg Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Ile Leu Leu Ile Tyr Trp Thr Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Gly Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
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Tyr Tyr Thr Ser Pro Trp Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile
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Lys
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<211> 17
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Lys Ser Ser Gln Ser Leu Leu Tyr Ser Arg Asn Gln Lys Asn Tyr Leu
1 5 10 15
Ala
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<211> 7
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<400> 7
Trp Thr Ser Thr Arg Glu Ser
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<210> 8
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Gln Gln Tyr Tyr Thr Ser Pro Trp Thr
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<210> 9
<211> 120
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<400> 9
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Ser Phe Thr Gly Tyr
20 25 30
Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Ile Ser Ser Ser Thr Ala His
65 70 75 80
Met Glu Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ser Val Thr Thr Val Gly Tyr Pro Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 10
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Gly Phe Ser Phe Thr Gly Tyr Asn Met Asn
1 5 10
<210> 11
<211> 17
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Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
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<211> 11
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<400> 12
Ser Val Thr Thr Val Gly Tyr Pro Met Asp Tyr
1 5 10
<210> 13
<211> 113
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Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly
1 5 10 15
Gln Lys Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Tyr Asn His Lys Asn Cys Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Ser Phe Ala Ser Ala Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys His Gln
85 90 95
His Tyr Thr Ser Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 14
<211> 17
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Lys Ser Ser Gln Ser Leu Leu Asn Ser Tyr Asn His Lys Asn Cys Leu
1 5 10 15
Ala
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<400> 15
Phe Ala Ser Ala Arg Glu Ser
1 5
<210> 16
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His Gln His Tyr Thr Ser Pro Leu Thr
1 5
<210> 17
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Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Gly Ile Trp Ala Gly Gly Asn Thr Gln Ser His Ser Ala Leu Met
50 55 60
Ser Arg Val Ser Ile Ser Lys Asp Ile Ser Lys Arg Gln Val Phe Leu
65 70 75 80
Lys Met Asp Arg Leu Gln Thr Asp Asp Ser Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Phe Tyr Gly Asn Phe Asp Val Trp Gly Ala Gly Thr Thr
100 105 110
Val Thr Val Ser Ser
115
<210> 18
<211> 10
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Gly Phe Ser Leu Thr Ala Tyr Gly Val His
1 5 10
<210> 19
<211> 16
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Gly Ile Trp Ala Gly Gly Asn Thr Gln Ser His Ser Ala Leu Met Ser
1 5 10 15
<210> 20
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Ala Leu Phe Tyr Gly Asn Phe Asp Val
1 5
<210> 21
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Asp Ile Val Met Thr Gln Ser Gln Arg Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Ser Val Asp Thr Ser
20 25 30
Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Glu Ala Leu Ile
35 40 45
Phe Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Asn Leu Glu Met Arg
100 105
<210> 22
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Lys Ala Ser Gln Ser Val Asp Thr Ser Val Ala
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Ser Ala Ser Tyr Arg Tyr Ser
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Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr
1 5
<210> 25
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Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ile Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ser His Pro Ser Asp Ser Glu Ser Arg Leu Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Pro Tyr Tyr Gly Ser Ser Ala Trp Phe Ala Asp Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ala
115 120
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Gly Tyr Ser Phe Thr Ile Tyr Trp Met His
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Met Ser His Pro Ser Asp Ser Glu Ser Arg Leu Asn Gln Lys Phe Lys
1 5 10 15
Asp
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Pro Tyr Tyr Gly Ser Ser Ala Trp Phe Ala Asp
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Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly
1 5 10 15
Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
85 90 95
His Tyr Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Arg
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Lys Ser Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu
1 5 10 15
Ala
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Phe Ala Ser Thr Arg Glu Ser
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<210> 32
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Gln Gln His Tyr Ser Phe Pro Leu Thr
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<210> 33
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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Ala Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Ser Asn
20 25 30
Tyr Ile Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Lys Pro Thr Asn Gly Val Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Gly Phe Ser Leu Leu Arg Leu Gln Gly Tyr Ala Met Asp Asp
100 105 110
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 34
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Gly Tyr Thr Phe Asn Ser Asn Tyr Ile Tyr
1 5 10
<210> 35
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Glu Ile Lys Pro Thr Asn Gly Val Thr Asn Phe Asn Glu Lys Phe Lys
1 5 10 15
Thr
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Gly Phe Ser Leu Leu Arg Leu Gln Gly Tyr Ala Met Asp Asp
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<210> 37
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Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Thr Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Ser Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Ser Ile Thr
65 70 75 80
Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Phe Cys Gln Gln Ser Asn
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Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
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Arg Ala Ser Glu Ser Val Glu Thr Tyr Gly Asn Ser Phe Met His
1 5 10 15
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Arg Ala Ser Ser Leu Glu Ser
1 5
<210> 40
<211> 9
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<400> 40
Gln Gln Ser Asn Glu Asp Pro Tyr Thr
1 5
<210> 41
<211> 121
<212> PRT
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<400> 41
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Phe Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Gly Pro Arg Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ala Ser Asn Ala Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Tyr Gly Thr Ser Tyr Pro Asn Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 42
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Gly Phe Asn Ile Lys Asp Thr Tyr Ile His
1 5 10
<210> 43
<211> 17
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<400> 43
Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Gly Pro Arg Phe Gln
1 5 10 15
Gly
<210> 44
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Asn Tyr Gly Thr Ser Tyr Pro Asn Ala Met Asp Tyr
1 5 10
<210> 45
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Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Thr Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Ser Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Thr
65 70 75 80
Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Phe Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 46
<211> 15
<212> PRT
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Arg Ala Ser Glu Ser Val Glu Thr Tyr Gly Asn Ser Phe Met His
1 5 10 15
<210> 47
<211> 7
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Arg Ala Ser Ser Leu Glu Ser
1 5
<210> 48
<211> 9
<212> PRT
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Gln Gln Ser Asn Glu Asp Pro Tyr Thr
1 5
<210> 49
<211> 117
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Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Gly Ile Trp Ala Gly Gly Ser Thr Gln Ser Asn Ser Ala Leu Met
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Arg Met Asn Ser Leu Gln Thr Asp Asp Ser Ala Met Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Phe Tyr Gly Asn Phe Asp Val Trp Gly Ala Gly Thr Thr
100 105 110
Val Thr Val Ser Ser
115
<210> 50
<211> 10
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<400> 50
Gly Phe Ser Leu Thr Ala Tyr Gly Val His
1 5 10
<210> 51
<211> 16
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<400> 51
Gly Ile Trp Ala Gly Gly Ser Thr Gln Ser Asn Ser Ala Leu Met Ser
1 5 10 15
<210> 52
<211> 9
<212> PRT
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Ala Leu Phe Tyr Gly Asn Phe Asp Val
1 5
<210> 53
<211> 107
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Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Ser Val Asp Thr Ser
20 25 30
Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Glu Ala Leu Ile
35 40 45
Phe Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys
100 105
<210> 54
<211> 11
<212> PRT
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Lys Ala Ser Gln Ser Val Asp Thr Ser Val Ala
1 5 10
<210> 55
<211> 7
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<400> 55
Ser Ala Ser Tyr Arg Tyr Ser
1 5
<210> 56
<211> 9
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<400> 56
Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr
1 5
<210> 57
<211> 120
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Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Asp Pro Ser Asp Ser Tyr Thr Arg Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met His Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Ser Gly Ile Gly Asn Ser Phe Ile Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 58
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 58
Gly Tyr Thr Phe Thr Thr Tyr Trp Met His
1 5 10
<210> 59
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 59
Val Ile Asp Pro Ser Asp Ser Tyr Thr Arg Tyr Asn Gln Lys Phe Lys
1 5 10 15
Asp
<210> 60
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 60
Ser Gly Ile Gly Asn Ser Phe Ile Met Asp Tyr
1 5 10
<210> 61
<211> 106
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 61
Gln Val Val Leu Thr Gln Ser Pro Ala Ile Met Pro Ala Phe Pro Gly
1 5 10 15
Glu Lys Val Thr Ile Ser Cys Ser Ala Ser Ser Ser Val Ile Tyr Met
20 25 30
Phe Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Thr Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Tyr Pro Leu Thr
85 90 95
Phe Gly Val Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 62
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 62
Ser Ala Ser Ser Ser Val Ile Tyr Met Phe
1 5 10
<210> 63
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 63
Arg Thr Ser Asn Leu Ala Ser
1 5
<210> 64
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 64
Gln Gln Tyr His Ser Tyr Pro Leu Thr
1 5
<210> 65
<211> 116
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 65
Glu Met Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Asp Met Ser Trp Ile Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Gln Ser Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ser Leu Asn Trp Gly Phe Asp His Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 66
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 66
Gly Phe Thr Phe Ser Thr Tyr Asp Met Ser
1 5 10
<210> 67
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 67
Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys
1 5 10 15
Gly
<210> 68
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 68
Leu Asn Trp Gly Phe Asp His
1 5
<210> 69
<211> 113
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 69
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Ala Arg Phe Ile Gly Asp Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
85 90 95
His Tyr Asn Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 70
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 70
Lys Ser Ser Gln Ser Leu Leu Asn Ser Asn Asn Gln Lys Asn Tyr Leu
1 5 10 15
Ala
<210> 71
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 71
Phe Ala Ser Thr Arg Glu Ser
1 5
<210> 72
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 72
Gln Gln His Tyr Asn Pro Pro Trp Thr
1 5
<210> 73
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 73
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Gly Ile Trp Ala Gly Gly Asn Thr Gln Ser His Ser Ala Leu Met
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Ile Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Arg Met Asn Ser Leu Gln Thr Asp Asp Ser Ala Met Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Phe Tyr Gly Asn Phe Asp Val Trp Gly Ala Gly Thr Thr
100 105 110
Val Thr Val Ser Ser
115
<210> 74
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 74
Gly Phe Ser Leu Thr Ala Tyr Gly Val His
1 5 10
<210> 75
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 75
Gly Ile Trp Ala Gly Gly Asn Thr Gln Ser His Ser Ala Leu Met Ser
1 5 10 15
<210> 76
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 76
Ala Leu Phe Tyr Gly Asn Phe Asp Val
1 5
<210> 77
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 77
Asp Ile Val Met Thr Gln Ser Gln Arg Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Ser Val Asp Thr Ser
20 25 30
Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Glu Ala Leu Ile
35 40 45
Phe Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys
100 105
<210> 78
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 78
Lys Ala Ser Gln Ser Val Asp Thr Ser Val Ala
1 5 10
<210> 79
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 79
Ser Ala Ser Tyr Arg Tyr Ser
1 5
<210> 80
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 80
Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr
1 5
<210> 81
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 81
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Val Ser Gly Asn Ala Phe Ser Leu Ser
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Phe
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Asn Tyr Tyr Gly Ser Ser Ser Trp Phe Val Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 82
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 82
Gly Asn Ala Phe Ser Leu Ser Trp Met Asn
1 5 10
<210> 83
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 83
Arg Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr Asn Gly Lys Phe Lys
1 5 10 15
Gly
<210> 84
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 84
Tyr Tyr Gly Ser Ser Ser Trp Phe Val Tyr
1 5 10
<210> 85
<211> 112
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 85
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Tyr Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 86
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 86
Arg Ser Ser Gln Ser Leu Val His Ser Tyr Gly Asn Thr Tyr Leu His
1 5 10 15
<210> 87
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 87
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 88
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 88
Ser Gln Ser Thr His Phe Pro Trp Thr
1 5
<210> 89
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 89
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr
20 25 30
Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Gly Ile Trp Ala Gly Gly Ser Thr Lys Ser Asn Ser Ala Leu Met
50 55 60
Ser Ser Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Ser Ala Met Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Phe Tyr Gly Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr
100 105 110
Val Thr Val Ser Ser
115
<210> 90
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 90
Gly Phe Ser Leu Thr Ala Tyr Gly Val His
1 5 10
<210> 91
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 91
Gly Ile Trp Ala Gly Gly Ser Thr Lys Ser Asn Ser Ala Leu Met Ser
1 5 10 15
<210> 92
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 92
Ala Leu Phe Tyr Gly Tyr Phe Asp Val
1 5
<210> 93
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 93
Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Ser Val Asp Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Glu Ala Leu Ile
35 40 45
Phe Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Val Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys
100 105
<210> 94
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 94
Lys Ala Ser Gln Ser Val Asp Thr Asn Val Ala
1 5 10
<210> 95
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 95
Ser Ala Ser Tyr Arg Tyr Ser
1 5
<210> 96
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 96
Gln Gln Tyr Asn Val Tyr Pro Tyr Thr
1 5
<210> 97
<211> 330
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 97
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> 98
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 98
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 99
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 99
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 Ser Phe Thr Ile Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ser His Pro Ser Asp Ser Glu Ser Arg Leu Asn Gln Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Pro Tyr Tyr Gly Ser Ser Ala Trp Phe Ala Asp Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 100
<211> 113
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 100
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 Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
His Tyr Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Arg
<210> 101
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 101
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 Ser Phe Thr Ile Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ser His Pro Ser Asp Ser Glu Ser Arg Leu Asn Gln Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Pro Tyr Tyr Gly Ser Ser Ala Trp Phe Ala Asp Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 102
<211> 220
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 102
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 Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
His Tyr Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Arg Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210> 103
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 103
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 Phe Ser Phe Thr Gly Tyr
20 25 30
Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Leu Thr Val Asp Ile Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ser Val Thr Thr Val Gly Tyr Pro Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 104
<211> 113
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 104
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 Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Tyr Asn His Lys Asn Cys Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Ser Phe Ala Ser Ala Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys His Gln
85 90 95
His Tyr Thr Ser Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 105
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 105
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 Phe Ser Phe Thr Gly Tyr
20 25 30
Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Leu Thr Val Asp Ile Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ser Val Thr Thr Val Gly Tyr Pro Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 106
<211> 220
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 106
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 Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Tyr Asn His Lys Asn Cys Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Ser Phe Ala Ser Ala Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys His Gln
85 90 95
His Tyr Thr Ser Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220

Claims (18)

1. An antibody or antigen-binding fragment thereof that binds human MUC17, comprising:
(a) Heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequence of said HCDR1 selected from the group consisting of SEQ ID NOs: 2. 10, 18, 26, 34, 42, 50, 58, 66, 74, 82 and 90, the amino acid sequence of said HCDR2 is selected from the group consisting of SEQ ID NO: 3. 11, 19, 27, 35, 43, 51, 59, 67, 75, 83 and 91, and the amino acid sequence of HCDR3 is selected from the group consisting of SEQ ID NO: 4. 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, and 92; and
(b) Light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, said LCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6. 14, 22, 30, 38, 46, 54, 62, 70, 78, 86 and 94, the amino acid sequence of LCDR2 is selected from the group consisting of SEQ ID NOs: 7. 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, and 95, and the amino acid sequence of LCDR3 is selected from the group consisting of SEQ ID NOs: 8. 16, 24, 32, 40, 48, 56, 64, 72, 80, 88 and 96.
2. The antibody or antigen-binding fragment thereof that binds to human MUC17 as claimed in claim 1,
(1) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 2. 3 and 4, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively as set forth in SEQ ID NOs: 6. 7 and 8;
(2) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 10. 11 and 12, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 14. 15 and 16;
(3) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 18. 19 and 20, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 22. 23 and 24;
(4) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 26. 27 and 28, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 30. 31 and 32;
(5) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 34. 35 and 36, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 38. 39 and 40;
(6) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 42. 43 and 44, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively set forth in SEQ ID NOs: 46. 47 and 48;
(7) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 50. 51 and 52, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 54. 55 and 56;
(8) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 58. 59 and 60, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 62. 63 and 64;
(9) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 66. 67 and 68, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively set forth in SEQ ID NOs: 70. 71 and 72;
(10) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 74. 75 and 76, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 78. 79 and 80;
(11) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 82. 83 and 84, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 86. 87 and 88; or (b)
(12) The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown in SEQ ID NO: 90. 91 and 92, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NOs: 94. 95 and 96.
3. The antibody or antigen-binding fragment thereof that binds to human MUC17 of claim 1 or 2, wherein the antibody is a monoclonal antibody or a polyclonal antibody; preferably, the antibody is a monoclonal antibody.
4. The antibody or antigen-binding fragment thereof that binds to human MUC17 of claim 1 or 2, wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.
5. The antibody or antigen-binding fragment thereof that binds human MUC17 of claim 1 or 2, wherein the antigen-binding fragment comprises a Fab fragment, a F (ab ') 2 fragment, a Fab' fragment, an Fv fragment, or a single chain Fv (scFv) fragment.
6. The antibody or antigen-binding fragment thereof that binds to human MUC17 as claimed in claim 1 or 2,
the antibody or antigen-binding fragment thereof that binds human MUC17 comprises a heavy chain variable region (VH) and a light chain variable region (VL),
(1) The amino acid sequence of VH is selected from the group consisting of SEQ ID NO: 1. 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 99 and 103; and
(2) The amino acid sequence of VL is selected from the group consisting of SEQ ID NO: 5. 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 100 and 104.
7. The antibody or antigen-binding fragment thereof that binds to human MUC17 as claimed in claim 6,
(1) The amino acid sequence of the VH is shown in SEQ ID NO:1, wherein the amino acid sequence of the VL is shown in SEQ ID NO:5 is shown in the figure;
(2) The amino acid sequence of the VH is shown in SEQ ID NO:9, the amino acid sequence of the VL is shown in SEQ ID NO: 13;
(3) The amino acid sequence of the VH is shown in SEQ ID NO:17, the amino acid sequence of the VL is shown in SEQ ID NO: 21;
(4) The amino acid sequence of the VH is shown in SEQ ID NO:25, said VL comprises the amino acid sequence set forth in SEQ ID NO:29, and a polypeptide comprising the amino acid sequence shown in seq id no;
(5) The amino acid sequence of the VH is shown in SEQ ID NO:33, the amino acid sequence of the VL is shown in SEQ ID NO: shown at 37;
(6) The amino acid sequence of the VH is shown in SEQ ID NO:41, wherein the amino acid sequence of the VL is shown in SEQ ID NO: 45;
(7) The amino acid sequence of the VH is shown in SEQ ID NO:49, the amino acid sequence of the VL is set forth in SEQ ID NO: 53;
(8) The amino acid sequence of the VH is shown in SEQ ID NO:57, the amino acid sequence of the VL is shown in SEQ ID NO: indicated at 61;
(9) The amino acid sequence of the VH is shown in SEQ ID NO:65, the amino acid sequence of the VL is shown in SEQ ID NO: 69;
(10) The amino acid sequence of the VH is shown in SEQ ID NO:73, the amino acid sequence of the VL is shown in SEQ ID NO: indicated at 77;
(11) The amino acid sequence of the VH is shown in SEQ ID NO:81, the amino acid sequence of the VL is shown in SEQ ID NO: indicated at 85;
(12) The amino acid sequence of the VH is shown in SEQ ID NO:89, the amino acid sequence of the VL is shown in SEQ ID NO:93, shown in the figure; .
(13) The amino acid sequence of the VH is shown in SEQ ID NO:99, the amino acid sequence of the VL is shown in SEQ ID NO: 100; or (b)
(14) The amino acid sequence of the VH is shown in SEQ ID NO:103, the amino acid sequence of the VL is shown in SEQ ID NO: 104.
8. The antibody or antigen-binding fragment thereof that binds human MUC17 of claim 6 or 7, wherein the antibody or antigen-binding fragment thereof that binds human MUC17 comprises a heavy chain constant region selected from an IgG1, igG2, igG3, or IgG4 heavy chain constant region and a light chain constant region selected from a kappa or lambda light chain constant region; preferably, the heavy chain constant region is an IgG1 heavy chain constant region and the light chain constant region is a kappa light chain constant region; preferably, the amino acid sequence of the heavy chain constant region is as set forth in SEQ ID NO:97, the amino acid sequence of the light chain constant region is shown in SEQ ID NO: shown at 98.
9. The antibody or antigen-binding fragment thereof that binds to human MUC17 as claimed in claim 1,
the antibody or antigen binding fragment thereof that binds human MUC17 comprises a heavy chain and a light chain selected from the group consisting of:
(1) The amino acid sequence of the heavy chain is shown in SEQ ID NO:101, wherein the amino acid sequence of the light chain is shown in SEQ ID NO: 102; and
(2) The amino acid sequence of the heavy chain is shown in SEQ ID NO:105, the amino acid sequence of the light chain is shown in SEQ ID NO: shown at 106.
10. An isolated polynucleotide molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-9 that binds human MUC 17.
11. An expression vector comprising the polynucleotide molecule of claim 10.
12. A host cell comprising the expression vector of claim 11.
13. A method of preparing an antibody or antigen-binding fragment thereof that binds to human MUC17 according to any one of claims 1 to 9, comprising the steps of:
(a) Culturing the host cell of claim 12 under expression conditions so as to express an antibody or antigen-binding fragment thereof that binds human MUC 17;
(b) Isolating and purifying the antibody or antigen-binding fragment thereof that binds human MUC17 as described in step (a).
14. A pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-9 that binds human MUC 17; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.
15. The pharmaceutical composition of claim 14, further comprising one or more additional therapeutic agents.
16. Use of an antibody or antigen-binding fragment thereof that binds to human MUC17 according to any one of claims 1-9, or a pharmaceutical composition according to claim 14 or 15, in the manufacture of a medicament for cancer or tumour.
17. The use of claim 16, wherein the cancer or tumor is selected from the group consisting of: pancreatic cancer, colorectal cancer, colon cancer, colorectal cancer, small intestine cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, gastroesophageal cancer, or a combination thereof.
18. An immunoconjugate, the immunoconjugate comprising:
(a) The antibody or antigen-binding fragment thereof that binds to human MUC17 of any one of claims 1-9; and
(b) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
CN202111500224.5A 2021-12-09 2021-12-09 Antibody binding to human MUC17, preparation method and application thereof Pending CN116284408A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024188319A1 (en) * 2023-03-15 2024-09-19 三生国健药业（上海）股份有限公司 Anti-muc17, anti-cd3 and anti-cd28 trispecific antibody

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024188319A1 (en) * 2023-03-15 2024-09-19 三生国健药业（上海）股份有限公司 Anti-muc17, anti-cd3 and anti-cd28 trispecific antibody

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