CN115521378A - PD-L1 antibodies and uses thereof - Google Patents

PD-L1 antibodies and uses thereof Download PDF

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CN115521378A
CN115521378A CN202210630162.8A CN202210630162A CN115521378A CN 115521378 A CN115521378 A CN 115521378A CN 202210630162 A CN202210630162 A CN 202210630162A CN 115521378 A CN115521378 A CN 115521378A
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amino acid
antigen binding
binding protein
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CN115521378B (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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    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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    • C12N2800/00—Nucleic acids vectors
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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 has a K of about 8E-09M or less D The polypeptide specifically binds to human PD-L1 protein and can block the binding of PD-L1 and PD-1; the application also relates to the engineering of the Fc region in said 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 medicines, in particular to a PD-L1 antibody and application thereof.
Background
Programmed death receptor ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD 274) or B7 homologous protein 1 (B7-H1), is a 40KDa type I transmembrane protein that is commonly 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 signal channel is a very important co-inhibition signal path in immune reaction, negatively regulates T cell immune response, inhibits T cell activity and weakens the secretion of cell factors. Researches show 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, mastocytosis, malignant melanoma and the like, and can be expressed in bone marrow cells infiltrating tumor microenvironment to protect tumor cells from immune attack.
Various mechanisms of immunosuppression may prevent immunotherapy from being effective. Therefore, the development of an anti-PD-L1 antibody with high affinity and the capability of blocking a PD-L1/PD-1 signal pathway is urgently needed, and a brand-new solution can be brought to 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 a Biacore assay, with a K of about 8E-09M or less D Specifically binding to human PD-L1 protein; 2) Can block the combination of PD-1 and PD-L1.
In certain embodiments, the antigen binding protein comprises HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in SEQ ID No. 18.
In certain embodiments, the antigen binding protein comprises HCDR2, wherein the HCDR2 comprises the amino acid sequence set forth in SEQ ID No. 12.
In certain embodiments, the antigen binding protein comprises HCDR1, wherein 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 shown in SEQ ID NO: 67.
In certain embodiments, the antigen binding protein comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH as set forth in any one of SEQ ID No. 39 to SEQ ID No. 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 set forth in SEQ ID No. 18; the HCDR2 comprises an amino acid sequence shown as 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 the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO 59.
In certain embodiments, the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO 1 to SEQ ID NO 2.
In certain embodiments, the antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 60.
In certain embodiments, the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NO 4 to SEQ ID NO 11.
In certain embodiments, the antigen binding protein comprises H-FR3, the H-FR3 is positioned between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO 61.
In certain embodiments, the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NO 13 to SEQ ID NO 17.
In certain embodiments, the antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is linked directly or indirectly to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence set forth 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 to SEQ ID NO 20.
In certain embodiments, the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 59; the H-FR2 comprises an amino acid sequence shown in SEQ ID NO: 60; the H-FR3 comprises an amino acid sequence shown in SEQ ID NO. 61; and the H-FR4 comprises an 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, wherein the H-FR1 comprises the amino acid sequence set forth 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 an 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 the antigen binding protein comprise an amino acid sequence selected from any one of the following groups:
a) H-FR1: 1, H-FR2: 4, H-FR3: 13 and H-FR4: 19 in SEQ ID NO;
b) H-FR1: 2, H-FR2: 5, H-FR3: 14 and H-FR4: 20 is SEQ ID NO;
c) H-FR1: 2, H-FR2: 6, H-FR3: 15 and H-FR4: 20 is SEQ ID NO;
d) H-FR1: 2, H-FR2: 7, H-FR3: 16 and H-FR4: 20 is SEQ ID NO;
e) H-FR1: 2, H-FR2: 8, H-FR3: 17 and H-FR4: 20 in SEQ ID NO;
f) H-FR1: 2, H-FR2: 9, H-FR3: 14 and H-FR4: 20 is SEQ ID NO;
g) H-FR1: 2, H-FR2: 7, H-FR3: 14 and H-FR4: 20 in SEQ ID NO;
h) H-FR1: 2, H-FR2: 6, H-FR3: 16 and H-FR4: 20 in SEQ ID NO;
i) H-FR1: 2, H-FR2: 10, H-FR3: 14 and H-FR4: 20 is SEQ ID NO; and
j) H-FR1: 2, H-FR2: 11, H-FR3: 15 and H-FR4: 20 in SEQ ID NO.
In certain embodiments, the antigen binding protein comprises a heavy chain variable region VH comprising the amino acid sequence set forth 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 an LCDR3, the LCDR3 comprising the amino acid sequence set forth in SEQ ID No. 36.
In certain embodiments, the antigen binding protein comprises LCDR2, wherein the LCDR2 comprises the amino acid sequence set forth in SEQ ID No. 29.
In certain embodiments, the antigen binding protein comprises LCDR1, wherein the LCDR1 comprises the amino acid sequence set forth 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 of any one of SEQ ID No. 68.
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 No. 49 to SEQ ID No. 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 set forth in SEQ ID NO: 36; the LCDR2 comprises an amino acid sequence shown as SEQ ID NO. 29; and the LCDR1 comprises an amino acid sequence shown as SEQ ID NO. 24.
In certain embodiments, the antigen binding protein comprises L-FR1, the C-terminus of the L-FR1 is linked directly or indirectly to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence set forth 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, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and wherein the L-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 64.
In certain embodiments, the L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NO 25 to SEQ ID NO 28.
In certain embodiments, the antigen binding protein comprises L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and wherein the L-FR3 comprises the amino acid sequence set forth in SEQ ID NO. 65.
In certain embodiments, the L-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NO 30 to SEQ ID NO 35.
In certain embodiments, the antigen binding protein comprises L-FR4, the N-terminus of the L-FR4 is linked directly or indirectly to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence set forth in SEQ ID NO. 66.
In certain embodiments, the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO 37 to SEQ ID NO 38.
In certain embodiments, the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein the L-FR1 comprises the amino acid sequence set forth 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 in SEQ ID NO. 65; and the L-FR4 comprises an amino acid sequence shown in SEQ ID NO: 66.
In certain embodiments, the antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein the L-FR1 comprises the amino acid sequence set forth 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-SEQ ID NO 35; and the L-FR4 comprises an 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 comprise an amino acid sequence selected from any one of the following groups:
a) L-FR1: 21, L-FR2: 25, L-FR3: 30 and L-FR4: 37 is SEQ ID NO;
b) L-FR1: 22, L-FR2: 26, L-FR3: 31 and L-FR4: 38;
c) L-FR1: 23, L-FR2: 27, L-FR3: 32 and L-FR4: 38, SEQ ID NO;
d) L-FR1: 22, L-FR2: 27, L-FR3: 32 and L-FR4: 38, SEQ ID NO;
e) L-FR1: 22, L-FR2: 27, L-FR3: 34 and L-FR4: 38, SEQ ID NO;
f) L-FR1: 23, L-FR2: 27, L-FR3: 33 and L-FR4: 38, SEQ ID NO; and
g) L-FR1: 22, L-FR2: 28, L-FR3: 35 and L-FR4: 38 in SEQ ID NO.
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 No. 49 to SEQ ID No. 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: 39 and VL: 49 in SEQ ID NO;
b) VH: 40 and VL: 50 in SEQ ID NO;
c) VH: 41 and VL: 51 is SEQ ID NO;
d) VH: 42 and VL: 51 is SEQ ID NO;
e) VH: 43 and VL: 52, SEQ ID NO;
f) VH: 44 and VL: 51 is SEQ ID NO;
g) VH: 45 and VL: 53 in SEQ ID NO;
h) VH: 46 and VL: 54 in SEQ ID NO;
i) VH: 47 and VL: 52, SEQ ID NO; and
j) VH: 48 and VL: SEQ ID NO:55.
In certain embodiments, the antigen binding protein has an extended half-life as 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 of the isolated antibody binding protein comprises an IgG-derived constant region.
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 and residues are numbered according to the Kabat system.
In certain embodiments, the heavy chain constant region is mutated at position M252 and residues are numbered according to the Kabat system.
In certain embodiments, the heavy chain constant region is mutated at position S254 and residues are numbered according to the Kabat system.
In certain embodiments, the heavy chain constant region is mutated at position T256 and residues are numbered according to the Kabat system.
In certain embodiments, the Fc region in the heavy chain constant region comprises the N297A mutation.
In certain embodiments, the Fc region in the heavy chain constant region comprises the 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 NO 56 to SEQ ID NO 57.
In certain embodiments, the antigen binding protein comprises a light chain constant region, and the light chain constant region comprises a constant region derived from Ig κ or a constant region derived from Ig λ.
In certain embodiments, the light chain constant region comprises a constant region derived from human Ig κ.
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 SEQ ID NO:69-78, or a pharmaceutically acceptable salt thereof.
In certain embodiments, the antigen binding protein comprises a light chain LC comprising the amino acid sequence of SEQ ID NO:79 to 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 fragments, 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 the isolated antigen binding protein or the 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 making the isolated antigen binding protein or the polypeptide, the method comprising culturing the cell under conditions such that the isolated antigen binding protein or the 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 determining PD-L1, said method comprising using said isolated antigen binding protein or said polypeptide.
In another aspect, the present application provides a detection kit for PD-L1, comprising said isolated antigen binding protein or said polypeptide.
In another aspect, the 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 the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition in the manufacture 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, 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 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 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, 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 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, stomach 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 be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
Drawings
The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The drawings are briefly described as follows:
FIG. 1 shows ELISA assays for the inhibition of the binding of PD-L1 to PD-1 by the antigen binding proteins described herein.
FIG. 2 shows ELISA assays for the inhibition of the binding of PD-L1 to PD-1 by the Fc engineered antigen binding proteins described herein.
FIG. 3 shows the results of functional cellular assays for Fc engineered antigen binding proteins described herein.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of the present specification.
Definition of terms
In the present application, the term "isolated" generally refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or the substance may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" does not exclude the presence of other impurities which do not interfere with the activity of the substance, either mixed with artificial or synthetic substances.
In the present application, the term "antigen binding protein" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a particular antigen. For example, in the present application, the term "antigen binding protein" may include an "antibody" or an "antigen binding fragment". 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 include 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, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein. 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. The VH and VL regions can be further distinguished as hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions termed Framework Regions (FRs). 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 the antibody may mediate the 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 according to different systems. The system described by Kabat (Kabat et al, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987) and (1991)) provides not only a clear residue numbering system applicable to any variable region of an antigen binding fragment, but also precise residue boundaries defining the CDRs, which can be referred to as KaCDRs, chothia and coworkers (Chothia and Lesk, J.mol.biol.196:901-917 (1987) and Chothia et al, nature 342-877-883 (1989)) found that despite large diversity at the amino acid sequence level, certain sub-parts within the Kabat CDRs adopt nearly identical peptide backbone conformations, these sub-parts being designated L1, L2 and L3 or H1, H2 and H3, where "L" and "H" refer to light chain and heavy chain regions, respectively.
In the present application, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that function to specifically bind antigen. The antigen binding function of an antibody can 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 'or F (ab') 2, or a light chain comprising a fragment of Fv, scFv, dsFv, 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 the VH and CH domains; (4) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (5) dAb fragments consisting of VH domains (Ward et al, (1989) Nature341: 544-546); (6) An isolated Complementarity Determining Region (CDR), and (7) a combination of two or more isolated CDRs which may optionally be joined by a linker. For example, monovalent single chain molecules Fv (scFv) formed by pairing VL and VH can also be included (see Bird et al (1988) Science 242, 423-426; and Huston et al (1988) Proc. Natl. Acad. Sci.85: 5879-5883). For example, a class of antibody VHHs that lacks the antibody light chain and has only the variable region of the heavy chain can also be included (see, e.g., shenzhen et al, bioEngineers, 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 the 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 homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. For example, the monoclonal antibodies can be prepared by hybridoma technology or produced in bacterial, eukaryotic animal or plant cells using recombinant DNA methods. Monoclonal antibodies can also be obtained from phage antibody libraries using techniques such as those described in Clackson et al, nature, 352.
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 region of an antibody from one animal species (e.g., mouse, rat, etc.), while the constant portions are homologous to antibody sequences from another species (e.g., human). For example, to obtain a chimeric antibody, B cells or hybridoma cells of non-human origin can be used to produce the variable region, in combination with a constant region from a human. 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. Also, since the constant region of a chimeric antibody may be of human origin, the possibility of the antibody eliciting an immune response upon injection of the chimeric antibody may be lower than if the constant region were of non-human origin.
In this application, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of the xenogenous 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 (1986)) and variants thereof; including "remodeling" (remodeling), (Verhoeyen, et al, 1988science 239 1534-1536, riechmann, et al, 1988Nature 332, 323-337 Tempest, et al, bio/Technol 1991, 266-271), "hyperaddition" (hyperpolymerization), (Queen, et al, 1989Proc Natl Acad Sci USA 86. Other regions, such as the hinge region and constant region domains, may also be humanized if they are also derived from non-human sources.
In this application, the term "murine antibody" generally refers to an antibody in which the variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains constant regions, it is also derived from mouse germline immunoglobulin sequences. The murine antibodies of the present application may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences, for example may include mutations introduced by random or point mutations in vitro or by somatic mutations in vivo.
In the present application, the term "PD-L1" generally refers to programmed death ligand 1 protein, functional variants thereof, and/or functionally active fragments 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, autoimmunized cardio in PD-1 receptor-parameter science, 2001. Complexation of PD-L1 and PD-1 exerts immunosuppressive effects by inhibiting T-cell proliferation and production of cytokines IL-2 and IFN- γ (Freeman et al, engage of PD-1 immunogenic receptor by a novel B7 family member leaves to a novel regulation of lymphocyte activation, J.Exp.Med.2000, 192. 1027-1034 et al, PD-1. The term "PD-L1" encompasses any native 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, as well as any form of PD-L1 that results from processing in a cell. PD-L1 may exist 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 Q9NZQ 7. 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 include a domain that binds to an antigen binding protein of the present application.
The term "functionally active fragment" refers to a polypeptide having substantially the same amino acid sequence as a naturally occurring sequence or encoded by substantially the same nucleotide sequence and capable of having one or more of the activities of a naturally occurring sequence. In the context of this application, a functionally active fragment of any given sequence refers to a sequence in which the 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 a 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 a naturally occurring protein and/or polynucleotide, so 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 residues (or nucleotides) to a polypeptide or polynucleotide of the present application, provided that the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
In the present application, the term "analog" generally with respect to a polypeptide or polynucleotide 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.
Typically, amino acid substitutions, such as at least 1 (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions can be made 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 the present application, the term "homologue" generally refers to an amino acid sequence or a nucleotide sequence having a certain homology with a naturally occurring sequence. The term "homology" may be equivalent to sequence "identity". A homologous sequence can include an amino acid sequence that can 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, homologues will comprise the same active site etc. as the subject amino acid sequence. Homology may be considered in terms of similarity (i.e., amino acid residues with similar chemical properties/functions), or may be expressed in terms of sequence identity. In the present application, a sequence having a percent identity of any one of SEQ ID NOs of the referenced amino acid sequence or nucleotide sequence refers to a sequence having said percent identity over the entire length of the referenced SEQ ID NOs. To determine sequence identity, sequence alignments can be performed, which can be performed by various means known to those skilled in the art, e.g., using BLAST, BLAST-2, ALIGN, needlet, or Megalign (DNASTAR) software, etc. One skilled in the art will be able to determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment over the full length of the sequences being compared.
The 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 on the basis of 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 without an electrically polar head group having similar hydrophilicity values include asparagine, glutamine, serine, threonine, and tyrosine.
In the present application, the term "tumor" generally refers to a neoplasm formed by local tissue cell proliferation under the action 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 expression of PD-L1. The term "tumor associated with 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 expression of PD-L1" may be a tumor formed by upregulation of PD-L1 expression leading to disease progression or evasion of immune surveillance. The tumor associated with protein expression of PD-L1 may be a PD-L1 positive tumor. In a PD-L1 positive tumor, PD-L1 is expressed at about 1%,5%,10%,15%,20%,25%,30%,35%,40%,50%,60%,70%,80% or more of the protein on the surface of the tumor cell or in the tumor microenvironment as compared to normal cells.
In the present application, the term "solid tumor" generally refers to a solid mass that can be detected by clinical examination (e.g., X-ray film, CT scan, B-ultrasound 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 this application, the term "non-solid tumor" generally refers to tumors that are not visible or palpable by X-ray, CT scan, B-ultrasound, and palpation. For example, the non-solid tumor may comprise leukemia. For example, the non-solid tumor may comprise lymphoma. For example, the non-solid tumor may comprise multiple myeloma.
In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating (e.g., covalently linking via a linking molecule) the additional therapeutic agent to the isolated antigen binding protein, which conjugate can deliver the additional therapeutic agent to a target cell (e.g., a tumor cell) via specific binding of the isolated antigen binding protein to an antigen on the target cell. Furthermore, the antigen may also be secreted by the target cell and located in a space outside the target cell.
In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.
In the present application, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length, isolated from their natural environment or artificially 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 can transfer the inserted nucleic acid molecule into and/or between cells. The vector may include a vector mainly for inserting a DNA or RNA into a cell, a vector mainly for replicating a DNA or RNA, and a vector mainly for expression of transcription and/or translation of a DNA or RNA. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell. Typically, the vector will produce the desired expression product by culturing an appropriate cell containing the vector. In the present 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 may already contain a plasmid or vector comprising a nucleic acid molecule described herein, or that is capable of expressing a polypeptide described herein or an antigen binding protein described herein. The cell may comprise progeny of a single cell. Due to natural, accidental, or deliberate mutation, the progeny cells may not be identical in morphology or in genome to the original parent cell, but may be capable of expressing the polypeptide or antigen binding protein described herein. The cells can be obtained by in vitro transfection of cells using the vectors described herein. The cell may be prokaryotic (e.g., E.coli) or eukaryotic (e.g., yeast cell, such as COS cell, chinese Hamster Ovary (CHO) cell, heLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell). In some embodiments, the cell may be an immune cell. For example, the immune cell 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 the present application, the term "treatment" generally means: (i) Preventing the occurrence of a disease, disorder, or condition in a patient who may be predisposed to the disease, disorder, and/or condition, but has not yet been diagnosed as having the disease; (ii) Inhibiting the disease, disorder or condition, i.e., arresting its development; and (iii) ameliorating 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 the present application, the terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and generally refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it 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 (acetylation), acetylation, phosphorylation, or any other manipulation (e.g., binding to a labeling component). The term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine as well as the D and L optical isomers, as well as amino acid analogs and peptidomimetics.
In the present 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. The polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a coding or non-coding region of a gene or gene fragment, multiple loci (one locus) defined according to ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide 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 moiety.
In this application, the term "K D "(likewise," K D 'OR' K D ") generally refers to an" affinity constant "or an" equilibrium dissociation constant, "and refers to a value obtained at equilibrium, or by dividing the dissociation rate constant (kd) by the association rate constant (ka), in a titration measurement. Using the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (K) D ) Representing binding proteins (e.g., isolated antigen binding proteins described herein) to an antigen (e.g., PD-L1 protein)Binding affinity. 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 the sample at equilibrium in physiological buffer. For example, the K can be determined by Biacore (biomolecule interaction analysis) (e.g., instruments available from BIAcorenterationalaB, aGEHealthcarecompany, uppsala, sweden) D Other experimental approaches and instruments such as Octet detection may also be used. In addition, the K can also be measured using KinExA (dynamic exclusion assay) available from Sapidyne instruments (Boise, idaho) D Value, or measuring said K using Surface Plasmon Resonance (SPR) D The value is obtained. For example, the K can also be determined by an amine coupling kit D The value is obtained.
In this application, the term "and/or" should be understood to mean either one of the options or both of the options.
In the present application, the term "comprising" is generally intended to include the explicitly specified features, but not to exclude other elements. In some cases, "comprising" also covers the case where only the specified components are included. For example, inclusion is also denoted by the meaning of also "consisting of 8230%, \8230; composition.
In this application, the term "about" generally means varying by 0.5% -10% above or below the stated value, for example, varying by 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 stated value.
In this application, the term "comprising" is used in a generic sense to mean including, summarizing, containing or encompassing. In some cases, the meaning of "is", "consists of" \8230; "\8230".
Detailed Description
Isolated antigen binding proteins as described herein
In one aspect, the present application provides an isolated antigen binding protein that can have 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 can 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 a VH of a heavy chain variable region of an antibody, which VH may comprise the amino acid sequence shown in SEQ ID NO: 67.
For example, the VH may comprise the amino acid sequence set forth 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 classified in any form, and as long as the VH is identical to the amino acid sequence shown in any one of SEQ ID No. 39 to SEQ ID No. 48, the HCDR obtained by the classification in any form may fall 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 in contact with an antigen or an antigenic epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, kabat, chothia, IMGT, abM, consensus Kabat/Chothia, and the like. Such encoding systems are known in the art and may be found, for example, in http:// www. Bio in. Org. Uk/abs/index. Html # kabatnum. One skilled in the art can determine the CDR regions using different coding systems depending on the sequence and structure of the antibody. Using different coding systems, the CDR regions may differ. In the present application, the CDR encompasses CDR sequences divided according to any CDR division manner; variants thereof are also contemplated, the variants comprising the amino acid sequence of the CDR substituted, deleted and/or added with one or more amino acids. E.g., 1-30, 1-20 or 1-10, further e.g., 1, 2,3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or insertions; also encompassed are homologs thereof, which can 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 sequence of the CDR. In certain embodiments, the isolated antigen binding protein described herein is 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 HCDR3.
In the present application, the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown 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 the present 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 can be defined according to the Kabat coding system.
In the present application, the HCDR1 of the antigen binding protein can 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 can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 12; and the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 18. For example, the antigen binding protein may comprise the antibodies 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM or an antigen binding fragment thereof having the same HCDR3 (e.g., the same HCDR1-3 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 in SEQ ID NO. 59. For example, the H-FR1 and SEQ ID NO of the antigen binding protein59, there is an amino acid substitution (e.g., a conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: 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 Can be Q or V, X 7 Can be P or S, X 10 Can be D or E, X 11 Can be L or V, X 12 Can be K or V, X 16 Can be A or S, X 20 May be L or V.
In the present application, H-FR1 of the antigen-binding protein may comprise an 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 the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 60. For example, the H-FR2 of the antigen-binding protein has an amino acid substitution (e.g., a conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: 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 Can be K or R, X 5 Can be A or R, X 11 Can be D or E, X 13 May be I or M.
In the present application, H-FR2 of the antigen-binding protein may comprise an 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 H-FR3 of the antigen-binding protein has an amino acid substitution (e.g., a conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: 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 Can be A or V, X 3 Can be S or T, X 4 Can be I or L, X 10 Can be S or T, X 16 Can be E or Q, X 21 Can be R or T, X 25 May be S or T.
In the present application, H-FR3 of the antigen-binding protein may comprise an amino acid sequence shown in any one of SEQ ID NO 13 to SEQ ID NO 17.
In the present application, the H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 62. For example, the H-FR4 of the antigen-binding protein has an amino acid substitution (e.g., a conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: 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, H-FR4 of the antigen-binding protein may comprise an amino acid sequence shown in any one of SEQ ID NO 19 to SEQ ID NO 20.
In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 59; the H-FR2 can comprise an amino acid sequence shown as SEQ ID NO: 60; the H-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 61; and the H-FR4 can comprise an amino acid sequence shown in SEQ ID NO: 62.
In the present application, H-FR1 of the antigen-binding protein may comprise an 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 shown in any one of SEQ ID NO 4 to SEQ ID NO 11; the H-FR3 can comprise an amino acid sequence shown in any one of SEQ ID NO 13 to SEQ ID NO 17; and the H-FR4 may comprise an 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 can comprise an amino acid sequence shown in SEQ ID NO. 4; the H-FR3 can comprise an amino acid sequence shown as SEQ ID NO 13; and the H-FR4 can comprise an amino acid sequence shown in SEQ ID NO. 19. For example, the antigen binding protein can include antibody 13H6D3 or an antigen binding fragment thereof having the same H-FR1-4.
In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 5; the H-FR3 can comprise an amino acid sequence shown as 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 can comprise the antibody hu13H6D3 or an antigen binding fragment thereof having the same H-FR1-4.
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 can comprise an amino acid sequence shown as SEQ ID NO. 6; the H-FR3 can comprise an amino acid sequence shown as 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 can include antibody 5BM or an antigen binding fragment thereof having the same H-FR1-4.
In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 7; the H-FR3 can comprise an 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-FR1-4.
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 can comprise an amino acid sequence shown as SEQ ID NO. 8; the H-FR3 can comprise an amino acid sequence shown as 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-FR1-4.
In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 9; the H-FR3 can comprise an amino acid sequence shown as 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-FR1-4.
In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 7; the H-FR3 can comprise an amino acid sequence shown as 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 can include antibody AH00231 or an antigen binding fragment thereof having the same H-FR1-4.
In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 6; the H-FR3 can comprise an 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-FR1-4.
In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 10; the H-FR3 can comprise an 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-FR1-4.
In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 11; the H-FR3 can comprise an amino acid sequence shown as 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 can include the antibody CBM or an antigen binding fragment thereof having the same H-FR1-4.
In the present 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 an amino acid substitution (e.g., a conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of SEQ ID NO: 67: 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 Can be Q or V, X 7 Can be P or S, X 10 Can be D or E, X 11 Can be L or V, X 12 Can be K or V, X 16 Can be A or S, X 20 Can be L or V, X 37 Can be D, M or V, X 38 Can be K or R, X 40 Can be A or R, X 46 Can be D or E, X 48 Can be I or M, X 67 Can be A or V, X 68 Can be S or T, X 69 Can be I or L, X 75 Can be S or T, X 81 Can 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 an amino acid sequence shown in any one of SEQ ID NO:39 to SEQ ID NO: 48.
In the present 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 the present application, the antigen binding protein may comprise at least one CDR in an antibody light chain variable region VL, which may comprise the amino acid sequence set forth 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 to SEQ ID NO. 55. In the present application, the LCDR of the isolated antigen binding protein may be classified in any form, and as long as the VL is identical to the amino acid sequence shown in any one of SEQ ID No. 49 to SEQ ID No. 55, the LCDR obtained by the classification in any form may fall within the scope of the present application.
In the present application, the antigen binding protein may comprise a light chain variable region VL, which may comprise at least one, at least two, or at least three of LCDR1, LCDR2, and LCDR3.
In the present application, the LCDR3 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 36. For example, the LCDR3 of the antigen binding protein may be defined according to the Kabat numbering system.
In the present application, the LCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 29. For example, the LCDR2 of the antigen binding protein may be defined according to the Kabat numbering system.
In the present application, the LCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 24. For example, the LCDR1 of the antigen binding protein may be defined according to the Kabat numbering system.
For example, LCDR1 of an antigen binding protein described herein can comprise the amino acid sequence shown in SEQ ID NO. 24; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 29; and the LCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 36. For example, the antigen binding protein may comprise the antibodies 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM or an antigen binding fragment thereof having the same LCDR3 (e.g., having the same LCDR1-3 therewith).
For example, the VL of the antigen binding protein may comprise the framework regions L-FR1, L-FR2, L-FR3 and L-FR4.
In the present application, L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 63. For example, the L-FR1 of the antigen-binding protein has an amino acid substitution (e.g., a conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: 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 Can be P or T, X 15 Can be L or V, X 22 May be S or T.
In the present application, L-FR1 of the antigen-binding protein may comprise an amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO: 23.
In the present application, L-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 64. For example, the L-FR2 of the antigen-binding protein has an amino acid substitution (e.g., a conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: 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 Can be D or G, X 8 Can be G or K, X 9 Can be A or T, X 10 Can be P or V, X 12 May be F or L.
In the present application, L-FR2 of the antigen-binding protein may comprise an amino acid sequence shown in any one of SEQ ID NO. 25 to SEQ ID NO. 28.
In the present application, the L-FR3 of the antigen-binding protein may comprise65 in sequence as shown in SEQ ID NO. For example, the L-FR3 of the antigen-binding protein has an amino acid substitution (e.g., a conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: 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 Can be E or Q, X 24 Can be P or Q, X 27 Can be F or V, X 31 May be F or Y.
In the present application, L-FR3 of the antigen-binding protein may comprise an amino acid sequence shown in any one of SEQ ID NO. 30 to SEQ ID NO. 35.
In the present application, the L-FR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO 66. For example, the L-FR4 of the antigen-binding protein has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: x 7 。
FGGGTKX 7 EIK (SEQ ID NO: 66) wherein X 7 May be L or V.
In the present application, L-FR4 of the antigen-binding protein may comprise an amino acid sequence shown in any one of SEQ ID NO 37 to SEQ ID NO 38.
In the present application, L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 63; the L-FR2 can comprise an amino acid sequence shown as SEQ ID NO: 64; the L-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 65; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 66.
In the present application, L-FR1 of the antigen-binding protein may comprise an 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 shown in any one of SEQ ID NO 25 to SEQ ID NO 28; the L-FR3 may comprise an amino acid sequence shown in any one of SEQ ID NO 30 to SEQ ID NO 35; and the L-FR4 may comprise an amino acid sequence shown in any one of SEQ ID NO 37 to SEQ ID NO 38.
In the present application, L-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 21; the L-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 25; the L-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 30; and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO 37. For example, the antigen binding protein may comprise the antibody 13H6D3 or an antibody having the same L-FR1-4 as it does.
In the present application, L-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 22; the L-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 26; the L-FR3 can comprise an amino acid sequence shown as 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 can include the antibody hu13H6D3 or an antibody having the same H-FR1-4.
In the present application, L-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the L-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 27; the L-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 32; and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO 38. For example, the antigen binding protein can include antibody 5BM or an antibody having the same L-FR1-4 as it.
In the present application, L-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the L-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 27; the L-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 32; and said 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.
In the present application, L-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the L-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 27; the L-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 33; and said 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.
In the present application, L-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the L-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 27; the L-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 32; and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO 38. For example, the antigen binding protein can include antibody AH00230 or an antibody having the same L-FR1-4.
In the present application, L-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 22; the L-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 27; the L-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 32; and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO 38. For example, the antigen binding protein can include antibody AH00231 or an antibody having the same H-FR1-4.
In the present application, L-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 22; the L-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 27; the L-FR3 can comprise an amino acid sequence shown as 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 can include antibody AH00232 or an antibody having the same L-FR1-4.
In the present application, L-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 23; the L-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 27; the L-FR3 can comprise an 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.
In the present application, L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 22; the L-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 28; the L-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 35; and said 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.
In the present application, the antigen binding protein may comprise a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO. 68. For example, the VL of the antigen binding protein has an amino acid substitution (e.g., a conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of SEQ ID NO: 68: 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 Can be P or T, X 15 Can be L or V, X 22 Can be S or T, X 41 Can be D or G, X 42 Can be G or K, X 43 Can be A or T, X 44 Can 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 Can be E or Q, X 80 Can 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 NO. 49 to SEQ ID NO. 55.
In the present application, the antigen binding protein may comprise a light chain constant region, which may comprise a constant region derived from Ig κ or a constant region derived from Ig λ.
For example, the light chain constant region can include a constant region derived from Ig κ.
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 protein may comprise HCDR1-3 and LCDR1-3. For example, the HCDR1 of the antigen binding protein can 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 set forth in SEQ ID NO. 12; the HCDR3 of the antigen binding protein can comprise an amino acid sequence shown as SEQ ID NO. 18; the LCDR1 of the antigen binding protein can comprise an amino acid sequence shown as SEQ ID NO. 24; the LCDR2 of the antigen binding protein can comprise an amino acid sequence shown in SEQ ID NO. 29; the LCDR3 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 36. For example, the antigen binding protein may comprise the antibodies 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM or antigen binding fragments thereof having the same HCDR3 (e.g., having the same HCDR1-3 therewith) and LCDR3 (e.g., having the same LCDR1-3 therewith).
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 variable region of the light chain of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 12; the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 18; the LCDR1 can comprise an amino acid sequence shown as SEQ ID NO. 24; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 29; the LCDR3 can 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 can comprise an amino acid sequence shown in SEQ ID NO. 4; the H-FR3 can comprise an amino acid sequence shown as SEQ ID NO 13; the H-FR4 can comprise an amino acid sequence shown as SEQ ID NO. 19; the L-FR1 can include the amino acid sequence of SEQ ID NO 21; the L-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 25; the L-FR3 can comprise an 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 set forth in SEQ ID NO: 39. For example, the antigen binding protein may include antibody 13H6D3 or an antigen binding protein having the same heavy chain variable region as it. For example, the variable region of the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 49. For example, the antigen binding protein may include 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 variable region of the light chain of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 12; the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 18; the LCDR1 can comprise an amino acid sequence shown as SEQ ID NO. 24; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 29; the LCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 5; the H-FR3 can comprise an amino acid sequence shown in SEQ ID NO. 14; the H-FR4 can comprise an 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 can comprise an amino acid sequence shown as SEQ ID NO. 26; the L-FR3 can comprise an amino acid sequence shown as 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 variable region of the light chain 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 variable region of the light chain of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 12; the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 18; the LCDR1 can comprise an amino acid sequence shown as SEQ ID NO. 24; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 29; the LCDR3 can 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 can comprise an amino acid sequence shown as SEQ ID NO. 6; the H-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 15; the H-FR4 can comprise an amino acid sequence shown as SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 23; the L-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 27; the L-FR3 can comprise an amino acid sequence shown as 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 set forth 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 variable region of the light chain 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 variable region of the light chain of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 12; the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 18; the LCDR1 can comprise an amino acid sequence shown as SEQ ID NO. 24; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 29; the LCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 7; the H-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 16; the H-FR4 can comprise an 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 can comprise an amino acid sequence shown in SEQ ID NO. 27; the L-FR3 can comprise an 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 variable region of the light chain 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.
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 variable region of the light chain of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 12; the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 18; the LCDR1 can comprise an amino acid sequence shown as SEQ ID NO. 24; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 29; the LCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 8; the H-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 17; the H-FR4 can comprise an 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 can comprise an amino acid sequence shown in SEQ ID NO. 27; the L-FR3 can comprise an 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 set forth 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 variable region of the light chain 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 variable region of the light chain of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 12; the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 18; the LCDR1 can comprise an amino acid sequence shown as SEQ ID NO. 24; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 29; the LCDR3 can 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 can comprise an amino acid sequence shown as SEQ ID NO. 9; the H-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 14; the H-FR4 can comprise an amino acid sequence shown as SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 23; the L-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 27; the L-FR3 can comprise an amino acid sequence shown in SEQ ID NO. 32; the L-FR4 can 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 variable region of the light chain 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 variable region of the light chain of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 12; the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 18; the LCDR1 can comprise an amino acid sequence shown as SEQ ID NO. 24; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 29; the LCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 7; the H-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 14; the H-FR4 can comprise an amino acid sequence shown as SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 22; the L-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 27; the L-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 32; the L-FR4 can 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 variable region of the light chain 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 variable region of the light chain of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 12; the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 18; the LCDR1 can comprise an amino acid sequence shown as SEQ ID NO. 24; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 29; the LCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO 2; the H-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 6; the H-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 16; the H-FR4 can comprise an amino acid sequence shown as SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 22; the L-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 27; the L-FR3 can comprise an amino acid sequence shown in SEQ ID NO. 34; the L-FR4 can 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 set forth 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 variable region of the light chain 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 variable region of the light chain of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 12; the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 18; the LCDR1 can comprise an amino acid sequence shown as SEQ ID NO. 24; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 29; the LCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 2; the H-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 10; the H-FR3 can comprise an amino acid sequence shown in SEQ ID NO. 14; the H-FR4 can comprise an amino acid sequence shown as SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 23; the L-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 27; the L-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 33; the L-FR4 can 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 set forth 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 variable region of the light chain 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 variable region of the light chain of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 12; the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 18; the LCDR1 can comprise an amino acid sequence shown as SEQ ID NO. 24; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 29; the LCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 36. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO 2; the H-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 11; the H-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 15; the H-FR4 can comprise an amino acid sequence shown as SEQ ID NO. 20; the L-FR1 may comprise the amino acid sequence of SEQ ID NO. 22; the L-FR2 can comprise an amino acid sequence shown in SEQ ID NO. 28; the L-FR3 can comprise an 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 set forth 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 variable region of the light chain 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 the present 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 HCDR3.
In the present application, the HCDR3 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 18. For example, the HCDR3 sequence of the reference antibody can be defined according to the Kabat coding system.
In the present application, the HCDR2 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 12. For example, the HCDR2 sequence of the reference antibody can be defined according to the Kabat coding system.
In the present application, the HCDR1 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 3. For example, the HCDR1 sequence of the reference antibody can be defined according to the Kabat coding system.
For example, the HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 12; and the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 18. For example, the reference antibody can include the antibodies 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or antigen-binding fragments thereof having the same HCDR3 (e.g., having the same HCDR1-3 therewith).
In the present application, the reference antibody may comprise a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO 67. For example, the reference antibody comprises a VH having an amino acid substitution (e.g., a conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of SEQ ID NO: 67: 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 Can be Q or V, X 7 Can be P or S, X 10 Can be D or E, X 11 Can be L or V, X 12 Can be K or V, X 16 Can be A or S, X 20 Can be L or V, X 37 Can be D, M or V, X 38 Can be K or R, X 40 Can be A or R, X 46 Can be D or E, X 48 Can be I or M, X 67 Can be A or V, X 68 Can be S or T, X 69 Can be I or L, X 75 Can be S or T, X 81 Can 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 the present 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 the present application, the reference antibody can comprise at least one CDR in an antibody light chain variable region VL, which can comprise the amino acid sequence set forth 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 to SEQ ID NO. 55. In the present application, the LCDR of the reference antibody may be divided in any form, and 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, the LCDR obtained by dividing in any form may fall within the scope of the present application.
In the present application, the reference antibody may comprise a light chain variable region VL, which may comprise at least one, at least two, or at least three of LCDR1, LCDR2, and LCDR3.
In the present application, the LCDR3 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO: 36. For example, the LCDR3 of the reference antibody can be defined according to the Kabat numbering system.
In the present application, the LCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO. 29. For example, the LCDR2 of the reference antibody can be defined according to the Kabat numbering system.
In the present application, LCDR1 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 24. For example, the LCDR1 of the reference antibody can be defined according to the Kabat numbering system.
For example, LCDR1 of a reference antibody described herein can comprise the amino acid sequence shown in SEQ ID NO. 24; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 29; and the LCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 36. For example, the reference antibody can include the antibodies 13H6D3, hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM, or antigen-binding fragments thereof having the same LCDR3 (e.g., having the same LCDR1-3 therewith).
In the present application, the reference antibody may comprise a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO. 68. For example, the VL of the reference antibody has an amino acid substitution (e.g., a conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of SEQ ID NO: 68: 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 Can be P or T, X 15 Can be L or V, X 22 Can be S or T, X 41 Can be D or G, X 42 Can be G or K, X 43 Can be A or T, X 44 Can 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 Can be E or Q, X 80 Can 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 variable region of the light chain of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO. 49 to SEQ ID NO. 55.
In the present application, the reference antibody may comprise a light chain constant region, which may include a constant region derived from Ig κ or a constant region derived from Ig λ.
For example, the light chain constant region can include a constant region derived from Ig κ.
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 can comprise the amino acid sequence set forth in SEQ ID NO. 3; the HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO. 12; the HCDR3 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 18; the LCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO. 24; the LCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID No. 29; the LCDR3 of the reference antibody can comprise the amino acid sequence set forth 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3 therewith) and LCDR3 (e.g., having the same LCDR1-3 therewith).
Polypeptides and immunoconjugates
In another aspect, the present application provides one or more polypeptides that can 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., bispecific antibody).
In another aspect, the present application provides one or more immunoconjugates that can comprise an isolated antigen binding protein of the present application. In certain embodiments, the immunoconjugate may further comprise a pharmaceutically acceptable therapeutic agent, label, and/or 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 encoding 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 a portion thereof (e.g., one or more of HCDR1-3, heavy chain variable region).
For example, where the nucleic acid molecules each encode a portion of the antigen binding protein, the products encoded by the nucleic acid molecules taken together can form a functional (e.g., binding to PD-L1) isolated antigen binding protein of the present application.
The nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (ii) produced recombinantly by cloning, (iii) purified, e.g., fractionated by enzymatic cleavage and gel electrophoresis, or (iv) synthesized, e.g., by chemical synthesis. For example, the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA techniques.
In the present application, nucleic acids encoding the isolated antigen binding proteins described herein can be prepared by a variety of methods known in the art, including, but not limited to, the use of reverse transcription PCR and PCR to obtain the 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 of the nucleic acid molecules described herein. One or more of the nucleic acid molecules may be contained in each vector. 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 contain expression control elements that allow for the 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 that regulate gene transcription or mRNA translation, among others. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but typically comprises 5' non-transcribed sequences and 5' and 3' non-translated sequences, such as TATA box, capping sequences, CAAT sequences, etc., which are involved in initiation of transcription and translation, respectively. For example, the 5' non-transcribed expression control sequence can comprise a promoter region that can comprise a promoter sequence for transcription control functionally linked to the nucleic acid. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerase, human U6RNA promoter, CMV promoter, and artificial hybrid promoters thereof (e.g., CMV), wherein a certain portion of the promoter may be fused with a certain portion of the promoter of other cellular proteins (e.g., human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene, which may or may not contain additional introns. One or more of the nucleic acid molecules described herein can 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 may be administered to the patient (ex vivo), for example, by treating the cells with the virus in vitro and then administering the treated cells to the patient. 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, retroviral, lentiviral and adeno-associated viral approaches can be used to integrate gene transfer into the host genome, 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 a truncated 3' LTR, RRE, rev response element (cPPT), central Termination Sequence (CTS) and/or post-translational regulatory element (WPRE). The vectors described herein can be introduced into cells.
In another aspect, the present application provides a cell. The cell may comprise an isolated antigen binding protein as described herein, a polypeptide as described herein, an immunoconjugate as described herein, one or more nucleic acid molecules, and/or one or more vectors as described herein. For example, each or every cell may comprise one or more of the 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, a vector described herein can be introduced into the host cell, e.g., a prokaryotic cell (e.g., a bacterial cell), a CHO cell, an NS/0 cell, an HEK293T cell, a 293F cell, or an HEK293A cell, or other eukaryotic cell, such as a plant-derived cell, a fungal or yeast cell, and 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 comprise yeast cells. For example, the cells may comprise E.coli cells. For example, the cells may comprise mammalian cells. For example, the cells may comprise immune cells.
The cells may comprise 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 the present application, the pharmaceutically acceptable adjuvant may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counter ion, a metal complex, and/or a non-ionic surfactant. Except insofar as any conventional media or agent is incompatible with the cells described herein, any conventional media or agent is contemplated for use in the pharmaceutical compositions of the present application. In the present application, the pharmaceutically acceptable excipient may include an additive other than the main drug in the pharmaceutical preparation, and may also be referred to as an auxiliary material. For example, the excipients may include binders, fillers, disintegrants, lubricants in tablets. For example, the excipients may include wine, vinegar, medicinal juices, etc. in a traditional Chinese medicine pill. For example, the excipient may comprise a base portion of a semisolid formulation ointment, cream. For example, the excipients may include preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, tonicity adjusting agents, colorants in liquid formulations.
Kit, use and method
In another aspect, the present application provides a method for detecting or determining PD-L1, which method may comprise the use of the isolated antigen binding protein or the polypeptide.
In the present application, the methods may include in vitro methods, ex vivo methods, methods of non-diagnostic or non-therapeutic interest.
For example, the method may comprise a method for detecting the presence and/or amount of PD-L1 for non-diagnostic purposes, which may comprise the steps of:
1) Contacting a sample with an antigen binding protein of the present application; and
2) Detecting the presence and/or amount of said antigen binding protein bound by the sample to determine the presence and/or level of expression of PD-L1 in the sample obtained from the subject.
In another aspect, the present application provides a kit for PD-L1, which can include 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 methods may include in vitro methods, ex vivo methods, methods of non-diagnostic or non-therapeutic interest.
In another aspect, the present application provides a use of the isolated antigen binding protein or the polypeptide in the preparation of a kit for use in a method for detecting the presence and/or amount of PD-L1. For example, the methods may include in vitro methods, ex vivo methods, methods of non-diagnostic or non-therapeutic interest.
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 the present application, the method of modulating an immune response may include in vitro methods, ex vivo methods, methods of non-diagnostic or non-therapeutic interest.
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 the present application, the method of modulating an immune response may comprise an in vitro method, an ex vivo method, a method of non-diagnostic or non-therapeutic interest.
In another aspect, the present application provides an isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the pharmaceutical composition for preventing, alleviating and/or treating a disease or disorder.
In the present application, the disease or disorder may include a tumor. For example, the tumor may comprise a non-solid tumor. For example, the tumor may comprise a tumor associated with expression of PD-L1. The term "tumor associated with 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 expression of PD-L1" may be a tumor formed by upregulation of PD-L1 expression leading to disease progression or evasion of immune surveillance. The tumor associated with protein expression of PD-L1 may be a PD-L1 positive tumor. In a PD-L1 positive tumor, PD-L1 is expressed at about 1%,5%,10%,15%,20%,25%,30%,35%,40%,50%,60%,70%,80% or more of the protein on the surface of the tumor cell or in the tumor microenvironment as compared to normal cells.
In another aspect, the present application provides a use of the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition for the preparation of a medicament for the prevention, alleviation and/or treatment of a disease or disorder.
In the present application, the disease or disorder 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 expression of PD-L1. The term "tumor associated with 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 expression of PD-L1" may be a tumor formed by upregulation of PD-L1 expression leading to disease progression or evasion of immune surveillance. The tumor associated with protein expression of PD-L1 may be a PD-L1 positive tumor. In PD-L1 positive tumors, PD-L1 protein expression at the surface of the tumor cells or in the tumor microenvironment is about 1%,5%,10%,15%,20%,25%,30%,35%,40%,50%,60%,70%,80% or higher than that of normal cells. In another aspect, the present application provides a method of preventing and/or treating a disease or disorder comprising administering the isolated antigen binding protein, the isolated nucleic acid molecule, the vector, the cell, the pharmaceutical composition to a subject in need thereof.
In the present application, the disease or disorder may include a tumor. For example, the tumor may comprise a non-solid tumor. For example, the tumor may comprise a tumor associated with expression of PD-L1. The term "tumor associated with expression of PD-L1" generally refers to a tumor formed by altered expression of PD-L1 resulting in disease progression or evading immune surveillance. For example, the "tumor associated with expression of PD-L1" may be a tumor formed by upregulation of PD-L1 expression leading to disease progression or evasion of immune surveillance. The tumor associated with protein expression of PD-L1 may be a PD-L1 positive tumor. In PD-L1 positive tumors, PD-L1 protein expression at the surface of the tumor cells or in the tumor microenvironment is about 1%,5%,10%,15%,20%,25%,30%,35%,40%,50%,60%,70%,80% or higher than that of 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 can be used for other disease conditions that depend on immune responses, such as inflammation, immune diseases, and infectious diseases.
In the present application, the subject may include a human or a 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, non-human animals may also include any animal species other than humans, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals. The human may be caucasian, african, asian, amphibian, or other ethnic group, or a hybrid of ethnic groups. As another example, the person may be an elderly person, an adult, a teenager, a child, or an infant.
An effective amount in humans can be presumed from an effective amount in experimental animals. For example, freiich et al describe the dose correlation between animals and humans (in milligrams per square meter of body surface) (Freirich et al, cancer Chemother. Rep.50, 219 (1966)). The body surface area can be approximately determined from the height and weight of the patient. See, e.g., scientific Tables, geigy Pharmaceuticals, ardsley, N.Y.,537 (1970).
Without wishing to be bound by any theory, the following examples are merely intended to illustrate the antigen binding proteins, multispecific antibodies, bispecific antibodies, methods of preparation and uses, etc., 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 animal immunization
anti-PD-L1 antibodies were generated by immunizing mice intramuscularly with a gene gun (gene gun) encoding the human PDL1 plasmid, the feeding environment: SPF grade. Mice were purchased and raised in a laboratory environment for l weeks, and environmentally adapted mice were immunized with 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 serum antibody titers were selected for booster immunizations, after which the mice were sacrificed, splenocytes taken, and fused with myeloma cells. 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 disclosed in many documents in the art.
1.2 screening of antigen binding proteins
ELISA experiments for antigen binding proteins to inhibit the binding of PD-L1 to PD-1
PD1 protein (Sulfo-NHS-Biotin, pierce) was labeled with Biotin and diluted with PBS buffer; coating PD-L1 protein in 96-well plate (1 ug/mL,100 uL/well, 4 ℃ overnight); adding 200 uL/hole sealing liquid for sealing, and washing the plate for 3 times by using PBST; adding an anti-PD-L1 antibody (3-fold dilution from 300ug/mL to 0.005ug/mL,11 dilution points) and PD-1, [ B ] (1 ug/mL, in the linear range of PD1-PDL1 binding) to a 96-well plate, incubating for 60 minutes, and washing the plate 3 times with PBST; adding HRP-streptavidin, and detecting OD value by ELISA.
As a result, as shown in FIG. 1, the antigen-binding protein of the present application was able to effectively inhibit the binding of PD-L1 to PD-1, and IC50 values are shown in Table 1.
TABLE 1 ELISA test results for inhibition of PD-L1 binding 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: selection of recipient framework regions
The variable domain sequences of the parental antibodies were searched in the human germline database using NCBI Ig-Blast (http:// www.ncbi.nlm.nih.gov/projects/igblast /). A different human acceptor (i.e., a human variable structure with high homology to the parent antibody) was selected for each heavy and light chain. The CDRs of the human acceptor were replaced by murine CDRs, resulting in a humanized variable domain sequence.
2.2 construction and production of humanized antibodies
DNA sequences encoding the chimeric antibody and humanized IgG heavy and light chains were synthesized and inserted into the pTGE5 vector to construct an expression plasmid for full-length IgG. Expression of the chimeric antibody 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 obtain purified antigen binding proteins hu13H6D3, 5BM, AH00228, AH00229, AH00230, AH00231, AH00232, AH00233, CBM.
Example 3 affinity detection assay for humanized antibody for PD-L1
3.1 immobilization of anti-human Fc on CM5 sensor chip
Anti-human Fc was immobilized. The target protein immobilization level was set to 8000RU using HBS-EP + (10mM HEPES,150mM NaCl,3mM EDTA and 0.05% P20, pH 7.4) as a 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 fixed to flow cells 1 and 2 using the wizard approach. After fixation, the buffer flow rate was maintained at 10. Mu.L/min in order to maintain equilibrium.
3.2 SPR binding between antigen binding proteins and PD-L1
HBS-EP + was used as flow buffer, and capture of the antibody to be tested, injection of serially diluted PD-L1 and surface regeneration were included in each kinetic cycle. The antibody to be detected is captured on flow cell 2 and flow cell 1 is used as a reference. The diluted PD-L1 fraction was then injected into flow cells 1 and 2 (30. Mu.L/min) for a correlation time of 180s. The buffer flow was maintained for 900 seconds for dissociation. To remove the sample and analyte tested, regeneration was performed using a 30s injection of 10mM glycine-hydrochloric acid (pH 1.5).
3.3 data analysis
K was evaluated for each sample using Biacore T200 (GE Healthcare) evaluation software 1.0 D The value is obtained. As a result, as shown in table 2, the antigen binding protein of the present application can bind to PD-L1 with high affinity.
TABLE 2 affinity assay results for PD-L1 for humanized antibodies
Figure BDA0003679279370000381
Example 4Fc engineering to increase half-life and remove glycosylation sites
4.1Fc engineering
Three amino acids in the Fc were replaced with YTE to extend the half-life of IgG, with specific mutations M252Y, S254T, T256E. N297 was mutated to Alanin to remove the glycosylation site.
4.2 affinity detection of Fc-engineered antigen binding proteins for FcRn
(1) Immobilization of anti-his antibodies on CM5 sensor
Using HBS-EP + (pH 6.0) as the running buffer, enough 50mM NaOH, 50mmol/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 set to appropriate parameter values (wash 30 seconds, activate 420 seconds, inject ligand 600 seconds, lock 420 seconds), and the immobilization procedure immobilized the anti-his antibody on 16 flow cells of a CM5 chip via Biacore 8K. After immobilization, the buffer flow rate was kept at 10. Mu.L/min for equilibrium.
(2) SPR binding of test samples (antigen binding proteins) to FcRn
HBS-EP + (ph 6.0) was used as the running buffer, including capture FcRn, injection of serial dilutions of the test antibody, and surface regeneration in each kinetic cycle. FcRn of 0.4 μ g/mL was captured to flow cell 2 of each channel, and flow cell 1 of the same channel was used as a reference. Serial diluted samples (30 μ l/min) were then injected in both flow cells of each channel, and dissociated. To remove the sample and acceptor tested, interfacial regeneration was performed using a 30s injection of 10mM glycine-hydrochloric acid (pH 1.5).
(3) Data analysis
Evaluation of K for each sample Using Biacore8K evaluation software D The value is obtained. Results as shown in table 3, the affinity of the Fc engineered antigen binding protein for FcRn was improved. 5BM-A refers to the 5BM and YTE mutant Fc fusion after the antigen binding protein, 5BM-B refers to the 5BM and unmodified Fc fusion after the antigen binding protein, 230-A refers to AH00230 and YTE mutant Fc fusion after the antigen binding protein, 230-B refers to AH00230 and unmodified Fc fusion after the antigen binding protein.
4.3 affinity detection of Fc-engineered antigen binding proteins for PD-L1
The experimental procedure was as in example 3. Results as shown in table 4, the affinity of the Fc engineered antigen binding protein to PD-L1 was improved. Wherein, 5BM-A refers to the antigen binding protein obtained by fusing 5BM with YTE mutant Fc, and 230-A refers to the antigen binding protein obtained by fusing AH00230 with YTE mutant Fc.
TABLE 3 affinity of Fc-engineered antigen binding proteins for FcRn
Figure BDA0003679279370000401
TABLE 4 affinity of Fc engineered antigen binding proteins for PD-L1
Figure BDA0003679279370000402
4.4Fc engineered antigen binding proteins ELISA experiments inhibiting the binding of PD-L1 to PD-1
PD1 protein (Sulfo-NHS-Biotin, pierce) was labeled with Biotin and diluted with PBS buffer; coating PD-L1 protein in 96-well plates (1 ug/mL,100 uL/well, 4 ℃ overnight); adding 200 uL/hole sealing liquid for sealing, and washing the plate for 3 times by using PBST; adding an anti-PD-L1 antibody (3-fold dilution from 300ug/mL to 0.005ug/mL,11 dilution points) and PD-1[ B ] (1 ug/mL, in the linear range of PD1-PDL1 binding) to a 96-well plate, incubating for 60 min, and washing the plate 3 times with PBST; adding HRP-streptavidin, and detecting OD value by ELISA.
The results are shown in FIG. 2, where the Fc engineered antigen binding protein was able to effectively inhibit the binding of PD-L1 to PD-1. Wherein, 230-Ag-YTE represents antigen binding protein obtained by fusing AH00230 with Fc of YTE mutation and N297 mutation, and 5BM-Ag-YTE represents antigen binding protein obtained by fusing Fc of 5BM with YTE mutation and N297 mutation.
4.5 functional cellular assays for Fc engineered antigen binding proteins
After HEK293 cells expressing CD3 antibody fragment/IL-2 gene/PDL 1 and HUTT cells expressing HPD1 are cultured in a mixture, the IL-2 production can be inhibited by the combination of PD1 and PDL 1. PDL1 antibodies can 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 the secretion of IL-2. Wherein, 230-Ag-YTE represents antigen binding protein obtained by fusing AH00230 with Fc of YTE mutation and N297 mutation, and 5BM-Ag-YTE represents antigen binding protein obtained by fusing Fc of 5BM with YTE mutation and N297 mutation.
It should be noted that the above-mentioned contents only illustrate the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and it is obvious to those skilled in the art that several modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations fall within the protection scope of the claims of the present invention.
Sequence listing
<110> Nanjing Gisheng Australia Ma Bio-pharmaceuticals Co., ltd
<120> PD-L1 antibodies and uses thereof
<150> 2021108351839
<151> 2021-07-23
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
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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 (89)

1. An isolated antigen binding protein having one or more of the following properties:
a) In a Biacore assay with a K of about 8E-09M or less D Specifically binding to human PD-L1 protein;
b) Can block the combination of PD-1 and PD-L1.
2. The isolated antigen binding protein of claim 1, comprising an HCDR3, said HCDR3 comprising the amino acid sequence set forth in SEQ ID No. 18.
3. The isolated antigen-binding protein of any of claims 1-2, comprising an HCDR2, the HCDR2 comprising the amino acid sequence set forth in SEQ ID No. 12.
4. The isolated antigen binding protein of any of claims 1-3, comprising an HCDR1, said HCDR1 comprising an amino acid sequence set forth in SEQ ID NO. 3.
5. The isolated antigen binding protein of any of claims 1-4, comprising HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH of SEQ ID NO: 67.
6. The isolated antigen binding protein of any of claims 1-5, comprising HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH set forth in any of SEQ ID NO:39 to SEQ ID NO: 48.
7. The isolated antigen binding protein of any of claims 1-6, comprising a heavy chain variable region VH comprising the HCDR1, HCDR2, and HCDR3, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO 18; the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 12; and the HCDR1 comprises an amino acid sequence shown in SEQ ID NO. 3.
8. The isolated antigen binding protein of any of claims 1-7, comprising H-FR1, the C-terminus of said H-FR1 being directly or indirectly linked to the N-terminus of said HCDR1, and said H-FR1 comprising the amino acid sequence set forth in SEQ ID No. 59.
9. The isolated antigen binding protein of claim 8, wherein said H-FR1 comprises an amino acid sequence set forth in any one of SEQ ID No. 1 to SEQ ID No. 2.
10. The isolated antigen binding protein of any of claims 1-9, comprising H-FR2, said H-FR2 being located between said HCDR1 and said HCDR2, and said H-FR2 comprising the amino acid sequence set forth in SEQ ID No. 60.
11. The isolated antigen binding protein of claim 10, wherein said H-FR2 comprises an amino acid sequence set forth in any one of SEQ ID No. 4 to SEQ ID No. 11.
12. The isolated antigen binding protein of any of claims 1-11, comprising H-FR3, wherein said H-FR3 is located between said HCDR2 and said HCDR3, and wherein said H-FR3 comprises the amino acid sequence set forth in SEQ ID No. 61.
13. The isolated antigen binding protein of claim 12, wherein said H-FR3 comprises an amino acid sequence set forth in any one of SEQ ID No. 13 to SEQ ID No. 17.
14. The isolated antigen binding protein of any of claims 1-13, comprising H-FR4, the N-terminus of said H-FR4 being linked directly or indirectly to the C-terminus of said HCDR3, and said H-FR4 comprising the amino acid sequence set forth in SEQ ID No. 62.
15. The isolated antigen binding protein of claim 14, wherein said H-FR4 comprises an amino acid sequence set forth in any one of SEQ ID No. 19 to SEQ ID No. 20.
16. The isolated antigen binding protein of any of claims 1-15, comprising H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR1 comprising the amino acid sequence set forth in SEQ ID No. 59; the H-FR2 comprises an amino acid sequence shown in SEQ ID NO: 60; the H-FR3 comprises an amino acid sequence shown in SEQ ID NO. 61; and the H-FR4 comprises an amino acid sequence shown as SEQ ID NO: 62.
17. The isolated antigen binding protein of claim 16, wherein said H-FR1 comprises an amino acid sequence set forth 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 an amino acid sequence shown in any one of SEQ ID NO 19 to SEQ ID NO 20.
18. The isolated antigen binding protein of any of claims 16-17, wherein said H-FR1, H-FR2, H-FR3, and H-FR4 comprise an amino acid sequence selected from any one of the group consisting of:
a) H-FR1: 1, H-FR2: 4, H-FR3: 13 and H-FR4: 19 in SEQ ID NO;
b) H-FR1: 2, H-FR2: 5, H-FR3: 14 and H-FR4: 20 in SEQ ID NO;
c) H-FR1: 2, H-FR2: 6, H-FR3: 15 and H-FR4: 20 is SEQ ID NO;
d) H-FR1: 2, H-FR2: 7, H-FR3: 16 and H-FR4: 20 is SEQ ID NO;
e) H-FR1: 2, H-FR2: 8, H-FR3: 17 and H-FR4: 20 in SEQ ID NO;
f) H-FR1: 2, H-FR2: 9, H-FR3: 14 and H-FR4: 20 is SEQ ID NO;
g) H-FR1: 2, H-FR2: 7, H-FR3: 14 and H-FR4: 20 in SEQ ID NO;
h) H-FR1: 2, H-FR2: 6, H-FR3: 16 and H-FR4: 20 in SEQ ID NO;
i) H-FR1: 2, H-FR2: 10, H-FR3: 14 and H-FR4: 20 in SEQ ID NO; and
j) H-FR1: 2, H-FR2: 11, H-FR3: 15 and H-FR4: 20 in SEQ ID NO.
19. The isolated antigen binding protein of any of claims 1-18, comprising a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID No. 67.
20. The isolated antigen binding protein of claim 19, wherein said VH comprises the amino acid sequence set forth in any one of SEQ ID No. 39 to SEQ ID No. 48.
21. The isolated antigen binding protein of any of claims 1-20, comprising an LCDR3, said LCDR3 comprising the amino acid sequence set forth in SEQ ID No. 36.
22. The isolated antigen binding protein of any of claims 1-21, comprising an LCDR2, said LCDR2 comprising the amino acid sequence set forth in SEQ ID No. 29.
23. The isolated antigen binding protein of any of claims 1-22, comprising an LCDR1, the LCDR1 comprising the amino acid sequence set forth in SEQ ID No. 24.
24. The isolated antigen binding protein of any of claims 1-23, comprising LCDR1, LCDR2, and LCDR3 of the light chain variable region VL of any one of SEQ ID NOs 68.
25. The isolated antigen binding protein of any of claims 1-24, comprising LCDR1, LCDR2, and LCDR3 of the light chain variable region VL of any one of SEQ ID No. 49 to SEQ ID No. 55.
26. The isolated antigen binding protein of any of claims 1-25, comprising a light chain variable region VL comprising the LCDR1, LCDR2, and LCDR3, the LCDR3 comprising the amino acid sequence set forth in SEQ ID No. 36; the LCDR2 comprises an amino acid sequence shown as SEQ ID NO. 29; and the LCDR1 comprises an amino acid sequence shown in SEQ ID NO. 24.
27. The isolated antigen binding protein of any of claims 1-26, comprising L-FR1, the C-terminus of said L-FR1 being directly or indirectly linked to the N-terminus of said LCDR1, and said L-FR1 comprising the amino acid sequence set forth in SEQ ID No. 63.
28. The isolated antigen binding protein of claim 27, wherein said L-FR1 comprises an amino acid sequence set forth in any one of SEQ ID No. 21 to SEQ ID No. 23.
29. The isolated antigen binding protein of any of claims 1-28, comprising L-FR2, wherein said L-FR2 is located between said LCDR1 and said LCDR2, and wherein said L-FR2 comprises the amino acid sequence set forth in SEQ ID No. 64.
30. The isolated antigen binding protein of claim 29, wherein said L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID No. 25 to SEQ ID No. 28.
31. The isolated antigen binding protein of any of claims 1-30, comprising L-FR3, wherein said L-FR3 is located between said LCDR2 and said LCDR3, and wherein said L-FR3 comprises the amino acid sequence set forth in SEQ ID No. 65.
32. The isolated antigen binding protein of claim 31, wherein said L-FR3 comprises an amino acid sequence set forth in any one of SEQ ID No. 30 to SEQ ID No. 35.
33. The isolated antigen binding protein of any of claims 1-32, comprising L-FR4, wherein the N-terminus of said L-FR4 is linked directly or indirectly to the C-terminus of said LCDR3, and wherein said L-FR4 comprises the amino acid sequence set forth in SEQ ID No. 66.
34. The isolated antigen binding protein of claim 33, wherein said L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID No. 37 to SEQ ID No. 38.
35. The isolated antigen binding protein of any of claims 1-34, comprising L-FR1, L-FR2, L-FR3, and L-FR4, wherein the L-FR1 comprises the amino acid sequence set forth 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 an amino acid sequence shown in SEQ ID NO: 66.
36. The isolated antigen binding protein of claim 35, wherein said L-FR1 comprises an amino acid sequence set forth 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-SEQ ID NO 35; and the L-FR4 comprises an amino acid sequence shown in any one of SEQ ID NO 37 to SEQ ID NO 38.
37. The isolated antigen binding protein of any of claims 35-36, wherein said L-FR1, L-FR2, L-FR3, and L-FR4 comprise an amino acid sequence selected from any one of the group consisting of:
a) L-FR1: 21, L-FR2: 25, L-FR3: 30 and L-FR4: 37 is SEQ ID NO;
b) L-FR1: 22, L-FR2: 26, L-FR3: 31 and L-FR4: 38, SEQ ID NO;
c) L-FR1: 23, L-FR2: 27, L-FR3: 32 and L-FR4: 38, SEQ ID NO;
d) L-FR1: 22, L-FR2: 27, L-FR3: 32 and L-FR4: 38, SEQ ID NO;
e) L-FR1: 22, L-FR2: 27, L-FR3: 34 and L-FR4: 38, SEQ ID NO;
f) L-FR1: 23, L-FR2: 27, L-FR3: 33 and L-FR4: 38, SEQ ID NO; and
g) L-FR1: 22, L-FR2: 28, L-FR3: 35 and L-FR4: 38 in SEQ ID NO.
38. The isolated antigen binding protein of any of claims 1-37, wherein said VL comprises the amino acid sequence set forth in SEQ ID No. 68.
39. The isolated antigen binding protein of claim 38, wherein the VL comprises an amino acid sequence set forth in any one of SEQ ID No. 49 to SEQ ID No. 55.
40. The isolated antigen binding protein of any of claims 1-39, comprising a VH and a VL comprising an amino acid sequence selected from any of the group consisting of:
a) VH: 39 and VL: 49 in SEQ ID NO;
b) VH: 40 and VL: 50 is SEQ ID NO;
c) VH: 41 and VL: 51 is SEQ ID NO;
d) VH: 42 and VL: 51 is SEQ ID NO;
e) VH: 43 and VL: 52, SEQ ID NO;
f) VH: 44 and VL: 51 is SEQ ID NO;
g) VH: 45 and VL: 53, SEQ ID NO;
h) VH: 46 and VL: 54 in SEQ ID NO;
i) VH: 47 and VL: 52, SEQ ID NO; and
j) VH: 48 and VL: SEQ ID NO:55.
41. The isolated antigen binding protein of any of claims 1-40, which has an extended half-life as compared to a wild-type antibody.
42. The isolated antigen binding protein of any of claims 1-41, which is capable of reducing Antibody Dependent Cellular Cytotoxicity (ADCC) as compared to a wild-type antibody.
43. The isolated antigen binding protein of any of claims 1-42, comprising a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region or an IgY-derived constant region.
44. The isolated antibody binding protein of claim 43, wherein the heavy chain constant region comprises an IgG-derived constant region.
45. The isolated antigen binding protein of any of claims 43-44, wherein the heavy chain constant region comprises a constant region derived from IgG1, igG2, igG3, or IgG 4.
46. The isolated antigen binding protein of any of claims 43-45, wherein the heavy chain constant region comprises an Fc region derived from IgG 1.
47. The isolated antigen binding protein of any of claims 43-46, wherein position N297 of the heavy chain constant region is mutated and residues numbered according to the Kabat system.
48. The isolated antigen binding protein of any of claims 43-47, wherein the heavy chain constant region is mutated at position M252, and residues are numbered according to the Kabat system.
49. The isolated antigen binding protein of any of claims 43-48, wherein the heavy chain constant region has a mutation at position S254 and residues numbered according to the Kabat system.
50. The isolated antigen binding protein of any of claims 43-49, wherein the heavy chain constant region is mutated at position T256 and residues numbered according to the Kabat system.
51. The isolated antigen binding protein of any of claims 43-50, wherein the Fc region in the heavy chain constant region comprises an N297A mutation.
52. The isolated antigen binding protein of any of claims 43-51, wherein the Fc region in the heavy chain constant region comprises the M252Y mutation.
53. The isolated antigen binding protein of any of claims 43-52, wherein the Fc region in the heavy chain constant region comprises the S254T mutation.
54. The isolated antigen binding protein of any of claims 43-53, wherein the Fc region in the heavy chain constant region comprises a T256E mutation.
55. The isolated antigen binding protein of any of claims 43-54, wherein the heavy chain constant region comprises an amino acid sequence set forth in any one of SEQ ID NO 56 to SEQ ID NO 57.
56. The isolated antigen binding protein of any one of claims 1-55, comprising a light chain constant region, and the light chain constant region comprises a constant region derived from Ig kappa or a constant region derived from Ig lambda.
57. The isolated antigen binding protein of claim 56, wherein the light chain constant region comprises a constant region derived from human Ig kappa.
58. The isolated antigen binding protein of any of claims 56-57, wherein the light chain constant region comprises the amino acid sequence set forth in SEQ ID No. 58.
59. The isolated antigen binding protein of any of claims 1-58, comprising a heavy chain HC comprising the amino acid sequence of SEQ ID NO:69-78, or a pharmaceutically acceptable salt thereof.
60. The isolated antigen binding protein of any of claims 1-59, comprising a light chain LC comprising the amino acid sequence of SEQ ID NO:79 to 85.
61. The isolated antigen binding protein of any of claims 1-60, comprising an antibody or antigen binding fragment thereof.
62. The isolated antigen binding protein of claim 61, wherein said antigen binding fragment is selected from the group consisting of: fab, fab ', F (ab) 2, fv fragments, F (ab') 2, scFv, di-scFv, VHH and/or dAb.
63. The isolated antigen binding protein of any of claims 61-62, wherein said 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.
64. A polypeptide comprising the isolated antigen binding protein of any one of claims 1-63.
65. An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-63 or the polypeptide of claim 64.
66. An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-63, or the polypeptide of claim 64.
67. A vector comprising the isolated nucleic acid molecule of claim 66.
68. A cell comprising the isolated antigen binding protein of any one of claims 1-63, the polypeptide of claim 64, the immunoconjugate of claim 65, the isolated nucleic acid molecule of claim 66, and/or the vector of claim 67.
69. A method of making the isolated antigen binding protein of any one of claims 1-63 or the polypeptide of claim 64, the method comprising culturing the cell of claim 68 under conditions such that the isolated antigen binding protein of any one of claims 1-63 or the polypeptide of claim 64 is expressed.
70. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-63, the polypeptide of claim 64, the immunoconjugate of claim 65, the isolated nucleic acid molecule of claim 66, the vector of claim 67, the cell of claim 68, and/or a pharmaceutically acceptable adjuvant and/or excipient.
71. A method for detecting or determining PD-L1, the method comprising using the isolated antigen binding protein of any one of claims 1-63 or the polypeptide of claim 64.
72. A detection kit for PD-L1 comprising the isolated antigen binding protein of any one of claims 1-63 or the polypeptide of claim 64.
73. Use of the isolated antigen binding protein of any one of claims 1-63 or the polypeptide of claim 64 in the preparation of a kit for detecting the presence and/or amount of PD-L1.
74. A method of inhibiting the interaction of PD-1 with PD-L1, which comprises administering to a subject in need thereof an effective amount of the isolated antigen binding protein of any one of claims 1-63, the polypeptide of claim 64, the immunoconjugate of claim 65, the isolated nucleic acid molecule of claim 66, the vector of claim 67, and/or the cell of claim 68.
75. Use of the isolated antigen binding protein of any one of claims 1-63, the polypeptide of claim 64, the immunoconjugate of claim 65, the isolated nucleic acid molecule of claim 66, the vector of claim 67, the cell of claim 68, and/or the pharmaceutical composition of claim 70 in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder.
76. The use of claim 75, wherein the disease or condition comprises a tumor.
77. The use of claim 76, wherein the tumor comprises a solid tumor.
78. The use of any one of claims 76-77, wherein the tumor comprises a non-solid tumor.
79. The use of any one of claims 76-78, wherein the tumor comprises breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and/or liver cancer.
80. The isolated antigen binding protein of any one of claims 1-63, the polypeptide of claim 64, the immunoconjugate of claim 65, the isolated nucleic acid molecule of claim 66, the vector of claim 67, the cell of claim 68, and/or the pharmaceutical composition of claim 70 for use in the prevention, amelioration, and/or treatment of a disease or disorder.
81. The use of claim 80, wherein the disease or condition comprises a tumor.
82. The use of claim 81, wherein the tumor comprises a solid tumor.
83. The use of any one of claims 81-82, wherein the tumor comprises a non-solid tumor.
84. The use of any one of claims 81-83, wherein the tumor comprises breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and/or liver cancer.
85. 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 of any one of claims 1-63, the polypeptide of claim 64, the immunoconjugate of claim 65, the isolated nucleic acid molecule of claim 66, the vector of claim 67, and/or the cell of claim 68.
86. The method of claim 85, wherein the disease or disorder comprises a tumor.
87. The method of claim 86, wherein the tumor comprises a solid tumor.
88. The method of any one of claims 86-87, wherein the tumor comprises a non-solid tumor.
89. The method of any one of claims 86-88, wherein the tumor comprises breast cancer, lung cancer, gastric cancer, intestinal cancer, renal 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
EP3133086B1 (en) * 2008-09-26 2018-08-01 Dana-Farber Cancer Institute, Inc. Human anti-pd-1, pd-l1, and pd-l2 antibodies and uses thereof
JP6909153B2 (en) * 2014-08-05 2021-07-28 アポロミクス インコーポレイテッド Anti-PD-L1 antibody
EP3565839A4 (en) * 2017-01-05 2021-04-21 Gensun Biopharma Inc. Checkpoint regulator antagonists
EP3732203A4 (en) * 2017-12-28 2021-12-15 Nanjing Legend Biotech Co., Ltd. Antibodies and variants thereof against pd-l1
CN111278861B (en) * 2018-01-10 2022-05-27 江苏恒瑞医药股份有限公司 PD-L1 antibody, antigen binding fragment thereof and medical application
TW202035445A (en) * 2018-10-10 2020-10-01 美商帝洛斯療法股份有限公司 Anti-lap antibody variants and uses thereof
CN115038718A (en) * 2019-11-08 2022-09-09 先声（上海）医药有限公司 Antibodies against human programmed death ligand-1 (PD-L1) and uses 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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