CN110023333B - High affinity soluble PD-1 molecules - Google Patents

High affinity soluble PD-1 molecules Download PDF

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CN110023333B
CN110023333B CN201780074723.8A CN201780074723A CN110023333B CN 110023333 B CN110023333 B CN 110023333B CN 201780074723 A CN201780074723 A CN 201780074723A CN 110023333 B CN110023333 B CN 110023333B
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李懿
李艳艳
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Xiangxue Life Science Technology Guangdong Co ltd
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Abstract

The present invention provides a high affinity soluble PD-1 molecule, in particular a programmed death receptor PD-1 molecule, which is mutated on the basis of a wild-type PD-1 molecule, and which has at least 2 times the affinity of the PD-1 molecule to its ligand PDL-1 molecule as the wild-type PD-1 molecule to the wild-type PDL-1 molecule. Meanwhile, the PD-1 molecule can effectively improve the killing capacity of lymphocytes. In addition, the invention provides nucleic acids encoding the PD-1 molecules of the invention, as well as complexes of the PD-1 molecules of the invention. The PD-1 molecules of the invention can be used alone or in combination with other molecules.

Description

High affinity soluble PD-1 molecules
Technical Field
The present invention relates to the field of biotechnology, and more particularly to high affinity soluble Programmed Death receptor (PD-1) molecules capable of recognizing the Programmed Death receptor ligand PDL-1 (Programmed Death Ligand-1, PDL-1) molecules with high affinity. The invention relates to a preparation method and application of the molecule.
Background
PD-1 is an immunosuppressive receptor expressed on activated T-cells and B-cells, and its ligand is PDL-1 or PDL-2.PD-1 belongs to the B7 family and is an Ig superfamily type I transmembrane glycoprotein with a size of 50-55 kD; it was found by structural and biochemical analysis that PD-1 exists as a monomer due to the lack of membrane proximal cysteine residues, consisting of the extracellular IgV region, the transmembrane region and the intracellular region (Xuewu Zhang and Almo, immunity,2004, 20, 337-347). PD-1 interacts with the ligand PDL-1 and plays an important role in the negative regulation of the immune response. Many tumor cell lines and tumor cells highly express PDL-1 molecules (Konishi J et al, clin. Cancer res.,2004, 10 (15): 5094-5100), which, when bound to lymphocyte surface PD-1 molecules, attenuate the body's anti-tumor immune response (Radziewicz H et al, J Virol,2007, 81 (6): 2545-2553), resulting in the occurrence of tumor immune escape. Research shows that in cervical cancer and liver cancer, nearly half of tumors infiltrate CD8 + T cells express PD-1 molecules that, when bound to PDL-1 expressed by tumor cells, can lead to depletion and apoptosis of CTL cells (Dong H et al, J Mol Med (Berl), 2003, 81 (5): 281-287;Karim R et al, clin Cancer Res,2009, 15 (20): 6341-6347;Zhao Q et al, eur J Immunol,2011, 41 (8): 2314-2322).
In order to solve the tumor immune escape problem, blocking the interaction between PD-1 on the surface of lymphocytes and PDL-1 on the surface of tumor cells can improve the immunity of the lymphocytes, thus helping the immune system to clear the tumor cells. In view of this problem, researchers have conducted a great deal of research. In a murine model of invasive pancreatic cancer, T.Nomi et al (Clin.cancer Res.2007, 13:2151-2157) demonstrated therapeutic efficacy in blocking PD-1 interaction with PDL-1. Those skilled in the art have focused on the study of the interaction of PDL-1 with PD-1 in an effort to find effective ways to increase lymphocyte killing capacity.
Disclosure of Invention
The object of the present invention is to provide a PD-1 molecule having a higher affinity for PDL-1 molecules.
It is still another object of the present invention to provide a method for preparing the above high affinity PD-1 molecule and its use.
In a first aspect of the invention, there is provided a PD-1 molecule comprising a mutation in the amino acid sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the amino acid sequence of the PD-1 molecule is based on SEQ ID No.:1, and performing mutation of one or more amino acid residues or insertion of an amino acid residue to the amino acid sequence shown in SEQ ID No.1 to obtain the PD-1 molecule.
In another preferred embodiment, the amino acid sequence of the PD-1 molecule has at least 90% (preferably at least 92%; more preferably at least 94%) sequence identity to the amino acid sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the affinity of the PD-1 molecule for a PDL-1 molecule is at least 2-fold greater than the affinity of a wild-type PD-1 molecule for a PDL-1 molecule; preferably at least 10 times; more preferably at least 100 times; most preferably at least 200 times.
In another preferred embodiment, the affinity of the PD-1 molecule for a PDL-1 molecule is at least 500-fold greater than the affinity of a wild-type PD-1 molecule for a PDL-1 molecule; preferably at least 1000 times; more preferably at least 2000 times.
In another preferred example, the mutated amino acid residue position in the PD-1 molecule is one or more of amino acid residues from 30 to 60, and/or from 85 to 105, wherein the amino acid residue numbering is as shown in SEQ ID NO. 1.
In another preferred embodiment, the mutated amino acid residue position in the PD-1 molecule is one or more of amino acid residues 31-37, 40-48, 56, and/or 89-103, wherein the amino acid residue numbering is as shown in SEQ ID NO. 1.
In another preferred embodiment, the number of mutated amino acid residue positions is n, wherein 1.ltoreq.n.ltoreq.15; preferably, 2.ltoreq.n.ltoreq.11; more preferably, 2.ltoreq.n.ltoreq.6, e.g.n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
In another preferred embodiment, the mutated amino acid residue position in the PD-1 molecule comprises one or more of 91G, 31V, 33N, 35Y, 37M, 40S, 41N, 42Q, 43T, 48A, 56P, 89L, 92A, 93I, 95L, 97P, 98K, 99A, 100Q, 101I, 103E, wherein the amino acid residue numbering is as set forth in SEQ ID NO. 1.
In another preferred embodiment, the mutated amino acid residue position in the PD-1 molecule comprises 91G, wherein the amino acid residue numbering is as set forth in SEQ ID NO. 1.
In another preferred embodiment, the mutated amino acid residue position in the PD-1 molecule comprises 99A, wherein the amino acid residue numbering is as set forth in SEQ ID NO. 1.
In another preferred embodiment, the mutated amino acid residue position in the PD-1 molecule further comprises 41N, 42Q, 43T, 48A, 95L, 97P, 98K and/or 100Q, wherein the amino acid residue numbering is as shown in SEQ ID NO. 1.
In another preferred embodiment, the mutated PD-1 molecule comprises one or more amino acid residues selected from the group consisting of: 91A, 91S, 91V or 91T;31T;33L;35N or 35M;37V, 37L or 37E;40A or 40T;41G, or 41L;42N;43V or 43G;48G or 48S;56L;89M;92V or 92Y;93L;95W or 95F;97G;98R, 98Y or 98P;99P, 99V, 99I or 99F;100S or 100W;101V; and 103D; wherein the amino acid residue number is represented by SEQ ID NO. 1.
In another preferred embodiment, the mutated PD-1 molecule comprises 91V or 91S.
In another preferred embodiment, the mutated PD-1 molecule further comprises 99I or 99P.
In another preferred embodiment, the PD-1 molecule comprises: 91V and 99I; or (b)
91S, 98Y and 99I; or (b)
41L, 42N, 43G, 48S, 91V and 99P; or (b)
41G, 43V, 48G, 91V and 99P,
wherein the amino acid residue number is represented by SEQ ID NO. 1.
In another preferred embodiment, the amino acid sequence of the PD-1 molecule is selected from SEQ ID NO.39, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41 or 43.
In another preferred embodiment, the amino acid sequence of the PD-1 molecule is selected from SEQ ID No.39, 5, 11, 13, 15 or 43;
In another preferred embodiment, the PD-1 molecule has the amino acid sequence SEQ ID NO.39.
In another preferred embodiment, the PD-1 molecule is soluble.
In another preferred embodiment, the PD-1 molecule has a conjugate attached to its C or N terminus.
In another preferred embodiment, the conjugate that binds to the PD-1 molecule is a T cell receptor, preferably the T cell receptor is a high affinity T cell receptor.
In a preferred embodiment, the PD-1 molecule increases anti-CD3 mAb and anti-CD28 mAb mediated PBMC proliferation by more than 15%, preferably 18% to 20%; and/or
The PD-1 molecule promotes IFN- γ release at a rate of about 20%; preferably, the rate of promoting IFN-gamma release is increased to 40-50%.
In a second aspect of the invention there is provided a fusion protein comprising a PD-1 molecule according to the first aspect of the invention.
In another preferred embodiment, the fusion protein further comprises IgG4.
In a third aspect of the invention, there is provided a multivalent PD-1 complex comprising at least two PD-1 molecules, and wherein at least one PD-1 molecule is a PD-1 molecule according to the first aspect of the invention; or the multivalent PD-1 complex comprises at least one fusion protein according to the second aspect of the invention.
In a fourth aspect of the invention, there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a PD-1 molecule according to the first aspect of the invention, a fusion protein according to the second aspect of the invention, or a multivalent PD-1 complex according to the third aspect of the invention, or a complement thereof.
In a fifth aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the fourth aspect of the invention.
In a sixth aspect of the invention, there is provided a host cell comprising a vector according to the fifth aspect of the invention or a nucleic acid molecule according to the fourth aspect of the invention integrated with an exogenous source in a chromosome; or alternatively
The host cell contains or expresses the PD-1 molecule of the first aspect of the invention, the fusion protein of the second aspect of the invention, or the multivalent PD-1 complex of the third aspect of the invention.
In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a PD-1 molecule according to the first aspect of the invention, or a fusion protein according to the second aspect of the invention, or a PD-1 complex according to the third aspect of the invention.
In an eighth aspect of the invention, there is provided a method of treating a disease comprising administering to a subject in need thereof an amount of a PD-1 molecule according to the first aspect of the invention, a fusion protein according to the second aspect of the invention, or a PD-1 complex according to the third aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention.
In another preferred embodiment, the disease is a tumor.
In a ninth aspect of the invention there is provided the use of a PD-1 molecule according to the first aspect of the invention, a fusion protein according to the second aspect of the invention, or a PD-1 complex according to the third aspect of the invention for the manufacture of a medicament for the treatment of a tumour.
In a tenth aspect of the present invention, there is provided a method for preparing the PD-1 according to the first aspect of the present invention, comprising the steps of:
(i) Culturing the host cell of the sixth aspect of the invention, thereby expressing the PD-1 molecule of the first aspect of the invention;
(ii) Separating or purifying the PD-1 molecule.
In a first aspect of the invention, there is provided a PD-1 molecule comprising a mutation in the amino acid sequence shown in SEQ ID NO. 1.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is a SDS-PAGE gel of purified wild type PD-1 protein M, protein molecular weight Mark.
FIG. 2 is a BIAcore profile of binding of a wild-type PDL-1 molecule to a PD-1 molecule.
FIG. 3 is a flow assay for PD-1, L5B7 recognizing H1299 cell surface PD-L1, showing that L5B7 recognizes H1299 cell surface PDL-1 more than PD-1. And (3) injection: a, anti-PDL-1 antibody (2.5 ul/sample) recognizes PDL-1 on the surface of H1299 cells; b, a flow assay for identifying PDL-1 on the surface of H1299 cells at different concentrations of PD-1 and L5B7 (0.02 mg/ml, 0.04mg/ml and 0.08 mg/ml), wherein the amount of SA-PE is 0.5 ul/sample; c, when the concentration is 0.08mg/ml, the control group, PD-1, L5B7 recognizes the flow histogram of PDL-1.
FIG. 4a is a schematic representation of PD-1 mutants and their eukaryotic expression (schematic representation of PD-1-IgG4 fusion proteins, note: dimer theoretical molecular weight around 80kD, actual molecular weight around 125kD after glycosylation).
FIG. 4b is an electrophoresis pattern of an 8% SDS-PAGE gel of the expression supernatant after 48 hours. And (3) injection: m, protein molecular weight Mark. 1. 2, PD-1-IgG4, L5B7-IgG4 Non-Reducing electrophoresis results; 3. FIG. 4, schematic representation of the result of the Reducing electrophoresis of PD-1-IgG4 and L5B7-IgG 4.
Fig. 4c is a functional verification graph that promotes ImmTAC-IG4 mediated killing. Shows that PD-1-IgG4, L5B7-IgG4 promotes LDH release and CD25 and CD107a flow detection of ImmTAC-IG4 mediated killing. And (3) injection: a, LDH release, has been formulated into a kill ratio. And B, when the killing ratio is 1:1, the flow detection diagram of the expression condition of CD25 and CD107a of the CD8T cells in the killing reaction system detects that the dosage of the flow antibody of the CD25 and the CD107a is 2.5 ul/sample.
FIG. 5 shows that the high affinity PD-1 mutants promote proliferation of stimulated PBMC; wherein a-F in fig. 5a shows in sequence a flow chart of high affinity mutants L1B2, L2B12, L2F8, L2F10, L5B7, L45 promoting proliferation of stimulated PBMCs; 5b is a statistical plot of the rate of proliferation of stimulated PBMC promoted by each high affinity PD-1 mutant.
FIG. 6 shows that high affinity PD-1 mutants promote IFN-gamma release from stimulated PBMC; wherein FIG. 6a shows the results of an Elispot assay to detect IFN-gamma release from high affinity PD-1 mutation-promoted stimulated PBMC; 6b is a statistical plot of the IFN-y release rate of stimulated PBMC promoted by each high affinity PD-1 mutant.
Detailed Description
Through extensive and intensive studies, the present invention has unexpectedly found that a soluble PD-1 molecule having a high affinity for PDL-1 molecules can effectively enhance the killing ability of lymphocytes. Accordingly, the present invention provides a soluble high affinity PD-1 molecule having at least twice the affinity for PDL-1 as a wild-type PD-1 molecule.
Specifically, the PD-1 molecule of the invention contains a mutation in the amino acid sequence shown in SEQ ID NO. 1. More specifically, the amino acid sequence of the PD-1 molecule has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO. 1.
Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
Terminology
Wild type PD-1 molecule: the wild type PD-1 molecule refers to an extracellular region of the wild type PD-1 molecule, and the amino acid sequence and the nucleotide sequence of the wild type PD-1 molecule are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2:
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.1, wild type PD-1)
The amino acid sequence and the nucleotide sequence of PDL-1 are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4:
PBMC: peripheral Blood Mononuclear Cells (PBMCs) are blood cells, such as lymphocytes or monocytes, with rounded nuclei. These blood cells are key components of the immune system to combat infection and to adapt to the intruder. The lymphocyte population consisted of T cells (CD 4 and CD8 positive about 75%), B cells and NK cells (pooled about 25%).
High affinity T cell receptor: refers to a T cell receptor having an increased affinity for its ligand over the corresponding wild-type T cell receptor and its ligand. For example, screening by a yeast screening system resulted in a single chain autoreactive murine 2C TCR with improved stability, which had an affinity for the ligand that was increased by about 100-fold over the wild type (9 nM) (Holler, P.D.et al Natl. Acad.Sci. USA.2000.97, 5387-5392).
Tumor: is meant to include all types of cancerous cell growth or oncogenic processes, metastatic tissue or malignant transformed cells, tissues or organs, regardless of the type of pathology or stage of infection. Examples of tumors include, without limitation: solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignant tumors of different organ systems, such as sarcomas, squamous cell carcinoma of the lung and cancers. For example: infected prostate, lung, breast, lymph, gastrointestinal (e.g., colon), and genitourinary tract (e.g., kidney, epithelial cells), and pharyngeal head. Lung squamous cancers include malignant tumors, e.g., most colon, rectum, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine and esophagus. Metastatic lesions of the cancers described above may likewise be treated and prevented with the methods and compositions of the present invention.
A pharmaceutically acceptable carrier: also known as excipients or stabilizers, are used in dosages and concentrations which are non-toxic to the cells or individuals to which they are exposed. Frequently, the physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid; a low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrins; complexing agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as tween (tm), polyethylene glycol (PEG), and pluronic (tm).
Detailed Description
PD-1 (Programmed Death-1) is an immunosuppressive receptor expressed on activated T-cells and B-cells, PDL-1 being its ligand. PD-1 belongs to the B7 family and is an Ig superfamily type I transmembrane glycoprotein with a size of 50-55 kD; it was found by structural and biochemical analysis that PD-1 exists as a monomer due to the lack of membrane proximal cysteine residues, consisting of an extracellular IgV region, a transmembrane region, and an intracellular region. PD-1 interacts with its ligand PDL-1 (Programmed Death Ligand-1), playing an important role in the negative regulation of immune responses. Through extensive and intensive studies, the present invention has unexpectedly found that a soluble PD-1 molecule having a high affinity for PDL-1 molecules can effectively enhance the killing ability of lymphocytes. Accordingly, the present invention provides a soluble high affinity PD-1 molecule having at least twice the affinity for PDL-1 as a wild-type PD-1 molecule.
The binding affinity of the PD-1 molecules described above to PDL-1 (with dissociation equilibrium constant K) can be determined by any suitable method D Inversely proportional) and binding half-life (expressed as T 1/2 ). It will be appreciated that doubling the affinity will result in K D Halving. T (T) 1/2 Calculated as In2 divided by dissociation rate (K off ). Thus, K is off Doubling can result in T 1/2 Halving. The binding affinity or binding half-life is preferably measured several times, e.g. 3 times or more, using the same assay protocol, and the results averaged. In a preferred embodiment, these assays are performed using the surface plasmon resonance (BIAcore) method of the present examples.
The method detects dissociation equilibrium constant K of the wild PD-1 molecules to the PDL-1 molecules in the invention D 2.815E-06M, its BIAcore binding profile is shown in FIG. 2. Will result in K due to the doubling of affinity D Halving, so if the dissociation equilibrium constant K of the high affinity PD-1 molecule for the PDL-1 molecule is detected D 1.408E-06M, this indicates that the high affinity PDL-1 molecule has a 2-fold affinity for PD-1 than the wild-type PD-1 molecule. K is well known to those skilled in the art D The conversion relationship between the units of values, i.e., 1m=1000 μΜ,1μΜ=1000 nm, 1nm=1000 pM.
In a preferred embodiment of the invention, the affinity of the PD-1 molecules of the invention to a PDL-1 molecule is at least 2-fold greater than the affinity of the wild-type PD-1 molecule to the PDL-1 molecule as measured by the preferred assay method of the invention; preferably at least 10 times; more preferably, at least 50 times; most preferably at least 100 times.
In another preferred embodiment, the affinity of the PD-1 molecule for a PDL-1 molecule is at least 500-fold greater than the affinity of a wild-type PD-1 molecule for a PDL-1 molecule; preferably at least 1000 times; more preferably at least 2000 times.
Specifically, the high affinity PD-1 molecules of the invention have an affinity K for PDL-1 D 1.408E-06M; preferably, 1.0E-06 M.ltoreq.K D Less than or equal to 5.0E-06M; more preferably, 1.0E-08 M.ltoreq.K D Less than or equal to 5.0E-07M; most preferably 1.0E-08M≤K D ≤1.0E-11M。
The high affinity PD-1 molecules of the invention contain one or more mutations in the amino acid sequence shown in SEQ ID NO. 1. Specifically, the PD-1 molecule has at least 90% (preferably at least 92%; more preferably at least 94%) sequence identity to the amino acid sequence shown in SEQ ID NO. 1.
More specifically, the mutated amino acid residue positions in the high affinity PD-1 molecules of the invention include one or more of 91G, 31V, 33N, 35Y, 37M, 40S, 41N, 42Q, 43T, 48A, 56P, 89L, 92A, 93I, 95L, 97P, 98K, 99A, 100Q, 101I, 103E, wherein the amino acid residue numbering is as set forth in SEQ ID NO. 1. Based on the technical disclosure of the present invention, and in particular the disclosed sequences, it will be understood by the person skilled in the art that the single letter in the amino acid residue position described above is an amino acid residue representing the position prior to mutation. Thus, the above-mentioned "amino acid residue positions" can also be written simply as positions 91, 31, 33, 35, 37, 40, 41, 42, 43, 48, 56, 89, 92, 93, 95, 97, 98, 99, 100, 101, 103, wherein the amino acid residue numbers are as shown in SEQ ID NO. 1.
In another preferred embodiment, the mutated PD-1 molecule comprises one or more amino acid residues selected from the group consisting of: 91A, 91S, 91V or 91T;31T;33L;35N, 35M;37V, 37L or 37E;40A, 40T;41G, 41L;42N;43V, 43G;48G, 48S;56L;89M;92V, 92Y;93L;95W, 95F;97G;98R, 98Y or 98P;99P, 99V, 99I or 99F;100S, 100W;101V;103D wherein the amino acid residue numbering is shown in SEQ ID NO. 1.
In another preferred embodiment, the amino acid sequence of the PD-1 molecule is selected from the group consisting of SEQ ID No.39, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41 or 43;
the coding nucleotide sequences correspond to: SEQ ID NOS.40, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42 and 44:
to obtain soluble high affinity PD-1 molecules, the wild type PD-1 molecules used in the present invention do not contain a transmembrane region. Thus, in a preferred embodiment of the invention, the PD-1 molecule is soluble.
Mutations may be made using any suitable method, including but not limited to those based on Polymerase Chain Reaction (PCR), cloning based on restriction enzymes, or Ligation Independent Cloning (LIC) methods. Many standard molecular biology textbooks detail these methods. For more details on Polymerase Chain Reaction (PCR) mutagenesis and cloning in accordance with restriction enzymes see Sambrook and Russell, (2001) molecular cloning-laboratory Manual (Molecular Cloning-A Laboratory Manual) (third edition) CSHL Press. More information on LIC methods can be found (Rashtchian, currOpinBiotechnol,1995,6 (1): 30-6).
Methods of producing high affinity PD-1 molecules of the invention may be, but are not limited to, screening a diverse library of phage particles displaying such PD-1 molecules for PD-1 having high affinity for PD-1, as described in the literature (Li, et al, nature Biotech,2005, 23 (3): 349-354).
It will be appreciated that genes expressing the wild type PD-1 of the invention or genes expressing the slightly modified wild type PD-1 of the invention can be used to prepare template strands. The changes required to produce the high affinity PD-1 of the invention are then introduced into the DNA encoding the template strand.
The PD-1 molecules of the invention may also be provided in the form of multivalent complexes. The multivalent PD-1 of the invention comprises a multimer of two, three, four or more molecules of the PD-1 of the invention bound, e.g., dimers may be prepared from IgG FC segments, or tetrameric domains of p53 to produce tetramers, or complexes of a plurality of PD-1 of the invention bound to another molecule.
The high affinity PD-1 molecules of the invention may be used alone or may be covalently or otherwise bound to a conjugate, preferably covalently. The conjugate is preferably a T cell receptor, more preferably the T cell receptor is a high affinity T cell receptor.
The high affinity PD-1 molecules of the invention can also be used in combination with other molecules to produce effective synergism. Preferably, the other molecule is ImmTAC or HATac. Both molecules are capable of redirecting T cells, thereby acting to kill target cells. The ImmTAC molecule is a fusion molecule of a soluble double-stranded TCR molecule containing an artificial interchain disulfide bond between the alpha beta constant regions and an anti-CD 3 antibody, as described in detail in the literature (Joanne Oates, bent K.Jakobsen, novel bi-specific agents for targeted cancer thrapy.Onco immunology,2013,2:2, e 22891). The HATac molecule is a High Affinity T cell activating core (High Affinity T-cell activation core), wherein one form of the fusion molecule of a soluble single chain TCR molecule, which can be formed by linking a hydrophobic core mutated α and β chain variable domains, with an anti-CD 3 antibody, is described in particular in WO2014/206304.
The invention also relates to nucleic acid molecules encoding the PD-1 of the invention. The nucleic acid molecules of the invention may be in the form of DNA or RNA. The DNA may be a coding strand or a non-coding strand. For example, the nucleic acid sequence encoding a TCR of the invention may be identical to or degenerate from the nucleic acid sequences shown in the figures of the invention. By way of example, a "degenerate variant" as used herein refers to a nucleic acid sequence encoding a protein sequence having SEQ ID NO.1, but differing from the sequence of SEQ ID NO. 2.
The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be generally obtained by, but not limited to, PCR amplification, recombinant methods or artificial synthesis. At present, it is already possible to obtain the DNA sequence encoding the PD-1 of the invention (or a fragment or derivative thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.
The invention also relates to vectors comprising the nucleic acid molecules of the invention, and host cells genetically engineered with the vectors or coding sequences of the invention.
The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the PD-1 of the invention or the PD-1 complex of the invention.
The present invention also provides a method of treating a disease comprising administering to a subject in need of treatment an appropriate amount of a PD-1 of the invention, or a PD-1 complex of the invention, or a pharmaceutical composition of the invention; in particular, the PD-1 molecules of the invention are used in combination with other molecules, preferably ImmTAC or HATac.
It should be understood that, in this document, the amino acid names are identified by international single english letters, and the amino acid names corresponding to the amino acid names are abbreviated as "three english letters: ala (A), arg (R), asn (N), asp (D), cys (C), gln (Q), glu (E), gly (G), his (H), ile (I), leu (L), lys (K), met (M), phe (F), pro (P), ser (S), thr (T), trp (W), tyr (Y), val (V); in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. The addition of one or several amino acids at the C-terminal and/or N-terminal will not generally alter the structure and function of the protein.
The invention also includes PD-1 molecules that have been slightly modified from PD-1 of the invention. Modified (typically without altering the primary structure) forms include: chemically derivatized forms of PD-1 of the invention, such as acetylated or carboxylated. Modifications also include glycosylation, such as those resulting from glycosylation modification during synthesis and processing or during further processing steps of the PD-1 of the invention. Such modification may be accomplished by exposing PD-1 to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are PD-1 modified to increase its proteolytic resistance or to optimize its solubility properties.
The PD-1 or PD-1 complexes of the invention may be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The PD-1, multivalent PD-1 complexes of the invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such kits (but not required) include instructions for use. Which may comprise a plurality of said unit dosage forms. In addition, PD-1 of the invention may be used alone or in combination or coupling with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to such agent carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and do not have excessive toxicity after administration. Such vectors are well known to those of ordinary skill in the art. A sufficient discussion of pharmaceutically acceptable excipients can be found in the pharmaceutical science of Remington (s Pharmaceutical Sciences, mack Pub.Co., N.J.1991)). Such vectors include (but are not limited to): saline, buffers, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
The pharmaceutically acceptable carrier in the therapeutic composition may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. In general, the therapeutic compositions may be formulated as an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for incorporation into a solution or suspension, and a liquid carrier prior to injection. Once formulated into the compositions of the present invention, they may be administered by conventional routes including, but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably parenteral including subcutaneous, intramuscular, or intravenous. The subject to be prevented or treated may be an animal; especially humans.
When the pharmaceutical composition of the present invention is used for actual treatment, various different dosage forms of the pharmaceutical composition can be employed according to the use condition. Preferably, injection, oral preparation and the like are exemplified. These pharmaceutical compositions may be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonides), preservatives, wetting agents, emulsifying agents, dispersing agents, stabilizers and cosolvents, and the formulation process may be carried out in a conventional manner according to dosage forms.
The pharmaceutical compositions of the present invention may also be administered in the form of a slow release formulation. For example, PD-1 of the invention may be incorporated into a pellet or microcapsule supported on a slow release polymer, which is then surgically implanted into the tissue to be treated. Examples of the slow release polymer include ethylene-vinyl acetate copolymer, polyhydroxymethacrylate (polyhydroxymethacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer, and the like, and preferably biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer.
When the pharmaceutical composition of the present invention is used for actual treatment, the PD-1 or the PD-1 complex of the present invention as an active ingredient can be appropriately determined according to the weight, age, sex, and symptom degree of each patient to be treated, and a reasonable amount is ultimately decided by a physician.
The invention has the main advantages that:
(1) The invention provides PD-1 molecules with high affinity for PDL-1.
(2) The high-affinity PD-1 molecule can effectively improve the killing capacity of lymphocytes.
The following specific examples further illustrate the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address specific conditions in the examples below, is generally followed by conventional conditions, for example those described in the laboratory Manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL Press, or by the manufacturer's recommendations (Sambrook and Russell et al, molecular cloning). Percentages and parts are by weight unless otherwise indicated.
EXAMPLE 1 expression, renaturation and purification of wild type PD-1
The extracellular amino acid sequence and the nucleotide sequence of the wild type PD-1 are SEQ ID NO.1 and SEQ ID NO. 2 respectively, and a target gene carrying the extracellular sequence of the wild type PD-1 is subjected to double digestion by Nco I and Not I and is connected with a pET28a vector (Novagen) subjected to double digestion by Nco I and Not I, and the vector is optimized and provided with a biotin tag. The ligation product was transformed into E.coli DH 5. Alpha (Vazyme), plated on LB plate containing kanamycin, cultured upside down at 37℃overnight, positive clones were picked up for PCR screening, positive recombinants were sequenced, and after confirming the correct sequence, the extracted recombinant plasmid was transformed into E.coli Rosetta strain (TIANGEN) for expression.
The Rosetta colony containing the recombinant plasmid pET28a-PD-1 is inoculated into LB culture medium containing kanamycin, cultured at 37 ℃ until the OD600 is 0.6-0.8, added with IPTG to a final concentration of 0.7mM, and cultured at 37 ℃ for 4 hours. Cell pellet was harvested by centrifugation at 6000g for 15min, cell pellet was lysed by BugbusterMaster Mix (Merck), inclusion bodies were recovered by centrifugation at 6000g for 15min, washed with Bugbuster (Merck) to remove cell debris and membrane components, and collected by centrifugation at 6000g for 15 min. The inclusion bodies were dissolved in a buffer (50 mM Tris-HCl,200mM NaCl,2mM EDTA,6M guanidine HCl,pH 8.1), the insoluble matter was removed by high-speed centrifugation, and the supernatant was quantified by BCA method and then sub-packaged, and stored at-80℃for use.
To 7mg of the solubilized PD-1 inclusion body protein, 2mL of buffer (50 mM Tris-H was addedCl,200mM NaCl,2mM EDTA,6M guanidine HCl,pH 8.1), DTT was added to a final concentration of 20mM and treated at 37℃for 1h. The PD-1 mixture thus treated was added dropwise to 100mL of a renaturation buffer (50mM HEPES,pH 7.5, 500mM L-arginine,9mM glutathione,1mM glutathione disulfide,24mM NaCl,1mM KCl), stirred at 4℃for 30 minutes, and the renaturation buffer was then filled to a retention of 3.5K D The dialysis bag was placed in 2L of pre-chilled water and stirred slowly overnight at 4 ℃. After 24 hours, the dialysate was changed to 2L of pre-chilled buffer (10 mM Tris-HCl, pH 8.5), dialysis was continued for 24 hours at 4℃and then the dialysate was changed to the same fresh buffer for 24 hours, the sample was filtered through a 0.45 μm filter, vacuum degassed and fed to an anion exchange column (HiTrap Q HP, GE Healthcare). The protein was purified using a linear gradient of 0-1M NaCl from 10mM Tris-HCl pH 8.5 and the collected fractions were analyzed by SDS-PAGE. Based on the analysis results, the target PD-1 fraction was collected, concentrated and further purified by a gel filtration column (Superdex 75 10/300,GE Healthcare), and the target fraction was also subjected to SDS-PAGE analysis, and the results are shown in FIG. 1.
Example 2 characterization in combination
BIAcore analysis
Binding activity of wild-type PD-1 molecules to PDL-1 was detected using the BIAcore T200 real-time assay system. The coupling process was completed by adding anti-streptavidin antibody (GenScript) to coupling buffer (10 mM sodium acetate buffer, pH 4.77), then flowing the antibody through CM5 chips previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally blocking the unreacted activated surface with ethanolamine in hydrochloric acid solution at a coupling level of about 15,000 RU.
The low concentration of streptavidin was allowed to flow over the surface of the antibody-coated chip, followed by biotinylated PD-1 over the detection channel and the other channel as a reference channel, and then 0.05mM biotin was allowed to flow over the chip at a flow rate of 10. Mu.L/min for 2min, blocking the remaining binding sites for streptavidin. The affinity was determined by single cycle kinetic analysis, PD-1 was diluted to several different concentrations with HEPES-EP buffer (10mM HEPES,150mMNaCl,3mM EDTA,0.005%P20,pH 7.4), and flowed sequentially over the chip surface at a flow rate of 30. Mu.L/min for a binding time of 120s each sample injection and allowed to dissociate for 600s after the end of the last sample injection. After each round of assay, the chip was regenerated with 10mM GLY-HCl at pH 1.75. Kinetic parameters were calculated using BIAcore Evaluation software.
The amino acid sequence and nucleotide sequence of PDL-1 used in this example are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, and the expression, renaturation and purification processes are the same as those of the wild-type PDL-1 in example 1. The biotinylation process is as follows:
a. biotinylation
Purified PDL-1 molecules were concentrated using Millipore ultrafiltration tubes while the buffer was replaced with 10mM Tris pH 8.0, and then biotinylated reagent 0.05MBicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. Mu. M D-Biotin, 100. Mu.g/ml BirA enzyme (GST-BirA), the mixture incubated overnight at room temperature and SDS-PAGE was performed to determine whether biotinylation was complete.
b. Purification of biotinylated complexes
Biotinylated PDL-1 molecules were concentrated to 500. Mu.l using a Millipore ultrafiltration tube, biotinylated PDL-1 was purified by gel filtration chromatography, a Superdex 75/300 gel filtration column (GE general electric company) was pre-equilibrated with filtered PBS, 500. Mu.l of the concentrated biotinylated PDL-1 molecules were loaded, then eluted with PBS at a flow rate of 1ml/min, the collected fractions were subjected to SDS-PAGE analysis, fractions containing the target protein were pooled according to the results, concentrated using a Millipore ultrafiltration tube, protein concentration was determined by the BCA method (Thermo), and split-packs of biotinylated PDL-1 molecules were stored at-80 ℃.
K for binding affinity of wild-type PD-1 molecule to PDL-1 molecule was detected by the procedure described above in this example D The BIAcore binding pattern was as shown in FIG. 2, with a value of 2.815E-06M.
Example 3 production of high affinity PD-1 molecules
The extracellular sequence of wild type PD-1 described in example 1 was used as a template strand according to Li et al, (2005) Nature Biotech 23 (3): 349-354) to perform high affinity PD-1 screening. Phage libraries after several rounds of screening all had stronger binding signals to PD-1, from which the monoclonal was picked and sequence analyzed.
The high affinity PD-1 molecules of the invention were expressed, renatured and purified as described in example 1 and their affinity to PDL-1 molecules was determined as described in example 2. The affinity of the high affinity PD-1 molecules obtained in the present invention to PDL-1 molecules is at least 2-fold higher than that of the wild-type PD-1 molecules to PDL-1 molecules, and the amino acid sequences and affinity values to PDL-1 molecules are shown in Table 1 below.
TABLE 1 BIAcore results of high affinity clones on PDL-1 molecules
Example 4L 5B7 ability to recognize PD-L1 on the surface of H1299 cells was higher than PD-1
Biacore results showed that PD-1 mutants with increased affinity were indeed obtained after screening, but whether this change in affinity would affect its binding to PDL-1 on the cell surface under physiological conditions was still experimentally confirmed. Therefore, we selected PDL-1 expressing positive H1299 cells, added different concentrations of biotinylated PD-1, L5B7 proteins, and analyzed the ability of PD-1, L5B7 to recognize cell surface PDL-1 by flow cytometry.
FIG. 3 shows that the recognition ability of PDL-1 is gradually enhanced as the concentration of the added PD-1, L5B7 proteins is increased; under the same concentration conditions, the L5B7 protein has higher ability to recognize PDL-1 than PD-1, and the change of the recognition ability is probably caused by higher affinity of L5B7, which is consistent with biochemical results.
Example 5 high affinity PD-1 molecule bivalent fusion protein production
a construction of expression vector
In order to increase the stability and potency of PD-1 and its high affinity mutants in vivo, eukaryotic expression takes the form of fusion expression with IgG 4. PD-1 protein eukaryotic expression sequences are optimized and synthesized by Souzhou province biotechnology limited company, connected to pGZFUSE plasmid vector through EcoR I and Nhe I sites after the completion of the splice with IgG4 overlay PCR, and transferred into Top 10 strain in a chemical way (mutant clones with other affinities are obtained through mutation). Inoculating into 200ml LB culture medium according to the ratio of 1:1000, culturing at 37 ℃ overnight, collecting bacteria on the next day, and extracting a large amount of plasmids. OD260/0D280 measured plasmid concentration, adjusted to 1mg/ml, split-packed and stored at-20 ℃. The large plasmid is ready for use. The schematic representation of the fusion expressed protein is shown in FIG. 4a.
b protein expression
And (3) constructing a eukaryotic expression vector according to the a, and after sequencing is correct, using a 293T adherent cell expression system for fusion protein expression. Day before transfection, 293T cells in logarithmic growth phase were plated in 10cm dishes, the next day with plasmid: lipo2000=1:2 (volume ratio) cells were transfected and replaced with fresh Freestyle after 4h TM 293 medium, and after 48h a small amount of supernatant was run on SDS-PAGE to identify expression as shown in FIG. 4b. After 72 hours, the supernatant was collected, filtered through a 0.22 μm filter, and diluted into 20-fold volume of 10mM Tris-HCl (pH=8.5) pre-chilled in advance, and anion and molecular sieve purification was performed. SDS-PAGE gel electrophoresis results show that: the fusion protein expressed by 293T adherent cells has high purity. Two purposes of further purification are that of replacing the solution in which the protein is located, preventing the influence of certain substances present during the expression process or in the culture medium itself on the subsequent experiments; secondly, the protein is concentrated, the purity is further increased, and the influence of the subsequent experiments caused by adding a large amount of protein or the existence of some hybrid proteins is avoided.
c fusion protein function identification
The ability of ImmTACs molecules to redirect T cell-specific killing of tumor cells has been reported in several studies (Jakobsen, 2013; oates et a1., 2015). The basic principle is that ImmTACs can simulate the key signals of T cell activation to play an effect function, on one hand, the MHC-peptide complex on the surface of tumor cells is identified through the high-affinity specific TCR, and on the other hand, the downstream signal path of T cell activation is activated through the anti-CD3 antibody end, so that the T cell specificity kills the tumor cells. Thus, the function of the PD-1/L5B7-IgG4 fusion protein evaluated in this study was assessed by whether the protein promoted the ImmTAC-IG4 molecule-mediated killing of Mel624 tumor cells by PBMC.
As a result, it was found that when the concentration of ImmTAC was 10 -9 M, E: T=5:1 and 1:1 (PBMC is effector cell, mel62 is target cell), PD-1-IgG4, L5B7-IgG4 fusion proteins can promote the LDH release of ImmTAC molecular oriented PBMC killing tumor cells, and L5B7-IgG4 fusion proteins promote LDH release higher than PD-1-IgG4 group; the cells in the reaction system are collected and subjected to flow detection when the killing ratio is 1:1, so that the expression of CD25 and CD107a of CD 8T cells of the PD-1-IgG4 and L5B7-IgG4 fusion protein groups is up-regulated compared with that of the non-protein group, the up-regulated proportion of the L5B7-IgG4 group is higher than that of the PD-1-IgG4 group, the CD25 is up-regulated from 10.9% to 13.2%, and the CD107a is up-regulated from 16.9% to 19.8%, and the result of the increase of LDH release is further verified. The PD-1-IgG4 and L5B7-IgG4 fusion proteins can promote the ImmTAC molecule-mediated PBMC to kill Mel624 tumor cells, and the promotion effect is further enhanced after the affinity is increased in accordance with the research results that the soluble PD-1 in other documents can promote tumor-specific T cell killing.
Example 6 proliferation assay of high affinity PD-1 molecules to promote activated PBMC
This experiment was performed to verify that the high affinity PD-1 molecules of the invention are able to promote the proliferation of activated PBMCs.
The fluorescent dye CFSE, also known as CFDA SE (5, 6-carboxyfluorescein diacetate, succinimidyl ester), i.e. hydroxyfluorescein diacetate succinimidyl ester, is a cell membrane permeable fluorescent dye with succinimidyl ester groups that bind specifically to cells and hydroxyfluorescein diacetate groups that have non-enzymatic hydrolysis, which makes CFSE a good cell marker. When the cells undergo division proliferation, the cytoplasmic proteins with fluorescence are equally distributed into the cells of the second generation, so that the fluorescence intensity is reduced to half compared with the cells of the first generation; similarly, the fluorescence intensity of the third generation cells obtained by division is reduced again than that of the second generation cells. The phenomenon can be detected and analyzed by a flow cytometry under the excitation light of 488nm, and the situation of cell division and proliferation can be further analyzed by detecting the continuous decrease of the fluorescence intensity of cells.
In this example, freshly isolated Peripheral Blood Mononuclear Cells (PBMC) were stained with CFDA-SE at a final concentration of 1. Mu.M, and then stained with RIPM-1640 containing 10% FBS to terminate the CFDA-SE twice. At 1.5 x 10 5 Each well was plated on a 96-well flat bottom plate and PBMC proliferation was stimulated by the addition of 15. Mu.g/ml anti-CD3 mAb and 7.5. Mu.g/ml anti-CD28 mAb. The results show that the high affinity PD-1 molecules not only significantly reduce the number of non-proliferating cells; also, from the fluorescence shift, the group fluorescence added with the high affinity PD-1 mutation shifted more leftward, indicating greater proliferation intensity (fig. 5 a). Statistics of the rate at which PD-1 protein and high affinity mutant promote proliferationEach high affinity mutant was found to promote 18% to 20% of anti-CD3 mAb and anti-CD28 mAb mediated proliferation of PBMC, but wild type PD-1 did not significantly promote proliferation of PBMC (FIG. 5 b).
Example 7 IFN-gamma Release assay of high affinity PD-1 molecules to promote PBMC
anti-CD3 mAb and anti-CD28 mAb can promote the proliferation of PBMC and promote the release of IFN-gamma from PBMC. In this example, after PBMC were stimulated with 60. Mu.g/ml anti-CD3 mAb and 30. Mu.g/ml anti-CD28 mAb, by enzyme-linked immunosorbent assay (Elispot), PD-1 monomeric protein was found not to promote IFN-gamma release from stimulated PBMC, but after addition of 5. Mu.g/ml L1B2, 5. Mu.g/ml L2B12, 5. Mu.g/ml L2F8, 5. Mu.g/ml L2F10, 5. Mu.g/ml L5B7, 5. Mu.g/ml L45 high affinity mutant protein, the spots for IFN-gamma release were detected to be significantly increased (FIG. 6 a) with a clear relationship between the increase in spots and affinity. Statistics of the rate at which different high affinity mutants promote IFN-gamma release The ratio of low affinity L1B2 to L2B12 promoting IFN-gamma release is about 20%, so that the affinity is further improved and the promotion is promotedThe rate of IFN-gamma release was increased to 40-50% (FIG. 6 b).
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Sequence listing
<110> Guangdong Xiangxue medical technology Co., ltd
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aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gctgcaattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 7
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 7
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Thr Leu
20 25 30
Leu Trp Met Arg Leu Ser Pro Thr Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 8
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt acgctgttgt ggatgcgtct tagcccgact 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt ggtgcaattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 9
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 9
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Leu Trp Met Arg Glu Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 10
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gttctgttgt ggatgcgtga gagcccgtcg 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt ggtgcaattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 11
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 11
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Gly Gln Gly Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 12
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 12
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
gggcaggggg ataagctggc ggcgttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt agtgcaattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 13
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 13
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Gly Gln Val Asp Lys Leu Ala Gly
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Pro Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 14
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 14
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
ggtcaggttg ataagctggc gggtttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gttgcaattt cactggctcc gaaaccccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 15
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 15
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Leu Asn Gly Asp Lys Leu Ala Ser
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Pro Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 16
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 16
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
cttaatggtg ataagctggc gtcgttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gttgcaattt cactggctcc gaaaccccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 17
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 17
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Met Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 18
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 18
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacatgtgt gttgcaattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 19
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 19
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Leu Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Thr Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 20
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 20
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagctggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt acggctattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 21
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 21
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ala Ala Leu Ser Trp Ala
85 90 95
Gly Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 22
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 22
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gctgcgctgt catgggctgg taaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 23
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 23
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 24
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 24
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt tcggctattt cattggctcc taaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 25
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 25
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 26
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 26
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gttgctattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 27
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 27
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Val Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 28
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 28
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt tcggttattt cattggctcc taaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 29
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 29
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Thr Ala Ile Ser Trp Ala
85 90 95
Gly Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 30
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 30
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt actgctattt catgggctgg gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 31
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 31
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Thr Tyr Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 32
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 32
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt acttatattt cactggctcc taaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 33
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 33
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Tyr Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 34
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 34
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gtgtatattt cacttgctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 35
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 35
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Val Ile Ser Phe Ala
85 90 95
Gly Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 36
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 36
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt tcggtgattt catttgctgg gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 37
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 37
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala
85 90 95
Pro Arg Val Ser Val Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 38
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 38
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt ggtgcaattt cactggctcc gcgtgttagt 300
gttaaagagt cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 39
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 39
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala
85 90 95
Pro Tyr Ile Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 40
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 40
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt agtgcaattt cactggctcc gtatattcag 300
attaaagagt cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 41
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 41
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala
85 90 95
Pro Pro Phe Trp Ile Lys Asp Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 42
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 42
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt ggtgcaattt cactggctcc gcctttttgg 300
attaaagatt cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 43
<211> 117
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 43
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ile Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 44
<211> 351
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 44
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gttgcaattt cactggctcc gaaaattcaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351

Claims (14)

1. A PD-1 molecule, wherein the amino acid sequence of the PD-1 molecule is SEQ ID No.39.
2. The PD-1 molecule of claim 1, wherein the PD-1 molecule is soluble.
3. The PD-1 molecule of claim 1, wherein the C-or N-terminus of the PD-1 molecule is conjugated to a conjugate.
4. The PD-1 molecule of claim 3, wherein the conjugate that binds to the PD-1 molecule is a T cell receptor.
5. The PD-1 molecule of claim 4, wherein the T cell receptor is a high affinity T cell receptor.
6. A fusion protein comprising the PD-1 molecule of any one of claims 1-5.
7. The fusion protein of claim 6, further comprising IgG4.
8. A multivalent PD-1 complex, wherein the multivalent PD-1 complex comprises at least two PD-1 molecules, and wherein at least one of the PD-1 molecules is the PD-1 molecule of any one of claims 1-5; or the multivalent PD-1 complex comprises at least one fusion protein of claim 6 or 7.
9. A nucleic acid molecule comprising a nucleic acid sequence encoding the PD-1 molecule of any one of claims 1-5, the fusion protein of claim 6 or 7, or the multivalent PD-1 complex of claim 8.
10. A vector comprising the nucleic acid molecule of claim 9.
11. A host cell comprising the vector of claim 10 or the nucleic acid molecule of claim 9 integrated into a chromosome; or alternatively
The host cell contains or expresses the PD-1 molecule of any one of claims 1-5, the fusion protein of claim 6 or 7, or the multivalent PD-1 complex of claim 8.
12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a PD-1 molecule according to any one of claims 1-5, or a fusion protein according to claim 6 or 7, or a PD-1 complex according to claim 7.
13. Use of a PD-1 molecule of any one of claims 1-5, a fusion protein of claim 6 or 7, or a PD-1 complex of claim 7, for the manufacture of a medicament for the treatment of a PD-1-related solid tumor.
14. A method of preparing the PD-1 of any one of claims 1-5, comprising the steps of:
(i) Culturing the host cell of claim 11, thereby expressing the PD-1 molecule of any one of claims 1-5;
(ii) Separating or purifying the PD-1 molecule.
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CN108794619B (en) * 2018-05-31 2021-09-17 郑州大学 High-affinity PD-1 protein mutant
CN114181296B (en) * 2018-06-07 2023-06-30 江苏东抗生物医药科技有限公司 Fusion protein of high-affinity PD-1 extracellular region mutant, and pharmaceutical composition and application thereof
CN111714618B (en) * 2019-03-22 2024-07-12 香雪生命科学技术（广东）有限公司 Combination of T cells and high affinity PD-1 fusion proteins
CN110590959B (en) * 2019-09-19 2021-01-05 北京伟杰信生物科技有限公司 Recombinant canine PD-1 fusion protein and preparation method and application thereof
CN110478472B (en) * 2019-09-29 2020-08-28 北京鼎成肽源生物技术有限公司 PD-1 sealant and application thereof

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WO2016023001A1 (en) * 2014-08-08 2016-02-11 The Board Of Trustees Of The Leland Stanford Junior University Multispecific high affinity pd-1 agents and methods of use
CN105985427A (en) * 2015-02-06 2016-10-05 广州市香雪制药股份有限公司 High-affinity NY-ESO T cell receptor

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