CN115521378B - PD-L1 antibodies and uses thereof - Google Patents

PD-L1 antibodies and uses thereof Download PDF

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CN115521378B
CN115521378B CN202210630162.8A CN202210630162A CN115521378B CN 115521378 B CN115521378 B CN 115521378B CN 202210630162 A CN202210630162 A CN 202210630162A CN 115521378 B CN115521378 B CN 115521378B
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CN115521378A (en
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康平
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Nanjing Jisheng Aoma Biomedical Co ltd
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Nanjing Jisheng Aoma Biomedical Co ltd
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86

Abstract

The present application relates to a PD-L1 antibody and uses thereof, the PD-L1 antibody having a K of about 8E-09M or less D The value is specifically bound with human PD-L1 protein and can block the binding of PD-L1 and PD-1; the application also relates to engineering of the Fc region in the antibodies; the application also provides immunoconjugates comprising the PD-L1 antibodies, methods of making the PD-L1 antibodies, and uses of the PD-L1 antibodies.

Description

PD-L1 antibodies and uses thereof
Technical Field
The application relates to the field of biological medicine, in particular to a PD-L1 antibody and application thereof.
Background
Programmed death receptor ligand 1 (PD-L1), also known as cluster 274 (CD 274) or B7-homologous protein 1 (B7-H1), is a type I transmembrane protein of 40kDa, typically expressed on activated T cells, B cells, monocytes, dendritic cells, macrophages and many non-hematopoietic cells.
PD-L1 binds to programmed death receptor 1 (PD-1) and B7-1 (CD 80). Wherein the PD-L1/PD-1 signaling pathway is a very important co-suppression signaling pathway in immune response, down-regulates T cell immune response, suppresses T cell activity, and reduces cytokine secretion. The research shows that PD-L1 can be expressed in a plurality of tumor tissues, including gastric cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, colon cancer, mastocytic tumor, malignant melanoma and the like, and can be expressed in bone marrow cells infiltrating the tumor microenvironment to protect the tumor cells from immune attack.
Various mechanisms of immunosuppression may prevent immunotherapy from being effective. Therefore, there is a need to develop anti-PD-L1 antibodies with high affinity that have the ability to block PD-L1/PD-1 signaling pathways, and can bring new solutions for the treatment of tumors and various immune system related diseases.
Disclosure of Invention
The present application provides an isolated antigen binding protein having one or more of the following properties: 1) In Biacore detection, at a K of about 8E-09M or less D The value specifically binds to human PD-L1 protein; 2) Can block the binding of PD-1 to PD-L1.
In certain embodiments, the antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 18.
In certain embodiments, the antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 12.
In certain embodiments, the antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 3.
In certain embodiments, the antigen binding protein comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH depicted in SEQ ID NO. 67.
In certain embodiments, the antigen binding protein comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH set forth in any one of SEQ ID NOs 39 through 48.
In certain embodiments, the antigen binding protein comprises a heavy chain variable region VH comprising the HCDR1, HCDR2 and HCDR3, the HCDR3 comprising the amino acid sequence of SEQ ID NO: 18; the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 12; and the HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 3.
In certain embodiments, the antigen binding protein comprises H-FR1, the C-terminus of said H-FR1 is directly or indirectly linked to the N-terminus of said HCDR1, and said H-FR1 comprises the amino acid sequence shown in SEQ ID NO. 59.
In certain embodiments, the H-FR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs 1 to 2.
In certain embodiments, the antigen binding protein comprises H-FR2, said H-FR2 is located between said HCDR1 and said HCDR2, and said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 60.
In certain embodiments, the H-FR2 comprises an amino acid sequence set forth in any one of SEQ ID NO. 4 through SEQ ID NO. 11.
In certain embodiments, the antigen binding protein comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 61.
In certain embodiments, the H-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NO. 13 through SEQ ID NO. 17.
In certain embodiments, the antigen binding protein comprises H-FR4, the N-terminus of H-FR4 is directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO. 62.
In certain embodiments, the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO. 19 through SEQ ID NO. 20.
In certain embodiments, the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising the amino acid sequence shown in SEQ ID NO. 59; the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 60; the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 61; and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO. 62.
In certain embodiments, the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising the amino acid sequence shown in any one of SEQ ID NO. 1 to SEQ ID NO. 2; the H-FR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 4 to SEQ ID NO. 11; the H-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO. 13 to SEQ ID NO. 17; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO. 19 to SEQ ID NO. 20.
In certain embodiments, the H-FR1, H-FR2, H-FR3 and H-FR4 in said antigen binding protein comprises an amino acid sequence selected from any one of the following groups:
a) H-FR1: SEQ ID NO:1, H-FR2: SEQ ID NO. 4, H-FR3: SEQ ID NO. 13 and H-FR4: SEQ ID NO. 19;
b) H-FR1: SEQ ID NO. 2, H-FR2: 5,H-FR3 of SEQ ID NO: SEQ ID NO. 14 and H-FR4: SEQ ID NO. 20;
c) H-FR1: SEQ ID NO. 2, H-FR2: 6,H-FR3 of SEQ ID NO: SEQ ID NO. 15 and H-FR4: SEQ ID NO. 20;
d) H-FR1: SEQ ID NO. 2, H-FR2: 7,H-FR3 of SEQ ID NO: SEQ ID NO. 16 and H-FR4: SEQ ID NO. 20;
e) H-FR1: SEQ ID NO. 2, H-FR2: 8,H-FR3 of SEQ ID NO: SEQ ID NO. 17 and H-FR4: SEQ ID NO. 20;
f) H-FR1: SEQ ID NO. 2, H-FR2: 9,H-FR3 of SEQ ID NO: SEQ ID NO. 14 and H-FR4: SEQ ID NO. 20;
g) H-FR1: SEQ ID NO. 2, H-FR2: 7,H-FR3 of SEQ ID NO: SEQ ID NO. 14 and H-FR4: SEQ ID NO. 20;
h) H-FR1: SEQ ID NO. 2, H-FR2: 6,H-FR3 of SEQ ID NO: SEQ ID NO. 16 and H-FR4: SEQ ID NO. 20;
i) H-FR1: SEQ ID NO. 2, H-FR2: SEQ ID NO. 10, H-FR3: SEQ ID NO. 14 and H-FR4: SEQ ID NO. 20; and
j) H-FR1: SEQ ID NO. 2, H-FR2: SEQ ID NO. 11, H-FR3: SEQ ID NO. 15 and H-FR4: SEQ ID NO. 20.
In certain embodiments, the antigen binding protein comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID No. 67.
In certain embodiments, the VH in the antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO 39 to SEQ ID NO 48.
In certain embodiments, the antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence depicted in SEQ ID NO. 36.
In certain embodiments, the antigen binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence depicted in SEQ ID NO. 29.
In certain embodiments, the antigen binding protein comprises LCDR1, said LCDR1 comprising the amino acid sequence depicted in SEQ ID NO. 24.
In certain embodiments, the isolated antigen binding protein comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL as set forth in any one of SEQ ID NOs 68.
In certain embodiments, the isolated antigen binding protein comprises LCDR1, LCDR2, and LCDR3 of the light chain variable region VL set forth in any one of SEQ ID NOS: 49 to 55.
In certain embodiments, the antigen binding protein comprises a light chain variable region VL comprising the LCDR1, LCDR2, and LCDR3, the LCDR3 comprising the amino acid sequence shown in SEQ ID No. 36; the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 29; and the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO. 24.
In certain embodiments, the antigen binding protein comprises L-FR1, the C-terminus of said L-FR1 is directly or indirectly linked to the N-terminus of said LCDR1, and said L-FR1 comprises the amino acid sequence shown in SEQ ID NO. 63.
In certain embodiments, the L-FR1 comprises an amino acid sequence set forth in any one of SEQ ID NO. 21 to SEQ ID NO. 23.
In certain embodiments, the antigen binding protein comprises L-FR2, said L-FR2 is located between said LCDR1 and said LCDR2, and said L-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 64.
In certain embodiments, the L-FR2 comprises an amino acid sequence set forth in any one of SEQ ID NO. 25 through SEQ ID NO. 28.
In certain embodiments, the antigen binding protein comprises L-FR3, said L-FR3 is located between said LCDR2 and said LCDR3, and said L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 65.
In certain embodiments, the L-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 30 to 35.
In certain embodiments, the antigen binding protein comprises L-FR4, the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO. 66.
In certain embodiments, the L-FR4 comprises an amino acid sequence set forth in any one of SEQ ID NOs 37 to 38.
In certain embodiments, the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR1 comprising the amino acid sequence shown in SEQ ID NO. 63; the L-FR2 comprises an amino acid sequence shown as SEQ ID NO. 64; the L-FR3 comprises an amino acid sequence shown as SEQ ID NO. 65; and the L-FR4 comprises the amino acid sequence shown as SEQ ID NO. 66.
In certain embodiments, the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR1 comprising the amino acid sequence shown in any one of SEQ ID NO. 21 to SEQ ID NO. 23; the L-FR2 comprises an amino acid sequence shown in any one of SEQ ID NO. 25 to SEQ ID NO. 28; the L-FR3 comprises an amino acid sequence shown in any one of SEQ ID NO. 30 to SEQ ID NO. 35; and the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:37 to SEQ ID NO: 38.
In certain embodiments, the L-FR1, L-FR2, L-FR3 and L-FR4 comprises an amino acid sequence selected from any one of the following groups:
a) L-FR1: SEQ ID NO. 21, L-FR2: SEQ ID NO. 25, L-FR3: SEQ ID NO. 30 and L-FR4: SEQ ID NO. 37;
b) L-FR1: SEQ ID NO. 22, L-FR2: SEQ ID NO. 26, L-FR3: SEQ ID NO. 31 and L-FR4: SEQ ID NO. 38;
c) L-FR1: SEQ ID NO. 23, L-FR2: SEQ ID NO. 27, L-FR3: SEQ ID NO. 32 and L-FR4: SEQ ID NO. 38;
d) L-FR1: SEQ ID NO. 22, L-FR2: SEQ ID NO. 27, L-FR3: SEQ ID NO. 32 and L-FR4: SEQ ID NO. 38;
e) L-FR1: SEQ ID NO. 22, L-FR2: SEQ ID NO. 27, L-FR3: SEQ ID NO. 34 and L-FR4: SEQ ID NO. 38;
f) L-FR1: SEQ ID NO. 23, L-FR2: SEQ ID NO. 27, L-FR3: SEQ ID NO. 33 and L-FR4: SEQ ID NO. 38; and
g) L-FR1: SEQ ID NO. 22, L-FR2: SEQ ID NO. 28, L-FR3: SEQ ID NO. 35 and L-FR4: SEQ ID NO. 38.
In certain embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO. 68.
In certain embodiments, the VL comprises an amino acid sequence set forth in any one of SEQ ID NOs 49 to 55.
In certain embodiments, the VH and VL in the antigen binding protein comprise amino acid sequences selected from any one of the following:
a) VH: SEQ ID NO 39 and VL: SEQ ID NO. 49;
b) VH: SEQ ID NO. 40 and VL: SEQ ID NO. 50;
c) VH: SEQ ID NO. 41 and VL: SEQ ID NO. 51;
d) VH: SEQ ID NO. 42 and VL: SEQ ID NO. 51;
e) VH: SEQ ID NO. 43 and VL: SEQ ID NO. 52;
f) VH: SEQ ID NO. 44 and VL: SEQ ID NO. 51;
g) VH: SEQ ID NO 45 and VL: SEQ ID NO. 53;
h) VH: SEQ ID NO 46 and VL: SEQ ID NO. 54;
i) VH: SEQ ID NO. 47 and VL: SEQ ID NO. 52; and
j) VH: SEQ ID NO 48 and VL: SEQ ID NO. 55.
In certain embodiments, the antigen binding protein has an extended half-life compared to a wild-type antibody.
In certain embodiments, the antigen binding protein is capable of reducing Antibody Dependent Cellular Cytotoxicity (ADCC) as compared to a wild-type antibody.
In certain embodiments, the antigen binding protein comprises a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region or an IgY-derived constant region.
In certain embodiments, the heavy chain constant region in the isolated antibody binding protein comprises a constant region derived from IgG.
In certain embodiments, the heavy chain constant region comprises a constant region derived from IgG1, igG2, igG3, or IgG 4.
In certain embodiments, the heavy chain constant region comprises an Fc region derived from IgG 1.
In certain embodiments, the heavy chain constant region is mutated at position N297, residues numbered according to the Kabat system.
In certain embodiments, the heavy chain constant region is mutated at position M252, residues numbered according to the Kabat system.
In certain embodiments, the heavy chain constant region is mutated at position S254, residues numbered according to the Kabat system.
In certain embodiments, the heavy chain constant region is mutated at position T256, residues numbered according to the Kabat system.
In certain embodiments, the Fc region in the heavy chain constant region comprises an N297A mutation.
In certain embodiments, the Fc region in the heavy chain constant region comprises an M252Y mutation.
In certain embodiments, the Fc region in the heavy chain constant region comprises the S254T mutation.
In certain embodiments, the Fc region in the heavy chain constant region comprises a T256E mutation.
In certain embodiments, the heavy chain constant region comprises the amino acid sequence set forth in any one of SEQ ID NOS: 56 to 57.
In certain embodiments, the antigen binding protein comprises a light chain constant region, and the light chain constant region comprises an igκ -derived constant region or an igλ -derived constant region.
In certain embodiments, the light chain constant region comprises a constant region derived from human igκ.
In certain embodiments, wherein the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 58.
In certain embodiments, the antigen binding protein comprises a heavy chain HC comprising the amino acid sequence of SEQ ID NO: 69-78.
In certain embodiments, the antigen binding protein comprises a light chain LC comprising the amino acid sequence of SEQ ID NO: 79-85.
In certain embodiments, the antigen binding protein comprises an antibody or antigen binding fragment thereof.
In certain embodiments, the antigen binding fragment is selected from the group consisting of: fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, di-scFv, VHH and/or dAb.
In certain embodiments, the antibody is selected from the group consisting of: monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, multispecific antibodies, bispecific antibodies, and fully human antibodies.
In another aspect, the present application provides one or more polypeptides comprising the isolated antigen binding protein.
In another aspect, the present application provides one or more immunoconjugates comprising said isolated antigen binding protein or said polypeptide.
In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the isolated antigen binding protein, or the polypeptide.
In another aspect, the present application provides one or more vectors comprising the isolated nucleic acid molecules.
In another aspect, the present application provides one or more cells comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule and/or the vector.
In another aspect, the present application provides a method of producing said isolated antigen binding protein or said polypeptide, said method comprising culturing said cell under conditions such that said isolated antigen binding protein or said polypeptide is expressed.
In another aspect, the present application provides one or more pharmaceutical compositions comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and/or a pharmaceutically acceptable adjuvant and/or excipient.
In another aspect, the present application provides a method for detecting or assaying PD-L1, comprising using the isolated antigen binding protein or the polypeptide.
In another aspect, the present application provides a detection kit for PD-L1, which comprises the isolated antigen binding protein or the polypeptide.
In another aspect, the present application provides the use of the isolated antigen binding protein or the polypeptide in the preparation of a kit for detecting the presence and/or amount of PD-L1.
In another aspect, the present application provides a method of inhibiting the interaction of PD-1 with PD-L1, comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and/or the cell.
In another aspect, the present application provides the use of said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said vector, said cell and/or said pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of a disease or disorder.
In certain embodiments, the disease or disorder comprises a tumor.
In certain embodiments, the tumor comprises a solid tumor.
In certain embodiments, the tumor comprises a non-solid tumor.
In certain embodiments, the tumor comprises breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and/or liver cancer.
In another aspect, the present application provides the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition for use in preventing, alleviating and/or treating a disease or disorder.
In certain embodiments, the disease or disorder comprises a tumor.
In certain embodiments, the tumor comprises a solid tumor.
In certain embodiments, the tumor comprises a non-solid tumor.
In certain embodiments, the tumor comprises breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and/or liver cancer.
In another aspect, the present application provides a method of preventing and/or treating a disease or disorder comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and/or the cell.
In certain embodiments, the disease or disorder comprises a tumor.
In certain embodiments, the tumor comprises a solid tumor.
In certain embodiments, the tumor comprises a non-solid tumor.
In certain embodiments, the tumor comprises breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and/or liver cancer.
Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the invention as described herein. Accordingly, the drawings and descriptions herein are to be regarded as illustrative in nature and not as restrictive.
Drawings
The specific features of the invention related to this application are set forth in the appended claims. The features and advantages of the invention that are related to the present application will be better understood by reference to the exemplary embodiments and the drawings that are described in detail below. The drawings are briefly described as follows:
FIG. 1 shows ELISA detection results of the antigen binding proteins described herein inhibiting the binding of PD-L1 to PD-1.
FIG. 2 shows ELISA detection results of inhibition of binding of PD-L1 to PD-1 by the Fc engineered antigen binding proteins described herein.
FIG. 3 shows the results of functional cell experiments with Fc engineered antigen binding proteins described herein.
Detailed Description
Further advantages and effects of the invention of the present application will become apparent to those skilled in the art from the disclosure of the present application, from the following description of specific embodiments.
Definition of terms
In this application, the term "isolated" generally refers to those obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide that has been isolated from the natural state and is of high purity is said to be isolated. The term "isolated" does not exclude the incorporation of artificial or synthetic substances, nor the presence of other impure substances that do not affect the activity of the substance.
In the present application, the term "antigen binding protein" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen. For example, in the present application, the term "antigen binding protein" may include "antibodies" or "antigen binding fragments". For example, the antibody may comprise an immunoglobulin of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and may comprise any molecule comprising an antigen binding portion thereof. The term "antibody" may include monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to murine antibodies, human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single chain antibodies (e.g., scFv), and antibody fragments that bind to an antigen (e.g., fab', VHH, and (Fab) 2 fragments). The term "antibody" may also include all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies, as well as any antigen-binding antibody fragment described herein and derivatives thereof. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region. VH and VL regions can be further distinguished as hypervariable regions called Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved, called Framework Regions (FR). Each VH and VL may be composed of three CDRs and four FR regions, which may be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen (e.g., human PD-L1). The constant region of an antibody may mediate binding of the immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). The exact boundaries of the CDRs have been defined differently from system to system. The system described by Kabat (Kabat et al Sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987) and (1991)) found that, despite the large diversity at the amino acid sequence level, certain subfractions within the Kabat CDRs take the almost identical peptide backbone conformation, these subfractions being designated L1, L2 and L3 or H1, H2 and H3, respectively, wherein "L" and "H" refer to the light and heavy chain regions, respectively, that may be designated as Chothia CDRs, which have the strict overlapping or overlapping properties with the boundaries of the Kabat (Kabat) 262 (1995) or with the amino acid sequence of the set of CDRs that may be defined by the specific group of Kabat CDRs, which may overlap with the boundaries of the specific CDRs (60) or by the specific group of Kabat (1995) and by the additional amino acid sequence of the amino acid sequence of CDRs that may be defined by the amino acid sequence of the CDRs (National Institutes of Health, bethesda, md. (1987) and (1991)).
In this application, the term "antigen binding fragment" generally refers to one or more fragments of an antibody that function to specifically bind an antigen. The antigen binding function of an antibody may be achieved by a full-length fragment of the antibody. The antigen binding function of an antibody can also be achieved by: a heavy chain comprising a fragment of Fv, scFv, dsFv, fab, fab 'or F (ab') 2, or a light chain comprising a fragment of Fv, scFv, dsFv, fab, fab 'or F (ab') 2. (1) Fab fragments, typically monovalent fragments consisting of VL, VH, CL and CH domains; (2) A F (ab') 2 fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (3) an Fd fragment consisting of VH and CH domains; (4) Fv fragments consisting of the VL and VH domains of the antibody single arm; (5) dAb fragments consisting of VH domains (Ward et al, (1989) Nature 341:544-546); (6) The isolated Complementarity Determining Regions (CDRs) and (7) may optionally be a combination of two or more isolated CDRs joined by a linker. For example, monovalent single chain molecules Fv (scFv) formed by the pairing of VL and VH (see Bird et al (1988) Science 242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci.85:5879-5883) may also be included. For example, a class of antibodies VHH that lacks the antibody light chain but only the heavy chain variable region may also be included (see, e.g., kang Xiaozhen et al, bioengineering journal 2018,34 (12): 1974-1984). The "antigen binding portion" may also include an immunoglobulin fusion protein comprising a binding domain selected from the group consisting of: (1) A binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) An immunoglobulin heavy chain CH2 constant region fused to a hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to a CH2 constant region.
In the present application, the term "monoclonal antibody" generally refers to a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that occur in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. For example, the monoclonal antibodies can be prepared by hybridoma technology or produced in bacterial, eukaryotic, or plant cells by using recombinant DNA methods. Monoclonal antibodies can also be obtained from phage antibody libraries using techniques such as those described by Clackson et al, nature,352:624-628 (1991) and Marks et al, mol. Biol.,222:581-597 (1991).
In the present application, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy or light chain amino acid sequence is homologous to a corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class, while the remaining segments of the chain are homologous to corresponding sequences in another species. For example, the variable regions of both the light and heavy chains are derived from the variable regions of antibodies from one animal species (e.g., mouse, rat, etc.), while the constant portion is homologous to the antibody sequences from another species (e.g., human). For example, to obtain chimeric antibodies, non-human B cells or hybridoma cells can be used to produce variable regions, and the constant regions combined therewith are from humans. The variable region has the advantage of being easy to prepare and its specificity is not affected by the source of the constant region with which it is combined. Meanwhile, since the constant region of a chimeric antibody may be derived from human, the chimeric antibody may be less likely to elicit an immune response upon injection than an antibody using a constant region of non-human origin.
In the present application, the term "humanized antibody" generally refers to a chimeric antibody that contains less sequence from a non-human immunoglobulin, thereby reducing immunogenicity of a xenogeneic antibody when introduced into humans, while maintaining the full antigen binding affinity and specificity of the antibody. For example, CDR grafting (Jones et al, nature 321:522 (1986)) and variants thereof may be used; non-human binding domains are humanised by technical means including "remodeling" (reshaping), (Verhoeyen, et al, 1988Science 239:1534-1536;Riechmann,et al, 1988Nature 332:323-337;Tempest,et al), "high addition" (hyperchimerisation), "Queen, et al, 1989Proc Natl Acad Sci USA86:10029-10033; co, et al, 1991Proc Natl Acad Sci USA88:2869-2873; co, et al, 1992J Immunol 148:1149-1154) and" veneering "(Mark, et al," "Derivation of therapeutically active humanized and veneered anti-CD18 anti-bodies," In: metal B W, dalton B J, eds.cell adhesion: molecular definition to therapeutic potential New York: plasma Press, 1994:291-312), surface reconstruction (US patent US 5639641). If other regions, such as hinge and constant region domains, are also derived from non-human sources, these regions may also be humanized.
In this application, the term "murine antibody" generally refers to antibodies in which the variable region framework and CDR regions are derived from the germline immunoglobulin sequences of the mouse. Furthermore, if the antibody comprises constant regions, it is also derived from the mouse germline immunoglobulin sequence. The murine antibodies of the present application may comprise amino acid residues not encoded by the mouse germline immunoglobulin sequences, and may include, for example, mutations introduced by random or point mutation in vitro or by somatic mutation in vivo.
In the present application, the term "PD-L1" generally refers to a programmed death ligand 1 protein, a functional variant thereof and/or a functionally active fragment thereof. PD-L1 is also known as cluster of differentiation 274 (CD 274) or B7 homolog 1 (B7-H1) and is a protein encoded by the CD274 gene (in humans). PD-L1 binds to its receptor, such as programmed death 1 (PD-1), which PD-1 is expressed in activated T cells, B cells and macrophages (Ishida et al 1992EMBO J,11:3887-3395;Okazaki et al, autoimmune dilated cardiomyopathy in PD-1 receptor-specific mico.science, 2001; 291:319-22). Complexation of PD-L1 and PD-1 exert immunosuppressive effects by inhibiting T cell proliferation and production of cytokines IL-2 and IFN-gamma (Freeman et al, energy of PD-1immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation,J.Exp.Med.2000,192:1027-1034;Carter et al, PD-1: PD-Linhibitory pathway affects both CD (+) and CD8 (+) T cells and is overcome by IL-2.Eur. J. Immunol.2002, 32:634-643). The term "PD-L1" encompasses any natural PD-L1 of any vertebrate origin, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full length", unprocessed PD-L1, and any form of PD-L1 produced by processing in a cell. PD-L1 may be present as a transmembrane protein or as a soluble protein. The term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants. The amino acid sequence of an exemplary full-length human PD-L1 protein can be found under UniProt accession number Q9 NZQ. For example, the "functionally active fragment" may include a fragment that retains the endogenous function of at least one naturally occurring protein (e.g., binds to an antigen binding protein described herein). For example, the "functionally active fragment" may comprise a domain that binds to an antigen binding protein of the present application.
The term "functionally active fragment" refers to a polypeptide having or encoded by a substantially identical amino acid sequence to a naturally occurring sequence and capable of having one or more activities of the naturally occurring sequence. In the context of the present application, a functionally active fragment of any given sequence refers to a sequence in which a particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one endogenous function. The sequence encoding the functionally active fragment may be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in the naturally occurring protein and/or polynucleotide, as long as the original functional activity is maintained.
In the present application, the term "derivative" generally refers to any substitution, variation, modification, substitution, deletion, and/or addition of one (or more) amino acid residue(s) (or nucleotide (s)) to a polypeptide or polynucleotide of the present application, so long as the resulting polypeptide or polynucleotide substantially retains at least one endogenous function thereof.
In this application, the term "analog" generally refers to a polypeptide or polynucleotide, and includes any mimetic of a polypeptide or polynucleotide, i.e., a chemical compound that possesses at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics.
Generally, amino acid substitutions can be made, for example, at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more) amino acid substitutions, so long as the modified sequence substantially retains the desired activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogs.
In this application, the term "homolog" generally refers to an amino acid sequence or nucleotide sequence that has some homology to a naturally occurring sequence. The term "homology" may be equivalent to the sequence "identity". Homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence. Typically, the homologue will comprise the same active site or the like as the subject amino acid sequence. Homology may be considered in terms of similarity (i.e., amino acid residues having similar chemical properties/functions), or homology may be expressed in terms of sequence identity. In the present application, a sequence of any one of the mentioned amino acid sequences or nucleotide sequences of SEQ ID NOs having a percent identity refers to a sequence having said percent identity over the entire length of the mentioned SEQ ID NOs. To determine sequence identity, sequence alignments can be performed in a variety of ways known to those skilled in the art, e.g., using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. One skilled in the art can determine the appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment in the compared full-length sequences.
Proteins or polypeptides used in the present application may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids containing uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.
In this application, the term "tumor" generally refers to a neoplasm formed by local tissue cell proliferation under the influence of various tumorigenic factors. For example, the tumor may comprise a solid tumor. For example, the tumor may comprise a non-solid tumor. For example, the tumor may comprise a tumor associated with the expression of PD-L1. The term "tumor associated with the expression of PD-L1" generally refers to a tumor formed by altered expression of PD-L1 resulting in disease progression or evasion of immune surveillance. For example, the "tumor associated with the expression of PD-L1" may be a tumor formed by up-regulating the expression level of PD-L1 to cause disease progression or evade immune surveillance. The tumor associated with the protein expression of PD-L1 may be a PD-L1 positive tumor. In PD-L1 positive tumors, the protein expression of PD-L1 is about 1%,5%,10%,15%,20%,25%,30%,35%,40%,50%,60%,70%,80% or more on the surface of tumor cells or in the tumor microenvironment compared to normal cells.
In this application, the term "solid tumor" generally refers to a tangible tumor that can be detected clinically (e.g., by X-ray film, CT scan, B-mode or palpation). For example, the solid tumor may include breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and/or liver cancer.
In the present application, the term "non-solid tumor" generally refers to a tumor that is not visible or accessible by X-ray film, CT scan, B-mode, and palpation. For example, the non-solid tumor may include leukemia. For example, the non-solid tumor may include a lymphoma. For example, the non-solid tumor may include multiple myeloma.
In this application, the term "immunoconjugate" generally refers to a conjugate formed by conjugation (e.g., covalent attachment via a linker molecule) of the other therapeutic agent to the isolated antigen binding protein, which can deliver the other therapeutic agent to a target cell (e.g., a tumor cell) through specific binding of the isolated antigen binding protein to an antigen on the target cell. In addition, the antigen may also be secreted by the target cell and located in the extracellular space of the target cell.
In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
In this application, the term "nucleic acid molecule" generally refers to any length of isolated form of nucleotide, deoxyribonucleotide or ribonucleotide or analog thereof, either isolated from the natural environment or synthesized.
In the present application, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. The vector may transfer the inserted nucleic acid molecule into and/or between cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable cell. Typically, the vector will produce the desired expression product by culturing a suitable cell containing the vector. In this application, the vector may comprise a lentiviral vector.
In the present application, the term "cell" generally refers to an individual cell, cell line or cell culture that may or has contained a plasmid or vector comprising a nucleic acid molecule as described herein, or that is capable of expressing a polypeptide as described herein or an antigen binding protein as described herein. The cells may include progeny of a single cell. The progeny cells may not necessarily be identical in morphology or in genome to the original parent cell due to natural, accidental or deliberate mutation, but are capable of expressing the polypeptides or antigen binding proteins described herein. The cells may be obtained by transfecting the cells in vitro using the vectors described herein. The cells may be prokaryotic cells (e.g., E.coli) or eukaryotic cells (e.g., yeast cells, e.g., COS cells, chinese Hamster Ovary (CHO) cells, heLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells). In some embodiments, the cell may be an immune cell. For example, the immune cells may be selected from the group consisting of: t cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and/or peripheral blood mononuclear cells.
In this application, the term "treatment" generally refers to: (i) Preventing the occurrence of a disease, disorder, or condition in a patient who may be susceptible to the disease, disorder, and/or condition, but has not been diagnosed with the disease; (ii) Inhibiting the disease, disorder or condition, i.e., inhibiting its development; and (iii) alleviating the disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition.
In this application, the terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and generally refer to a polymer having amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may comprise: disulfide bond formation, glycosylation, lipidation (lipid), acetylation, phosphorylation, or any other manipulation (e.g., in combination with a labeling component). The term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine as well as D and L optical isomers, as well as amino acid analogs and peptidomimetics.
In this application, the terms "polynucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably and generally refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, multiple loci (one locus), exons, introns, messenger RNAs (mRNA), transfer RNAs, ribosomal RNAs, short interfering RNAs (siRNA), short hairpin RNAs (shRNA), micro-RNAs (miRNA), ribozymes, cdnas, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers defined according to ligation analysis. Polynucleotides may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure, if present, may be performed before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeled component.
In the present application, the term "K D "(likewise," K) D "or" K D ") generally refers to an" affinity constant "or" equilibrium dissociation constant "and refers to a value obtained at equilibrium in a titration measurement, or by dividing the dissociation rate constant (kd) by the association rate constant (ka). Using the binding rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (K) D ) Represents the binding affinity of a binding protein (e.g., an isolated antigen binding protein described herein) to an antigen (e.g., a PD-L1 protein). Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers. For example, the K can be determined by Biacore (analysis of biomolecular interactions) (e.g., an instrument available from BIAcoreInternationalAB, aGEHealthcarecompany, uppsala, sweden) D Values, other experimental pathways and instruments such as Octet detection may also be used. In addition, the K can also be determined using KinExA (kinetic exclusion assay) available from Sapidyneinstruments (Boise, idaho) D Value, or determination of the K using a Surface Plasmon Resonance (SPR) D Values. For example, the K can also be determined by an amine coupling kit D Values.
In this application, the term "and/or" is understood to mean either one of the selectable items or both of the selectable items.
In this application, the term "comprising" is generally intended to include the features specifically recited, but does not exclude other elements. In some cases, "comprising" also encompasses cases where only specified components are included. For example, the inclusion is also denoted as meaning "consisting of … …".
In this application, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.
In this application, the terms "comprises," "comprising," and "includes" are used in their plain, inclusive, and open-ended meaning. In some cases, the meaning of "as", "consisting of … …" is also indicated.
Detailed Description
Isolated antigen binding proteins as described herein
In one aspect, the present application provides an isolated antigen binding protein that can be at a K of about 8E-09M or less in a Biacore assay D Value (e.g. the K D Not greater than about 8E-09M, not greater than about 7.5E-09M, not greater than about 7E-09M, not greater than about 6.5E-09M, not greater than about 6E-09M, not greater than about 5.5E-09M, not greater than about 5E-09M, not greater than about 4.5E-09M, not greater than about 4E-09M, not greater than about 3.5E-09M, not greater than about 3E-09M, not greater than about 2.5E-09M, not greater than about 2E-09M, not greater than about 1.5E-09M, not greater than about 1E-09M, not greater than about 9E-10M, or not greater than about 8.5E-10M or less) specifically binds to human PD-L1 protein.
In the present application, the antigen binding protein may block the binding of PD-1 to PD-L1.
In one aspect, the present application provides an isolated antigen binding protein that may comprise at least one CDR in an antibody heavy chain variable region VH that may comprise the amino acid sequence shown in SEQ ID NO. 67.
For example, the VH may comprise the amino acid sequence shown in any one of SEQ ID NO 39 to SEQ ID NO 48. In the present application, the HCDR of the isolated antigen binding protein may be divided in any form, so long as VH is identical to the amino acid sequence shown in any one of SEQ ID NO:39 to SEQ ID NO:48, and the HCDR divided in any form falls within the scope of the present application.
The CDRs of an antibody, also known as complementarity determining regions, are part of the variable region. The amino acid residues of this region may be contacted with an antigen or epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, kabat, chothia, IMGT, abM, a combination of Kabat/Chothia et al. These coding systems are known in the art and can be found, for example, in http:// www.bioinf.org.uk/abs/index. The CDR regions can be determined by one skilled in the art using different coding systems depending on the sequence and structure of the antibody. Using different coding systems, CDR regions may differ. In this application, the CDRs encompass CDR sequences partitioned according to any CDR partitioning scheme; variants thereof are also contemplated, including amino acid substitutions, deletions and/or additions to the amino acid sequence of the CDRs. Such as 1-30, 1-20 or 1-10, and further such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or insertions; homologues thereof are also contemplated, which may be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequences of the CDRs. In certain embodiments, the isolated antigen binding proteins described herein are defined by the Kabat coding system.
In the present application, the antigen binding protein may comprise a heavy chain variable region VH, which may comprise at least one, two or three of HCDR1, HCDR2 and HCDR 3.
In this application, the HCDR3 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO. 18. For example, the HCDR3 sequence of the antigen binding protein may be defined according to the Kabat coding system.
In this application, the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 12. For example, the HCDR2 sequence of the antigen binding protein may be defined according to the Kabat coding system.
In this application, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 3. For example, the HCDR1 sequence of the antigen binding protein may be defined according to the Kabat coding system.
For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 18. For example, the antigen binding protein may include antibodies 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or antigen binding fragments having the same HCDR3 (e.g., the same HCDR 1-3) as it.
For example, the VH of the antigen binding protein may comprise the framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 59. For example, the antigen binding protein H-FR1 has amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: x is X 5 ，X 7 ，X 10 ，X 11 ，X 12 ，X 16 And X 20 。
QVQLX 5 QX 7 GAX 10 X 11 X 12 KPGX 16 SVKX 20 SCKASGYTFT (SEQ ID NO: 59), wherein X 5 May be Q or V, X 7 Can be P or S, X 10 May be D or E, X 11 May be L or V, X 12 May be K or V, X 16 Can be A or S, X 20 May be L or V.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 1 to SEQ ID NO. 2.
In the present application, the H-FR2 of said antigen binding protein may be packagedContains the amino acid sequence shown in SEQ ID NO. 60. For example, the antigen binding protein H-FR2 has amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: x is X 2 ，X 3 ，X 5 ，X 11 And X 13 。
WX 2 X 3 QX 5 PGQGLX 11 WX 13 G (SEQ ID NO: 60), wherein X 2 Can be D, M or V, X 3 May be K or R, X 5 May be A or R, X 11 May be D or E, X 13 May be I or M.
In the present application, the H-FR2 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 4 to SEQ ID NO. 11.
In the present application, the H-FR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 61. For example, the antigen binding protein H-FR3 has amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: x is X 2 ，X 3 ，X 4 ，X 10 ，X 16 ，X 21 And X 25 。
RX 2 X 3 X 4 TADKSX 10 STAYMX 16 LSSLX 21 SEDX 25 AVYYCAR (SEQ ID NO: 61), wherein X 2 May be A or V, X 3 Can be S or T, X 4 May be I or L, X 10 Can be S or T, X 16 May be E or Q, X 21 Can be R or T, X 25 May be S or T.
In the present application, the H-FR3 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 13 to SEQ ID NO. 17.
In this application, the H-FR4 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 62. For example, the antigen binding protein H-FR4 has amino acid substitutions at one or more amino acids selected from the group consisting of (e.g., a protective amino acid sequence as compared to the sequence set forth in SEQ ID NO:62Conservative amino acid substitutions, etc.): x is X 6 And X 7 。
WGQGTX 6 X 7 TVSS (SEQ ID NO: 62), wherein X 6 Can be M or T, X 7 May be L or V.
In the present application, the H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 19 to SEQ ID NO. 20.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 59; the H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 60; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 61; and the H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 62.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 1 to SEQ ID NO. 2; the H-FR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NO. 4 to SEQ ID NO. 11; the H-FR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NO. 13 to SEQ ID NO. 17; and the H-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NO. 19 to SEQ ID NO. 20.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 1; the H-FR2 may comprise an amino acid sequence shown in SEQ ID NO. 4; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 13; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 19. For example, the antigen binding protein may comprise antibody 13H6D3 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 14; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20. For example, the antigen binding protein may comprise antibody hu13H6D3 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 6; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 15; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20. For example, the antigen binding protein may comprise antibody 5BM or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 7; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 16; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20. For example, the antigen binding protein may comprise antibody AH00228 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 8; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 17; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20. For example, the antigen binding protein may comprise antibody AH00229 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 9; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 14; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20. For example, the antigen binding protein may comprise antibody AH00230 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 7; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 14; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20. For example, the antigen binding protein may comprise antibody AH00231 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 6; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 16; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20. For example, the antigen binding protein may comprise antibody AH00232 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 10; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 14; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20. For example, the antigen binding protein may comprise antibody AH00233 or an antigen binding fragment thereof having the same H-FR 1-4.
In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 11; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 15; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20. For example, the antigen binding protein may comprise an antibody CBM or an antigen binding fragment thereof having the same H-FR 1-4.
In this application, the antigen binding protein may comprise a heavy chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO. 67. For example, the antigen-binding protein comprises a VH having amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of SEQ ID NO: 67: x is X 5 ，X 7 ，X 10 ，X 11 ，X 12 ，X 16 ，X 20 ，X 37 ，X 38 ，X 40 ，X 46 ，X 48 ，X 67 ，X 68 ，X 69 ，X 75 ，X 81 ，X 86 ，X 90 ，X 115 And X 116 。
QVQLX 5 QX 7 GAX 10 X 11 X 12 KPGX 16 SVKX 20 SCKASGYTFTSNWMHWX 37 X 38 QX 40 PGQGLX 46 WX 48 GMIHPNSAIKYNEKFKSRX 67 X 68 X 69 TADKSX 75 STAYMX 81 LSSLX 86 SEDX 90 AVYYCARSYYGSSPYFFDYWGQGTX 115 X 116 TVSS (SEQ ID NO: 67), wherein X 5 May be Q or V, X 7 Can be P or S, X 10 May be D or E, X 11 May be L or V, X 12 May be K or V, X 16 Can be A or S, X 20 May be L or V, X 37 Can be D, M or V, X 38 May be K or R, X 40 May be A or R, X 46 May be D or E, X 48 Can be I or M, X 67 May be A or V, X 68 Can be S or T, X 69 May be I or L, X 75 Can be S or T, X 81 May be E or Q, X 86 Can be R or T, X 90 Can be S or T, X 115 Can be M or T, X 116 May be L or V.
In the present application, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO 39 to SEQ ID NO 48.
In this application, the antigen binding protein may comprise a heavy chain constant region, which may comprise an IgG-derived constant region or an IgY-derived constant region.
For example, the heavy chain constant region of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO. 56 to SEQ ID NO. 57.
In this application, the antigen binding protein may comprise at least one CDR in the variable region VL of an antibody light chain, which VL may comprise the amino acid sequence shown in SEQ ID NO. 68.
For example, the VL may comprise an amino acid sequence set forth in any one of SEQ ID NO. 49 through SEQ ID NO. 55. In the present application, the LCDR of the isolated antigen-binding protein may be divided in any form, so long as VL is identical to the amino acid sequence shown in any one of SEQ ID NOS.49 to 55, and the LCDR divided in any form falls within the scope of the present application.
In the present application, the antigen binding protein may include a light chain variable region VL, which may comprise at least one, at least two, or at least three of LCDR1, LCDR2, and LCDR 3.
In this application, LCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, LCDR3 of the antigen binding protein can be defined according to the Kabat numbering system.
In this application, LCDR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 29. For example, LCDR2 of the antigen binding protein can be defined according to the Kabat numbering system.
In this application, LCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 24. For example, LCDR1 of the antigen binding protein can be defined according to the Kabat numbering system.
For example, LCDR1 of the antigen-binding proteins described herein may comprise the amino acid sequence shown in SEQ ID NO. 24; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 29; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the antigen binding protein can include antibodies 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or antigen binding fragments having the same LCDR3 (e.g., the same LCDR 1-3) as it.
For example, the VL of the antigen binding protein may comprise framework regions L-FR1, L-FR2, L-FR3 and L-FR4.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 63. For example, the antigen binding protein L-FR1 has amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: x is X 7 ，X 8 ，X 15 And X 22 。
DIQMTQX 7 X 8 SSLSASX 15 GDRVTIX 22 C (SEQ ID NO: 63), wherein X 7 Can be S or T, X 8 May be P or T, X 15 Can be used forIs L or V, X 22 May be S or T.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 21 to SEQ ID NO. 23.
In this application, the L-FR2 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 64. For example, the antigen binding protein L-FR2 has amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: x is X 7 ，X 8 ，X 9 ，X 10 And X 12 。
WYQQKPX 7 X 8 X 9 X 10 KX 12 LIY (SEQ ID NO: 64), wherein X 7 May be D or G, X 8 Can be G or K, X 9 May be A or T, X 10 May be P or V, X 12 May be F or L.
In the present application, the L-FR2 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 25 to SEQ ID NO. 28.
In this application, the L-FR3 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 65. For example, the antigen binding protein L-FR3 has amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: x is X 4 ，X 15 ，X 16 ，X 21 ，X 23 ，X 24 ，X 27 And X 31 。
GVPX 4 RFSGSGSGTDX 15 X 16 LTISX 21 LX 23 X 24 EDX 27 ATYX 31 C (SEQ ID NO: 65), wherein X 4 Can be A or S, X 15 Can be F or Y, X 16 Can be F or T, X 21 Can be N or S, X 23 May be E or Q, X 24 May be P or Q, X 27 Can be F or V, X 31 May be F or Y.
In the present application, the L-FR3 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 30 to SEQ ID NO. 35.
In this application, the L-FR4 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 66. For example, the antigen binding protein L-FR4 has amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: x is X 7 。
FGGGTKX 7 EIK (SEQ ID NO: 66), wherein X 7 May be L or V.
In the present application, the L-FR4 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 37 to SEQ ID NO. 38.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 63; the L-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 64; the L-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 65; and the L-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 66.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO. 21 to SEQ ID NO. 23; the L-FR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NO. 25 to SEQ ID NO. 28; the L-FR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NO. 30 to SEQ ID NO. 35; and the L-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NO:37 to SEQ ID NO: 38.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 21; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 25; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 30; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 37. For example, the antigen binding protein may comprise antibody 13H6D3 or an antibody having the same L-FR1-4 as it.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 22; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 26; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 31; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the antigen binding protein may comprise antibody hu13H6D3 or an antibody having the same H-FR1-4 as it.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 32; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the antigen binding protein may comprise antibody 5BM or an antibody having the same L-FR1-4 as it.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 32; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the antigen binding protein may comprise antibody AH00228 or an antibody having the same H-FR1-4 as it does.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 33; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the antigen binding protein may comprise antibody AH00229 or an antibody having the same L-FR1-4 as it.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 32; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the antigen binding protein may comprise antibody AH00230 or an antibody having the same L-FR1-4 as it.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 22; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 32; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the antigen binding protein may comprise antibody AH00231 or an antibody having the same H-FR1-4 as it.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 22; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 34; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the antigen binding protein may comprise antibody AH00232 or an antibody having the same L-FR1-4 as it.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 33; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the antigen binding protein may comprise antibody AH00233 or an antibody having the same H-FR1-4 as it.
In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 22; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 28; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 35; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the antigen binding protein may comprise the antibody CBM or an antibody having the same H-FR1-4 as it.
In this application, the antigen binding protein may comprise a light chain variable region VL which may comprise the amino acid sequence shown in SEQ ID NO. 68. For example, the VL of the antigen-binding protein has amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: x is X 7 ，X 8 ，X 15 ，X 22 ，X 41 ，X 42 ，X 43 ，X 44 ，X 46 ，X 60 ，X 71 ，X 72 ，X 77 ，X 79 ，X 80 ，X 83 ，X 87 And X 104 。
DIQMTQX 7 X 8 SSLSASX 15 GDRVTIX 22 CRASQDISKYLNWYQQKPX 41 X 42 X 43 X 44 KX 46 LIY YTSRLHSGVPX 60 RFSGSGSGTDX 71 X 72 LTISX 77 LX 79 X 80 EDX 83 ATYX 87 CQQGDTLPWTFGGGT KX 104 EIK (SEQ ID NO: 68), wherein X 7 Can be S or T, X 8 May be P or T, X 15 May be L or V, X 22 Can be S or T, X 41 May be D or G, X 42 Can be G or K, X 43 May be A or T, X 44 May be P or V, X 46 Can be F or L, X 60 Can be A or S, X 71 Can be F or Y, X 72 Can be F or T, X 77 Can be N or S, X 79 May be E or Q, X 80 May be P or Q, X 83 Can be F or V, X 87 Can be F or Y, X 104 May be L or V.
In the present application, the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs 49 to 55.
In the present application, the antigen binding protein may comprise a light chain constant region, which may comprise an igκ -derived constant region or an igλ -derived constant region.
For example, the light chain constant region can include a constant region derived from igκ.
For example, the light chain constant region of the antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs 58.
In the present application, the antigen binding proteins may comprise HCDR1-3 and LCDR1-3. For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 12; the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 18; LCDR1 of the antigen-binding protein may comprise an amino acid sequence shown as SEQ ID NO. 24; LCDR2 of the antigen-binding protein may comprise an amino acid sequence set forth in SEQ ID NO. 29; LCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the antigen binding protein may include antibodies 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or antigen binding fragments having the same HCDR3 (e.g., the same HCDR 1-3) and LCDR3 (e.g., the same LCDR 1-3) as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4. The light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 18; the LCDR1 may include an amino acid sequence shown in SEQ ID NO. 24; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 29; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 1; the H-FR2 may comprise an amino acid sequence shown in SEQ ID NO. 4; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 13; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 19; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 21; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 25; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 30; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 37. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 39. For example, the antigen binding protein may comprise antibody 13H6D3 or an antigen binding protein having the same heavy chain variable region as it. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 49. For example, the antigen binding protein may comprise antibody 13H6D3 or an antigen binding protein having the same light chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4. The light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 18; the LCDR1 may include an amino acid sequence shown in SEQ ID NO. 24; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 29; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 14; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 22; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 26; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 31; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 40. For example, the antigen binding protein may comprise antibody hu13H6D3 or an antigen binding protein having the same heavy chain variable region as it. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 50. For example, the antigen binding protein may comprise antibody hu13H6D3 or an antigen binding protein having the same light chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4. The light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 18; the LCDR1 may include an amino acid sequence shown in SEQ ID NO. 24; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 29; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 6; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 15; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 23; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 32; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 41. For example, the antigen binding protein may comprise antibody 5BM or an antigen binding protein having the same heavy chain variable region as it. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 51. For example, the antigen binding protein may comprise antibody 5BM or an antigen binding protein having the same light chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4. The light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 18; the LCDR1 may include an amino acid sequence shown in SEQ ID NO. 24; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 29; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 7; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 16; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 23; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 32; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 42. For example, the antigen binding protein may comprise antibody AH00228 or an antigen binding protein having the same heavy chain variable region as it. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 51. For example, the antigen binding protein may comprise antibody AH00228 or an antigen binding protein having the same light chain variable region as it does.
In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4. The light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 18; the LCDR1 may include an amino acid sequence shown in SEQ ID NO. 24; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 29; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 8; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 17; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 23; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 33; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 43. For example, the antigen binding protein may comprise antibody AH00229 or an antigen binding protein having the same heavy chain variable region as it. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 52. For example, the antigen binding protein may comprise antibody AH00229 or an antigen binding protein having the same light chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4. The light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 18; the LCDR1 may include an amino acid sequence shown in SEQ ID NO. 24; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 29; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 9; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 14; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 23; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 32; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 44. For example, the antigen binding protein may comprise antibody AH00230 or an antigen binding protein having the same heavy chain variable region as it. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 51. For example, the antigen binding protein may comprise antibody AH00230 or an antigen binding protein having the same light chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4. The light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 18; the LCDR1 may include an amino acid sequence shown in SEQ ID NO. 24; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 29; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 7; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 14; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 22; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 32; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 45. For example, the antigen binding protein may comprise antibody AH00231 or an antigen binding protein having the same heavy chain variable region as it. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 53. For example, the antigen binding protein may comprise antibody AH00231 or an antigen binding protein having the same light chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4. The light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 18; the LCDR1 may include an amino acid sequence shown in SEQ ID NO. 24; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 29; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 6; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 16; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 22; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 34; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 46. For example, the antigen binding protein may comprise antibody AH00232 or an antigen binding protein having the same heavy chain variable region as it. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 54. For example, the antigen binding protein may comprise antibody AH00232 or an antigen binding protein having the same light chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4. The light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 18; the LCDR1 may include an amino acid sequence shown in SEQ ID NO. 24; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 29; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 10; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 14; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 23; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 27; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 33; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 47. For example, the antigen binding protein may comprise antibody AH00233 or an antigen binding protein having the same heavy chain variable region as it. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 52. For example, the antigen binding protein may comprise antibody AH00233 or an antigen binding protein having the same light chain variable region as it.
In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4. The light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 18; the LCDR1 may include an amino acid sequence shown in SEQ ID NO. 24; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 29; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 11; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 15; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 22; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 28; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 35; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 38. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 48. For example, the antigen binding protein may comprise an antibody CBM or an antigen binding protein having the same heavy chain variable region as it. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 55. For example, the antigen binding protein may comprise an antibody CBM or an antigen binding protein having the same light chain variable region as it.
In this application, the isolated antigen binding protein may also compete for binding to the human PD-L1 protein with a reference antibody, which may comprise a heavy chain variable region VH, which may comprise at least one, two or three of HCDR1, HCDR2 and HCDR 3.
In this application, the HCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 18. For example, the HCDR3 sequence of the reference antibody may be defined according to the Kabat coding system.
In this application, the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 12. For example, the HCDR2 sequence of the reference antibody may be defined according to the Kabat coding system.
In this application, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 3. For example, the HCDR1 sequence of the reference antibody may be defined according to the Kabat coding system.
For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 12; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 18. For example, the reference antibody can include antibodies 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or antigen binding fragments thereof that have the same HCDR3 (e.g., the same HCDR1-3 as it).
In this application, the reference antibody may comprise a heavy chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO. 67. For example, the reference antibody comprises a VH having amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of SEQ ID No. 67: x is X 5 ，X 7 ，X 10 ，X 11 ，X 12 ，X 16 ，X 20 ，X 37 ，X 38 ，X 40 ，X 46 ，X 48 ，X 67 ，X 68 ，X 69 ，X 75 ，X 81 ，X 86 ，X 90 ，X 115 And X 116 。
QVQLX 5 QX 7 GAX 10 X 11 X 12 KPGX 16 SVKX 20 SCKASGYTFTSNWMHWX 37 X 38 QX 40 PGQGLX 46 WX 48 GMIHPNSAIKYNEKFKSRX 67 X 68 X 69 TADKSX 75 STAYMX 81 LSSLX 86 SEDX 90 AVYYCARSYYGSSPYFFDYWGQGTX 115 X 116 TVSS (SEQ ID NO: 67), wherein X 5 May be Q or V, X 7 Can be P or S, X 10 May be D or E, X 11 May be L or V, X 12 May be K or V, X 16 Can be A or S, X 20 May be L orV，X 37 Can be D, M or V, X 38 May be K or R, X 40 May be A or R, X 46 May be D or E, X 48 Can be I or M, X 67 May be A or V, X 68 Can be S or T, X 69 May be I or L, X 75 Can be S or T, X 81 May be E or Q, X 86 Can be R or T, X 90 Can be S or T, X 115 Can be M or T, X 116 May be L or V.
In the present application, the heavy chain variable region of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO 39 to SEQ ID NO 48.
In this application, the reference antibody may comprise a heavy chain constant region, which may comprise an IgG-derived constant region or an IgY-derived constant region.
For example, the heavy chain constant region of the reference antibody may comprise the amino acid sequences shown in SEQ ID NO. 56 to SEQ ID NO. 57.
In this application, the reference antibody may comprise at least one CDR in the antibody light chain variable region VL, which may comprise the amino acid sequence shown as SEQ ID NO. 68.
For example, the VL may comprise an amino acid sequence set forth in any one of SEQ ID NO. 49 through SEQ ID NO. 55. In the present application, the LCDR of the reference antibody may be divided in any form as long as VL is identical to the amino acid sequence shown in any one of SEQ ID NO:49 to SEQ ID NO:55, and the LCDR divided in any form falls within the scope of the present application.
In the present application, the reference antibody may include a light chain variable region VL, which may include at least one, at least two, or at least three of LCDR1, LCDR2, and LCDR 3.
In this application, the LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, LCDR3 of the reference antibody can be defined according to the Kabat numbering system.
In this application, the LCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 29. For example, LCDR2 of the reference antibody can be defined according to the Kabat numbering system.
In this application, the LCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 24. For example, LCDR1 of the reference antibody can be defined according to the Kabat numbering system.
For example, LCDR1 of a reference antibody described herein may comprise the amino acid sequence shown in SEQ ID NO. 24; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 29; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the reference antibody can include antibodies 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or antigen binding fragments having the same LCDR3 (e.g., having the same LCDR 1-3) as it.
In this application, the reference antibody may comprise a light chain variable region VL which may comprise the amino acid sequence shown in SEQ ID NO. 68. For example, the VL of the reference antibody has amino acid substitutions (e.g., conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: x is X 7 ，X 8 ，X 15 ，X 22 ，X 41 ，X 42 ，X 43 ，X 44 ，X 46 ，X 60 ，X 71 ，X 72 ，X 77 ，X 79 ，X 80 ，X 83 ，X 87 And X 104 。
DIQMTQX 7 X 8 SSLSASX 15 GDRVTIX 22 CRASQDISKYLNWYQQKPX 41 X 42 X 43 X 44 KX 46 LIYYTSRLHSGVPX 60 RFSGSGSGTDX 71 X 72 LTISX 77 LX 79 X 80 EDX 83 ATYX 87 CQQGDTLPWTFGGGTKX 104 EIK (SEQ ID NO: 68), wherein X 7 Can be S or T, X 8 May be P or T, X 15 May be L or V, X 22 Can be S or T, X 41 May be D or G, X 42 Can be G or K, X 43 May be A or T, X 44 May be P or V, X 46 Can be F or L, X 60 Can be A or S, X 71 Can be F or Y, X 72 Can be F or T, X 77 Can be N or S, X 79 May be E or Q, X 80 May be P or Q, X 83 Can be F or V, X 87 Can be F or Y, X 104 May be L or V.
In the present application, the light chain variable region of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NOs 49 to 55.
In this application, the reference antibody may comprise a light chain constant region, which may comprise an igκ -derived constant region or an igλ -derived constant region.
For example, the light chain constant region can include a constant region derived from igκ.
For example, the light chain constant region of the reference antibody comprises the amino acid sequence set forth in any one of SEQ ID NOs 58.
In the present application, the reference antibody may comprise HCDR1-3 and LCDR1-3. For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 3; the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 12; the HCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 18; LCDR1 of the reference antibody may comprise an amino acid sequence shown in SEQ ID NO. 24; LCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 29; LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the reference antibody may comprise antibodies 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or antigen binding fragments thereof that have the same HCDR3 (e.g., the same HCDR 1-3) and LCDR3 (e.g., the same LCDR 1-3) as it.
Polypeptides and immunoconjugates
In another aspect, the present application provides one or more polypeptides, which may comprise an isolated antigen binding protein of the present application. For example, the polypeptide may comprise a fusion protein. For example, the polypeptide can include a multispecific antibody (e.g., a bispecific antibody).
In another aspect, the present application provides one or more immunoconjugates that may comprise the isolated antigen binding proteins of the present application. In certain embodiments, the immunoconjugate may further comprise a pharmaceutically acceptable therapeutic agent, a label, and/or a detection agent.
Nucleic acids, vectors, cells and pharmaceutical compositions
In another aspect, the present application also provides an isolated one or more nucleic acid molecules that can encode an isolated antigen binding protein described herein. For example, each of the one or more nucleic acid molecules may encode the entire antigen binding protein, or may encode a portion thereof (e.g., one or more of HCDR1-3, heavy chain variable regions).
For example, when nucleic acid molecules individually encode a portion of the antigen binding protein, the products encoded by the nucleic acid molecules taken together may form an isolated antigen binding protein of the present application that is functional (e.g., can bind PD-L1).
The nucleic acid molecules described herein may be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, e.g. by Polymerase Chain Reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified, e.g. fractionated by cleavage and gel electrophoresis, or (iv) synthesized, e.g. by chemical synthesis. For example, the isolated nucleic acid may be a nucleic acid molecule prepared by recombinant DNA techniques.
In the present application, nucleic acids encoding the isolated antigen binding proteins can be prepared by a variety of methods known in the art, including, but not limited to, using reverse transcription PCR and PCR to obtain nucleic acid molecules of the isolated antigen binding proteins described herein.
In another aspect, the present application provides one or more vectors comprising one or more nucleic acid molecules described herein. Each vector may comprise one or more of the nucleic acid molecules. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, and the like. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequences may vary depending on the species or cell type function, but typically comprises 5' non-transcribed and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as TATA boxes, capping sequences, CAAT sequences, and the like. For example, a 5' non-transcriptional expression control sequence may comprise a promoter region that may comprise a promoter sequence for a transcriptional control functional attachment nucleic acid. The expression control sequences may also include enhancer sequences or upstream activator sequences. In this application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerase, the human U6RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (e.g., CMV), wherein a portion of the promoter may be fused to a portion of the promoter of other cellular proteins (e.g., human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene, which may or may not comprise additional introns. One or more nucleic acid molecules described herein may be operably linked to the expression control element.
The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering. For example, the vector may be an expression vector. For example, the vector may be a viral vector. The viral vector may be administered directly to the patient (in vivo) or the cells may be treated with the virus in an indirect form, e.g., in vitro, and then the treated cells are administered to the patient (ex vivo). Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York) and other virology and molecular biology manuals. Conventional virus-based systems may include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex viral vectors for gene transfer. In some cases, the gene transfer may be integrated into the host genome by retrovirus, lentivirus, and adeno-associated virus methods, allowing long-term expression of the inserted gene. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers. Lentiviral vectors may comprise a long terminal repeat 5'LTR and truncated 3' LTR, RRE, rev responsive element (cPPT), central Termination Sequence (CTS) and/or post-translational regulatory element (WPRE). The vectors described herein may be introduced into cells.
In another aspect, the present application provides a cell. The cell may comprise an isolated antigen binding protein described herein, the polypeptide, the immunoconjugate, one or more nucleic acid molecules, and/or one or more vectors described herein. For example, each or each cell may comprise one or more nucleic acid molecules or vectors described herein. For example, each or each cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into the host cell, such as a prokaryotic cell (e.g., a bacterial cell), CHO cell, NS/0 cell, HEK293T cell, 293F cell, or HEK293A cell, or other eukaryotic cell, such as a cell from a plant, a fungal or yeast cell, or the like. The vectors described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like. For example, the cells may include yeast cells. For example, the cells may include E.coli cells. For example, the cells may include mammalian cells. For example, the cells may include immune cells.
The cells may include immune cells. In some cases, the cells may include immune cells. For example, the cells may include T cells, B cells, natural Killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells.
In another aspect, the present application provides a pharmaceutical composition. The pharmaceutical composition may comprise an isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and/or a pharmaceutically acceptable adjuvant and/or excipient described herein. In this application, the pharmaceutically acceptable adjuvant may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and/or nonionic surfactants. Any conventional medium or agent is contemplated for use in the pharmaceutical compositions of the present application unless incompatible with the cells described herein. In this application, the pharmaceutically acceptable excipients may include additives other than the main drug in the pharmaceutical formulation, which may also be referred to as excipients. For example, the excipients may include binders, fillers, disintegrants, lubricants in the tablet. For example, the excipients may include wine, vinegar, juices, etc. in a traditional Chinese medicine pill. For example, the excipient may comprise a base portion of a semi-solid formulation ointment, cream. For example, the excipients may include preservatives, antioxidants, flavoring agents, fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants in liquid formulations.
Kit, use and method
In another aspect, the present application provides a method for detecting or assaying PD-L1, which may comprise using the isolated antigen binding protein or the polypeptide.
In this application, the methods may include in vitro methods, ex vivo methods, methods of non-diagnostic or non-therapeutic purpose.
For example, the method may comprise a method of detecting the presence and/or amount of PD-L1 for non-diagnostic purposes, which may comprise the steps of:
1) Contacting the sample with an antigen binding protein of the present application; and
2) Detecting the presence and/or amount of the antigen binding protein bound by the sample to determine the presence and/or expression level of PD-L1 in the sample obtained from the subject.
In another aspect, the present application provides a kit of PD-L1, which may comprise the use of the isolated antigen binding protein or the polypeptide.
In the present application, the kit may further comprise instructions describing a method for detecting the presence and/or amount of PD-L1. For example, the method may comprise an in vitro method, an ex vivo method, a method of non-diagnostic or non-therapeutic purpose.
In another aspect, the present application provides the use of the isolated antigen binding protein or the polypeptide in the preparation of a kit useful in a method of detecting the presence and/or amount of PD-L1. For example, the method may comprise an in vitro method, an ex vivo method, a method of non-diagnostic or non-therapeutic purpose.
In another aspect, the present application provides a method of modulating an immune response comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition, and/or a pharmaceutically acceptable therapeutic agent.
In this application, the method of modulating an immune response may include in vitro methods, ex vivo methods, methods of non-diagnostic or non-therapeutic purpose.
In another aspect, the present application provides a method of modulating an immune response comprising administering to a subject in need thereof an effective amount of the pharmaceutical combination, and/or a pharmaceutically acceptable therapeutic agent.
In this application, the method of modulating an immune response may include in vitro methods, ex vivo methods, methods of non-diagnostic or non-therapeutic purpose.
In another aspect, the present application provides an isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said vector, said pharmaceutical composition for use in preventing, alleviating and/or treating a disease or disorder.
In this application, the disease or condition may include a tumor. For example, the tumor may comprise a non-solid tumor. For example, the tumor may comprise a tumor associated with the expression of PD-L1. The term "tumor associated with the expression of PD-L1" generally refers to a tumor formed by altered expression of PD-L1 resulting in disease progression or evasion of immune surveillance. For example, the "tumor associated with the expression of PD-L1" may be a tumor formed by up-regulating the expression level of PD-L1 to cause disease progression or evade immune surveillance. The tumor associated with the protein expression of PD-L1 may be a PD-L1 positive tumor. In PD-L1 positive tumors, the protein expression of PD-L1 is about 1%,5%,10%,15%,20%,25%,30%,35%,40%,50%,60%,70%,80% or more on the surface of tumor cells or in the tumor microenvironment compared to normal cells.
In another aspect, the present application provides the use of said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said vector, said cell and/or said pharmaceutical composition for the preparation of a medicament for the prevention, alleviation and/or treatment of a disease or disorder.
In this application, the disease or condition may include a tumor. For example, the tumor may comprise a solid tumor. For example, the tumor may comprise a non-solid tumor. For example, the tumor may comprise a tumor associated with the expression of PD-L1. The term "tumor associated with the expression of PD-L1" generally refers to a tumor formed by altered expression of PD-L1 resulting in disease progression or evasion of immune surveillance. For example, the "tumor associated with the expression of PD-L1" may be a tumor formed by up-regulating the expression level of PD-L1 to cause disease progression or evade immune surveillance. The tumor associated with the protein expression of PD-L1 may be a PD-L1 positive tumor. In PD-L1 positive tumors, the protein expression of PD-L1 is about 1%,5%,10%,15%,20%,25%,30%,35%,40%,50%,60%,70%,80% or more on the surface of tumor cells or in the tumor microenvironment compared to normal cells. In another aspect, the present application provides a method of preventing and/or treating a disease or disorder comprising administering to a subject in need thereof said isolated antigen binding protein, said isolated nucleic acid molecule, said vector, said cell, said pharmaceutical composition.
In this application, the disease or condition may include a tumor. For example, the tumor may comprise a non-solid tumor. For example, the tumor may comprise a tumor associated with the expression of PD-L1. The term "tumor associated with the expression of PD-L1" generally refers to a tumor formed by altered expression of PD-L1 resulting in disease progression or evasion of immune surveillance. For example, the "tumor associated with the expression of PD-L1" may be a tumor formed by up-regulating the expression level of PD-L1 to cause disease progression or evade immune surveillance. The tumor associated with the protein expression of PD-L1 may be a PD-L1 positive tumor. In PD-L1 positive tumors, the protein expression of PD-L1 is about 1%,5%,10%,15%,20%,25%,30%,35%,40%,50%,60%,70%,80% or more on the surface of tumor cells or in the tumor microenvironment compared to normal cells. The pharmaceutical compositions and methods described herein may be used in conjunction with other types of cancer therapies, such as chemotherapy, surgery, radiation, gene therapy, and the like. The pharmaceutical compositions and methods described herein are useful for other immune response dependent disease conditions, such as inflammation, immune disorders, and infectious diseases.
In this application, the subject may include a human or non-human animal. For example, the non-human animal may be selected from the group consisting of: monkey, chicken, goose, cat, dog, mouse and rat. Furthermore, the non-human animal may also include any animal species other than human, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals. The person may be caucasian, african, asian, sphaleid, or other race, or a hybrid of various races. As another example, the person may be an elderly, adult, adolescent, child, or infant.
The effective amount in humans can be presumed from the effective amount in experimental animals. For example, freireich et al describe the relationship of animal and human dose (milligrams per square meter of body surface) (Freireich et al, cancer chemther. Rep.50, 219 (1966)). Body surface area may be approximately determined from the height and weight of the patient. See, e.g., scientific Tables, geigy Pharmaceuticals, ardsley, n.y.,537 (1970).
Without intending to be limited by any theory, the following examples are presented merely to illustrate the antigen binding proteins, multispecific antibodies, bispecific antibodies, methods of manufacture, uses, and the like of the present application and are not intended to limit the scope of the invention of the present application.
Examples
Example 1 preparation and screening of antigen binding proteins
1.1 immunization of animals
anti-PD-L1 antibodies were generated by intramuscular injection of immunized mice with a gene gun (gene gun) encoding the human PDL1 plasmid, feeding environment SPF grade. Mice were kept in a laboratory environment for one week after purchase, and the mice that had been acclimatized were immunized with a Cardiotoxin (Cardiotoxin) adjuvant by intramuscular injection with a gene gun (gene gun) encoding the human PDL1 plasmid. Multiple immunizations were performed, during which serum antibody levels were monitored, mice with high antibody titers in serum were selected for booster immunization, after which mice were sacrificed and spleen cells were taken and fused with myeloma cells. The spleen lymphocytes and myeloma cells Sp2/0 cells are fused to obtain hybridoma cells. The preparation of hybridomas can be carried out according to methods well known in the art.
1.2 screening of antigen binding proteins
ELISA experiments in which antigen binding proteins inhibit the binding of PD-L1 to PD-1
The PD1 protein (Sulfo-NHS-Biotin, pierce) was labeled with Biotin and diluted with PBS buffer; PD-L1 protein (1 ug/mL,100 uL/well, overnight at 4deg.C) was coated in 96-well plates; adding 200 uL/hole sealing liquid for sealing, and washing the plate 3 times by using PBST; anti-PD-L1 antibody (3-fold dilution to 0.005ug/mL,11 dilution points from 300 ug/mL) and PD-1[ B ] (1 ug/mL, in the linear range of PD1-PDL1 binding) were added to the 96-well plate, incubated for 60 min, and the plates were washed 3 times with PBST; HRP-streptavidin was added and OD was detected by ELISA.
The results are shown in FIG. 1, and the antigen binding proteins of the present application are capable of effectively inhibiting the binding of PD-L1 to PD-1, and the IC50 values are shown in Table 1.
TABLE 1 ELISA assay results for inhibition of binding of PD-L1 to PD-1 by antigen binding proteins
Cloning IC50(nM)
13H6D3 1.232
Example 2 humanized design of antigen binding proteins
2.1 humanization of antibodies by CDR grafting: selecting a recipient framework area
The human germline database was searched for the variable domain sequence of the parent antibody using NCBI Ig-Blast (http:// www.ncbi.nlm.nih.gov/subjects/igblast /). Different human receptors (i.e., human variable structures with high homology to the parent antibody) were selected for each heavy and light chain. The CDRs of the human recipient are replaced by murine CDRs, thereby forming a humanized variable domain sequence.
2.2 construction and production of humanized antibodies
DNA sequences encoding chimeric antibodies and humanized IgG heavy and light chains were synthesized and inserted into the pTGE5 vector to construct expression plasmids for full-length IgG. Expression of the chimeric antibodies was performed in 100ml HEK293 cells and the supernatant was purified using a protein A affinity column. Purified antibodies were exchanged into PBS buffer using PD-10 desalting column to give purified antigen binding proteins hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM.
Example 3 affinity detection experiment of humanized antibody for PD-L1
3.1 immobilization of antihuman Fc on CM5 sensor chip
Anti-human Fc was immobilized. The target protein immobilization level was set at 8000RU using HBS-EP+ (10mM HEPES,150mM NaCl,3mM EDTA and 0.05% P20, pH 7.4) as running buffer. Sufficient 50mM NaOH, 50mM N-hydroxysuccinimide (NHS), 200mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 20. Mu.g/mL anti-human Fc (diluted at 10mmol/L NaAc pH 4.5) and 1M ethanolamine were prepared and immobilized to flow cells 1 and 2 using a wizard procedure. To maintain equilibrium after fixation, the buffer flow rate was maintained at 10 μl/min.
3.2 SPR binding between antigen binding proteins and PD-L1
HBS-EP+ was used as flow buffer, including capture of the antibody tested, injection of serial dilutions of PD-L1 and surface regeneration in each kinetic cycle. The antibody to be tested is captured on the flow cell 2 and the flow cell 1 is used as a reference. The diluted PD-L1 fraction was then injected into flow units 1 and 2 (30. Mu.L/min) for a correlation time of 180s. The buffer stream was kept for 900 seconds for dissociation. To remove the samples and analytes tested, regeneration was performed using 30s injection of 10mM glycine-HCl (pH 1.5).
3.3 data analysis
K evaluation of each sample using Biacore T200 (GE Healthcare) evaluation software 1.0 D Values. The results are shown in table 2, where the antigen binding proteins of the present application can bind to PD-L1 with high affinity.
TABLE 2 affinity detection results of humanized antibodies for PD-L1
Example 4Fc engineering to increase half-life and remove glycosylation sites
4.1Fc engineering
Three amino acids in Fc were replaced with YTE to extend the half-life of IgG, and specific mutations were M252Y, S254T, T256E. N297 was mutated to Alanin to remove the glycosylation site.
4.2 affinity detection of FcRn by Fc engineered antigen binding proteins
(1) Immobilization of anti-his antibodies on CM5 sensor
Using HBS-EP+ (pH 6.0) as running buffer, enough 50mM NaOH,50 mmol/LN-hydroxysuccinimide (NHS), 200 mmol/L1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 15. Mu.g/mL anti-his antibody (diluted in 10mmol/L NaAc pH 4.5) and 1M ethanolamine were prepared, and appropriate parameters were set (washing for 30 seconds, activation for 420 seconds, ligand injection for 600 seconds, locking for 420 seconds), immobilization method immobilized anti-his antibody on 16 flow units of one CM5 chip by Biacore 8K. After immobilization, a buffer flow of 10. Mu.L/min was maintained for equilibration.
(2) SPR binding of test samples (antigen binding proteins) to FcRn
HBS-ep+ (ph 6.0) was used as running buffer, including FcRn capture, serial dilution of the test antibody injection and surface regeneration in each kinetic cycle. FcRn of 0.4 μg/mL was captured to flow cell 2 of each channel and flow cell 1 of the same channel was used as reference. Serial dilutions of the sample (30 μl/min) were then injected in the two flow cells of each channel and dissociated. To remove the tested samples and receptors, an interface regeneration was performed using 30s injection of 10mM glycine-HCl (pH 1.5).
(3) Data analysis
Evaluation of K for each sample using Biacore8K evaluation software D Values. The results are shown in table 3, with improved affinity of Fc engineered antigen binding proteins for FcRn. 5BM-Areferstoanantigenbindingproteinobtainedafterfusionof5BMwithaYTEmutantFc,5BM-Breferstoanantigenbindingproteinobtainedafterfusionof5BMwithanunmodifiedFc,230-AreferstoanantigenbindingproteinobtainedafterfusionofAH00230withaYTEmutantFc,and230-BreferstoanantigenbindingproteinobtainedafterfusionofAH00230withanunmodifiedFc.
4.3 affinity detection of Fc engineered antigen binding proteins for PD-L1
The experimental procedure is the same as in example 3. The results are shown in table 4, with improved affinity of Fc engineered antigen binding proteins for PD-L1. wherein5BM-Areferstoanantigenbindingproteinobtainedafterfusionof5BMwithaYTEmutantFc,and230-AreferstoanantigenbindingproteinobtainedafterfusionofAH00230withaYTEmutantFc.
Table 3 affinity of Fc engineered antigen binding proteins for FcRn
TABLE 4 affinity of Fc engineered antigen binding proteins for PD-L1
4.4 ELISA experiments in which the Fc-engineered antigen binding protein inhibited the binding of PD-L1 to PD-1
The PD1 protein (Sulfo-NHS-Biotin, pierce) was labeled with Biotin and diluted with PBS buffer; PD-L1 protein (1 ug/mL,100 uL/well, overnight at 4deg.C) was coated in 96-well plates; adding 200 uL/hole sealing liquid for sealing, and washing the plate 3 times by using PBST; anti-PD-L1 antibody (3-fold dilution to 0.005ug/mL,11 dilution points from 300 ug/mL) and PD-1[ B ] (1 ug/mL, in the linear range of PD1-PDL1 binding) were added to the 96-well plate, incubated for 60 min, and the plates were washed 3 times with PBST; HRP-streptavidin was added and OD was detected by ELISA.
As shown in fig. 2, the Fc-engineered antigen binding protein was able to effectively inhibit the binding of PD-L1 to PD-1. Wherein 230-Ag-YTE represents an antigen binding protein obtained by fusion of AH00230 with Fc mutated with YTE and N297, and 5BM-Ag-YTE refers to an antigen binding protein obtained by fusion of 5BM with Fc mutated with YTE and N297.
4.5 functional cell experiments of Fc engineered antigen binding proteins
After mixed culture of HEK293 cells expressing CD3 antibody fragment/IL-2 gene/PDL 1 and HUTT cells expressing HPD1, binding of PD1 and PDL1 inhibited IL-2 production. PDL1 antibodies block binding and increase IL-2 secretion. As shown in fig. 3, it can be seen that the antigen binding proteins of the present application significantly stimulate secretion of IL-2. Wherein 230-Ag-YTE represents an antigen binding protein obtained by fusion of AH00230 with Fc mutated with YTE and N297, and 5BM-Ag-YTE refers to an antigen binding protein obtained by fusion of 5BM with Fc mutated with YTE and N297.
It should be noted that the foregoing merely illustrates the technical idea of the present invention and is not intended to limit the scope of the present invention, and that a person skilled in the art may make several improvements and modifications without departing from the principles of the present invention, which fall within the scope of the claims of the present invention.
Sequence listing
<110> Nanjing Ji Chengao ma biomedical Co., ltd
<120> PD-L1 antibodies and uses thereof
<150> 2021108351839
<151> 2021-07-23
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<400> 41
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Asp Trp Ile
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 42
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 42
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 43
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 43
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Asp Trp Met
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 44
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 44
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Asp Trp Met
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 45
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 45
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 46
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 46
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Asp Trp Ile
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 47
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 47
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Asp Arg Gln Ala Pro Gly Gln Gly Leu Asp Trp Met
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 48
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 48
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 49
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 49
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Phe Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Phe Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 50
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 50
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 51
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 51
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Phe Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 52
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 52
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Phe Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 53
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 53
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Phe Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 54
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 54
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Phe Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 55
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 55
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 56
<211> 330
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 56
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 57
<211> 629
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 57
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 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
100 105 110
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
115 120 125
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
130 135 140
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
145 150 155 160
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
165 170 175
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
180 185 190
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
195 200 205
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
210 215 220
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro
225 230 235 240
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
245 250 255
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
260 265 270
Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val
275 280 285
Leu Thr Val Leu His Gln Asp Trp Leu Asn Lys Ser Cys Asp Lys Thr
290 295 300
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
305 310 315 320
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg
325 330 335
Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
340 345 350
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
355 360 365
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
370 375 380
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
385 390 395 400
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
405 410 415
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
420 425 430
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
435 440 445
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
450 455 460
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
465 470 475 480
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
485 490 495
Arg Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
500 505 510
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
515 520 525
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
530 535 540
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
545 550 555 560
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
565 570 575
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
580 585 590
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
595 600 605
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
610 615 620
Leu Ser Pro Gly Lys
625
<210> 58
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 58
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> 69
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 69
Gln Val Gln Leu Gln Gln Pro Gly Ala Asp Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Ala Ser Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met
65 70 75 80
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu 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 Asp Glu Leu 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> 70
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 70
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met 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 Asp Glu Leu 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> 71
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 71
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Asp Trp Ile
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met 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 Asp Glu Leu 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> 72
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 72
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met 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 Asp Glu Leu 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> 73
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 73
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Asp Trp Met
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met 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 Asp Glu Leu 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> 74
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 74
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Asp Trp Met
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met 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 Asp Glu Leu 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> 75
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 75
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met 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 Asp Glu Leu 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> 76
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 76
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Asp Trp Ile
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met 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 Asp Glu Leu 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> 77
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 77
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Asp Arg Gln Ala Pro Gly Gln Gly Leu Asp Trp Met
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met 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 Asp Glu Leu 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> 78
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 78
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
20 25 30
Trp Met His Trp Met Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile His Pro Asn Ser Ala Ile Lys Tyr Asn Glu Lys Phe Lys
50 55 60
Ser Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ser Tyr Tyr Gly Ser Ser Pro Tyr Phe Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met 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 Asp Glu Leu 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> 79
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 79
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Phe Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Phe Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 80
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 80
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 81
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 81
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Phe Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 82
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 82
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Phe Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 83
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 83
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Phe Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 84
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 84
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Phe Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 85
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 85
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Gly Asp Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210

Claims (28)

1. An isolated antigen binding protein, which is an antibody or antigen binding fragment thereof, having the following properties:
in Biacore detection, at 8E-09M or lessK D The value specifically binds to human PD-L1 protein;
Can block the combination of PD-1 and PD-L1;
the antigen binding protein comprises a heavy chain variable region VH comprising HCDR1, HCDR2, HCDR3; the HCDR1 is an amino acid sequence shown in SEQ ID NO. 3; the HCDR2 is an amino acid sequence shown in SEQ ID NO. 12; and HCDR3 is the amino acid sequence shown in SEQ ID NO. 18;
the antigen binding protein comprises a light chain variable region VL, wherein the VL comprises LCDR1, LCDR2 and LCDR3, and the LCDR1 of the antigen binding protein is an amino acid sequence shown as SEQ ID NO. 24; the LCDR2 is an amino acid sequence shown as SEQ ID NO. 29; and LCDR3 is the amino acid sequence shown in SEQ ID NO. 36.
2. The isolated antigen binding protein of claim 1, comprising a VH and a VL comprising an amino acid sequence selected from any one of the group consisting of:
VH: SEQ ID NO 39 and VL: SEQ ID NO. 49;
VH: SEQ ID NO. 40 and VL: SEQ ID NO. 50;
VH: SEQ ID NO. 41 and VL: SEQ ID NO. 51;
VH: SEQ ID NO. 42 and VL: SEQ ID NO. 51;
VH: SEQ ID NO. 43 and VL: SEQ ID NO. 52;
f) VH: SEQ ID NO. 44 and VL: SEQ ID NO. 51;
g) VH: SEQ ID NO 45 and VL: SEQ ID NO. 53;
h) VH: SEQ ID NO 46 and VL: SEQ ID NO. 54;
i) VH: SEQ ID NO. 47 and VL: SEQ ID NO. 52; and
j) VH: SEQ ID NO 48 and VL: SEQ ID NO. 55.
3. The isolated antigen binding protein of claim 1, which has an extended half-life compared to a wild-type antibody.
4. The isolated antigen binding protein of claim 1, which is capable of reducing Antibody Dependent Cellular Cytotoxicity (ADCC) as compared to a wild-type antibody.
5. The isolated antigen binding protein of claim 1, comprising a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region or an IgY-derived constant region.
6. The isolated antigen binding protein of claim 5, wherein the heavy chain constant region comprises a constant region derived from IgG1, igG2, igG3 or IgG 4.
7. The isolated antigen binding protein of claim 6, wherein the heavy chain constant region comprises an Fc region derived from IgG 1.
8. The isolated antigen binding protein of claim 3, wherein the heavy chain constant region is mutated at position N297, residues numbered according to the Kabat system; the Fc region in the heavy chain constant region comprises the N297A mutation.
9. The isolated antigen binding protein of claim 3, wherein the heavy chain constant region is mutated at position M252, residues numbered according to the Kabat system; the Fc region in the heavy chain constant region comprises an M252Y mutation.
10. The isolated antigen binding protein of claim 3, wherein the heavy chain constant region is mutated at position S254, residues numbered according to the Kabat system; the Fc region in the heavy chain constant region comprises the S254T mutation.
11. The isolated antigen binding protein of claim 3, wherein the heavy chain constant region is mutated at position T256, residues numbered according to the Kabat system; the Fc region in the heavy chain constant region comprises a T256E mutation.
12. The isolated antigen binding protein of any one of claims 6-11, wherein the heavy chain constant region comprises the amino acid sequence set forth in any one of SEQ ID NOs 56 to 57.
13. The isolated antigen binding protein of claim 1, comprising a light chain constant region, and the light chain constant region comprises an igκ -derived constant region or an igλ -derived constant region.
14. The isolated antigen binding protein of claim 13, wherein the light chain constant region comprises a constant region derived from human igκ.
15. The isolated antigen binding protein of claim 13, wherein said light chain constant region comprises the amino acid sequence set forth in SEQ ID No. 58.
16. The isolated antigen binding protein of claim 1, comprising a heavy chain HC and a light chain LC comprising an amino acid sequence selected from any one of the group consisting of seq id nos:
a) HC: SEQ ID NO 69 and LC: SEQ ID NO. 79;
b) HC: SEQ ID NO 70 and LC: 80 of SEQ ID NO;
c) HC: SEQ ID NO 71 and LC: SEQ ID NO. 81;
d) HC: SEQ ID NO 72 and LC: SEQ ID NO. 81;
e) HC: SEQ ID NO 73 and LC: SEQ ID NO. 82;
f) HC: SEQ ID NO 74 and LC: SEQ ID NO. 81;
g) HC: SEQ ID NO 75 and LC: 83 of SEQ ID NO;
h) HC: SEQ ID NO 76 and LC: SEQ ID NO. 84;
i) HC: SEQ ID NO 77 and LC: SEQ ID NO. 82; and
j) HC: SEQ ID NO 78 and LC: SEQ ID NO. 85.
17. The isolated antigen binding protein of claim 1, wherein the antigen binding fragment is selected from the group consisting of: fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, di-scFv.
18. The isolated antigen binding protein of claim 1, wherein the antibody is selected from the group consisting of: monoclonal antibodies, single chain antibodies, multispecific antibodies, bispecific antibodies.
19. An immunoconjugate comprising the isolated antigen binding protein of claim 1.
20. An isolated nucleic acid molecule encoding the isolated antigen binding protein of claim 1.
21. A vector comprising the isolated nucleic acid molecule of claim 20.
22. A cell comprising the isolated antigen binding protein of claim 1, the immunoconjugate of claim 19, the isolated nucleic acid molecule of claim 20 and/or the vector of claim 21.
23. A method of making the isolated antigen binding protein of claim 1, comprising culturing the cell of claim 22 under conditions such that the isolated antigen binding protein of claim 1 is expressed.
24. A pharmaceutical composition comprising the isolated antigen binding protein of claim 1, the immunoconjugate of claim 19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, the cell of claim 22, and/or a pharmaceutically acceptable adjuvant and/or excipient.
25. A PD-L1 detection kit comprising the isolated antigen binding protein of claim 1.
26. Use of the isolated antigen binding protein of claim 1 in the preparation of a kit for detecting the presence and/or amount of PD-L1.
27. Use of the isolated antigen binding protein of claim 1, the immunoconjugate of claim 19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, the cell of claim 22 and/or the pharmaceutical composition of claim 24 for the preparation of a medicament for the prevention and/or treatment of a solid tumor disease or disorder.
28. The use of claim 27, wherein the tumor is breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and/or liver cancer.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108337890A (en) * 2015-06-23 2018-07-27 纪念斯隆-凯特琳癌症中心 Novel PD-1 immunomodulators
CN110790839A (en) * 2018-08-03 2020-02-14 江苏恒瑞医药股份有限公司 anti-PD-1 antibody, antigen binding fragment thereof and medical application
WO2020127366A1 (en) * 2018-12-21 2020-06-25 Ose Immunotherapeutics Humanized anti-human-pd-1 antibody
CN111363041A (en) * 2016-03-23 2020-07-03 迈博斯生物医药(苏州)有限公司 Novel anti-PD-L1 antibodies

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101814408B1 (en) * 2008-09-26 2018-01-04 다나-파버 캔서 인스티튜트 인크. Human anti-pd-1, pd-l1, and pd-l2 antibodies and uses therefor
MX2017001597A (en) * 2014-08-05 2017-11-17 Cb Therapeutics Inc Anti-pd-l1 antibodies.
CN109071656B (en) * 2017-01-05 2021-05-18 璟尚生物制药公司 Checkpoint modulator antagonists
CN111542543B (en) * 2017-12-28 2023-12-22 南京传奇生物科技有限公司 Antibodies to PD-L1 and variants thereof
KR20200108306A (en) * 2018-01-10 2020-09-17 지앙수 헨그루이 메디슨 컴퍼니 리미티드 PD-L1 antibody or antigen-binding fragment thereof, and pharmaceutical use thereof
EP3863722A2 (en) * 2018-10-10 2021-08-18 Tilos Theapeutics, Inc. Anti-lap antibody variants and uses thereof
EP4056592A4 (en) * 2019-11-08 2024-03-20 Jiangsu Simcere Pharm Co Ltd Anti-human programmed cell death ligand-1 (pd-l1) antibody and use thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108337890A (en) * 2015-06-23 2018-07-27 纪念斯隆-凯特琳癌症中心 Novel PD-1 immunomodulators
CN111363041A (en) * 2016-03-23 2020-07-03 迈博斯生物医药(苏州)有限公司 Novel anti-PD-L1 antibodies
CN110790839A (en) * 2018-08-03 2020-02-14 江苏恒瑞医药股份有限公司 anti-PD-1 antibody, antigen binding fragment thereof and medical application
WO2020127366A1 (en) * 2018-12-21 2020-06-25 Ose Immunotherapeutics Humanized anti-human-pd-1 antibody

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