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CN111714618B - Combination of T cells and high affinity PD-1 fusion proteins - Google Patents

Combination of T cells and high affinity PD-1 fusion proteins Download PDF

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CN111714618B
CN111714618B CN201910224044.5A CN201910224044A CN111714618B CN 111714618 B CN111714618 B CN 111714618B CN 201910224044 A CN201910224044 A CN 201910224044A CN 111714618 B CN111714618 B CN 111714618B
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CN111714618A (en
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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 discloses combinations of T cells that specifically recognize NY-ESO-1 and high affinity PD-1 fusion proteins. The combination realizes the combination of TCR-T adoptive cell therapy and immunodetection point therapy, thereby solving the difficult problem that the high-affinity TCR-T adoptive immunotherapy can not overcome the inhibition effect mediated by PD-1 signaling pathway, achieving the excellent therapeutic effect which can not be achieved by single drug therapy and obviously limiting the growth of tumor cells.

Description

Combination of T cells and high affinity PD-1 fusion proteins
Technical Field
The invention relates to the field of cellular immunotherapy of tumors; in particular, the invention relates to a combination of T-cell receptor transduced T cells with a high affinity PD-1 fusion protein and the use of such a combination in the prevention and treatment of cancer.
Background
T cell receptor transduced T cells (TCR-T) adoptive immunotherapy is a rapidly evolving tumor treatment in recent years. The method transfers the T cells with specific reactivity to the target cell antigen into the patient so as to make the T cells act against the target cells. The T Cell Receptor (TCR) is a membrane protein on the surface of T cells that is capable of recognizing the corresponding antigenic short peptide on the surface of target cells. In the immune system, direct contact of T cells with Antigen Presenting Cells (APC) is initiated by binding of an antigen oligopeptide-specific TCR to a oligopeptide-major histocompatibility complex (pMHC complex). Then, other cell membrane surface molecules of both T cells and APC interact, causing a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells of different antigen specificities to exert immune effects on their target cells. In particular, when the affinity of the TCR for recognizing the pMHC complex is further improved, the target antigen on the surface of the tumor cell can be recognized more efficiently, and the tumor cell can be cleared more effectively.
The short peptide SLLMWITQC is derived from the NY-ESO-1 protein expressed by a variety of tumor cells (Chen et al, PNAS USA,94, 1914-1918). The HLA class I molecules of tumor cells present short peptides derived from NY-ESO-1, including SLLMWITQC. Thus, the SLLMWITQC-HLA A A2 complex provides a marker for targeting tumor cells by TCR. The TCR capable of combining with SLLMWITQC-HLA A A2 complex has high application value for treating tumors.
How T cells after adoptive transfer enter the tumor microenvironment is a challenge. Wherein the inhibitory pathway of the T cell negative co-stimulatory molecule is exploited by the tumor against the immune system, allowing it to escape from the monitoring and attack of the immune system of the body. PD-1 (Programmed desath 1) and its ligand PD-L1 (Programmed desath-ligand 1) belong to the CD28/B7 superfamily of proteins, are important immunosuppressive molecules, can intercept T cell activation signals, inhibit T cell proliferation and release of cytokines, play an important role in tumor immune escape, autoimmune diseases and viral infection processes (Keir ME et al, annu Rev Immunol,26, 677-704). In earlier studies, high affinity PD-1 molecules were found to recognize PD-L1 and PD-L2 on the surface of tumor cells with higher affinity than wild type PD-1 molecules, blocking the PD-1 signaling pathway, reversing PD-1 signaling pathway mediated T cell inhibition.
There is an urgent need in the art for a means of enhancing the killing effect of T cells after adoptive transfer on tumors, thereby enhancing the therapeutic effect of T cells.
Disclosure of Invention
The present invention aims to provide a combination which is capable of enhancing the killing effect of T cells against tumors after adoptive transfer.
It is also an object of the present invention to provide such a combination for the preparation of a medicament for the prevention and treatment of tumors and the use of such a combination for the prevention and treatment of cancer.
In a first aspect, the invention provides a combination comprising a T cell and a high affinity PD-1 fusion protein comprising a high affinity PD-1 molecule and an Fc fragment of an IgG molecule, wherein:
(i) The affinity of the high affinity PD-1 molecule to the PDL-1 molecule is at least 100-fold greater than the affinity of the wild-type PD-1 molecule to the PDL-1 molecule; and
(Ii) The amino acid sequence of the high affinity PD-1 molecule has at least 90% sequence identity to the wild type PD-1 amino acid sequence (SEQ ID NO: 1).
In a preferred embodiment, the amino acid sequence of the high affinity PD-1 molecule is 92% from the amino acid sequence shown in SEQ ID No. 1; preferably, at least 94% (e.g., 95%, 96%, 97%, 98%, and 99%) of the sequence is identical.
In a preferred embodiment, the affinity of the high affinity PD-1 molecule for PDL-1 molecule is at least 200-fold greater than the affinity of the wild-type PD-1 molecule for PDL-1 molecule; preferably at least 500 times; more preferably at least 1000 times; more preferably at least 2000 times.
In a preferred embodiment, the amino acid sequence of the PD-1 molecule is selected from SEQ ID NO 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.
In a preferred embodiment, the PD-1 molecule has an amino acid sequence selected from the group consisting of SEQ ID NO 15, 16, 28, 30 or 31.
In a specific embodiment, the high affinity PD-1 molecule of the high affinity PD-1 fusion protein is located N-terminal to the Fc fragment of an IgG molecule.
In specific embodiments, the Fc fragment of IgG is selected from the group consisting of the Fc fragment of human IgG1, igG2, igG3, or IgG 4; preferably, the Fc fragment of IgG 4.
In a preferred embodiment, the Fc fragment comprises a hinge region, CH2 and CH3 domains, preferably the Fc fragment does not comprise CH1 and CH4 domains, more preferably the Fc fragment has the amino acid sequence SEQ ID NO:32.
In specific embodiments, the amino acid sequence of the high affinity PD-1 fusion protein is selected from the group consisting of SEQ ID NO. 2, 3, 33, 34 or 35; preferably, the amino acid sequence of the high affinity PD-1 fusion protein is SEQ ID NO 2 or 3.
In a preferred embodiment, the fusion protein comprises two high affinity PD1 molecules.
In a preferred embodiment, two high affinity PD1 molecules in the fusion protein are linked by disulfide bonds to form a dimer structure; preferably, the disulfide bond is a disulfide bond of the hinge region of the Fc fragment of an IgG molecule.
In particular embodiments, the T cell contains a nucleic acid encoding a high affinity TCR and expresses the high affinity TCR.
In a specific embodiment, the T cell contains a nucleic acid encoding a high affinity TCR that specifically recognizes NY-ESO-1 and expresses a high affinity TCR that specifically recognizes NY-ESO-1.
In a specific embodiment, the high affinity TCR that specifically recognizes NY-ESO-1 has activity of binding to SLLMWITQC-HLA A A2 complex,
The high affinity TCR that specifically recognizes NY-ESO-1 comprises a TCR alpha chain variable domain comprising 3 CDR regions and a TCR beta chain variable domain, the reference sequence of the 3 CDR regions of the TCR alpha chain variable domain is as follows,
CDR1α：TSINN
CDR2α：IRSNERE
CDR3 a: ATDANGKII, and contains at least one of the following mutations:
And/or, the TCR.beta.chain variable domain comprises 3 CDR regions, the reference sequence of the 3 CDR regions of the TCR.beta.chain variable domain is as follows,
CDR1β：SGHDY
CDR2β：FNNNVP
CDR3 β: ASSLGSNEQY, and contains at least one of the following mutations:
Residues before mutation Residues after mutation
3 Rd N of CDR2 beta H
4 Th N of CDR2 beta G
CDR 2. Beta. Position 5V A
Position 6P of CDR2 beta V
3 Rd position S of CDR3 beta Q or M
CDR3 beta 4 th L R or K or M, and
CDR3 beta 6 th S A or G or P
In a specific embodiment, the alpha chain variable domain of the high affinity TCR that specifically recognizes NY-ESO-1 comprises an amino acid sequence having at least 90% sequence homology with the amino acid sequence set forth in SEQ ID NO. 4; and/or the β chain variable domain of the high affinity TCR that specifically recognizes NY-ESO-1 comprises an amino acid sequence having at least 90% sequence homology with the amino acid sequence set forth in SEQ ID No. 5.
In a specific embodiment, the alpha chain variable domain sequence of the high affinity TCR that specifically recognizes NY-ESO-1 is SEQ ID NO:6 and the beta chain variable domain sequence of the high affinity TCR that specifically recognizes NY-ESO-1 is SEQ ID NO:5.
In a preferred embodiment, the C-or N-terminus of the alpha and/or beta chain of the TCR is conjugated to a conjugate.
In a preferred embodiment, the conjugate that binds to the TCR is a detectable label, a therapeutic agent, a PK modifying moiety, or a combination of any of these.
In a preferred embodiment, the therapeutic agent that binds to the TCR is an anti-CD 3 antibody that is linked to the C-or N-terminus of the alpha or beta chain of the TCR.
In a specific embodiment, the combination is a pharmaceutical composition.
In a specific embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
In specific embodiments, the high affinity PD-1 fusion protein is expressed on T cells expressing the high affinity TCR that specifically recognizes NY-ESO-1.
In specific embodiments, the high affinity TCR that specifically recognizes NY-ESO-1 and the high affinity PD-1 fusion protein are expressed by T cells via the same vector or different vectors.
In a second aspect, the present invention provides the use of a combination according to the first aspect for the manufacture of a medicament for the treatment of a tumour, preferably a tumour with high expression of NY-ESO-1.
In a third aspect, the present invention provides a method of treating a disease comprising administering to a subject in need of treatment a combination according to the first aspect.
In a specific embodiment, the disease is a tumor that highly expresses NY-ESO-1.
In a fourth aspect, the invention also provides a combination of a T cell and a high affinity PD-1 molecule, wherein the T cell and the high affinity PD-1 molecule are as described in the first aspect above.
In a specific embodiment, the high affinity PD-1 molecules of the present invention are produced according to CN 107987153A. The entire contents of CN 107987153A are incorporated herein by reference.
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 shows the amino acid sequence of a wild-type PD-1 molecule.
FIG. 2 shows the amino acid sequence of the fusion protein L5B7-Fc of the high affinity PD1 molecule with the fragment of the IgG4 molecule.
FIG. 3 shows the amino acid sequence of the fusion protein L45-123-Fc of the high affinity PD1 molecule and the fragment of the IgG4 molecule.
FIGS. 4a and 4b show the wild-type TCR alpha and beta chain variable domain amino acid sequences capable of specifically binding to the SLLMWITQC-HLA A A2 complex, respectively; wherein the wild-type TCR β chain variable domain amino acid sequence shown in figure 4b is also the β chain variable domain amino acid sequence of a T cell transferred high affinity TCR of the invention.
FIG. 5 shows the alpha chain variable domain amino acid sequence of a high affinity TCR transferred into T cells in an embodiment of the invention.
FIGS. 6a and 6b show the amino acid sequence and nucleic acid sequence, respectively, of a wild-type TCR alpha chain that is capable of specifically binding to the SLLMWITQC-HLA A A2 complex.
FIGS. 7a and 7b show the amino acid and nucleic acid sequences, respectively, of a wild-type TCR beta chain capable of specifically binding to the SLLMWITQC-HLA A A2 complex.
FIG. 8 shows the efficiency of detecting high affinity TCR transfected T cells by flow cytometry.
Figure 9 shows the clearance of tumor cells by the combination of the invention as determined by a cytotoxic assay (LDH).
Detailed Description
The inventors have conducted extensive and intensive studies and have unexpectedly found that designing T cells, particularly T cells specifically recognizing NY-ESO-1, in combination with a high affinity PD-1 fusion protein to a novel combination enables the combination of TCR-T adoptive cell therapy and immunodetection point therapy, thereby solving the problem that high affinity TCR-T adoptive immunotherapy cannot overcome the inhibition mediated by PD-1 signaling pathway, achieving excellent therapeutic effects that cannot be achieved by monotherapy, and significantly limiting the growth of tumor cells. The present invention has been completed on the basis of this finding.
The combinations of the invention include T cells and high affinity PD-1 fusion proteins. The T cells are from a subject, such as a human. In one or more embodiments, the T cells are present in the form of Peripheral Blood Mononuclear Cells (PBMCs) that include T cells. In one or more embodiments, the T cell and the high affinity PD-1 fusion protein can be administered separately, sequentially or simultaneously.
Based on the teachings of the present invention, one skilled in the art will recognize that the combination of the present invention may be a composition, such as a pharmaceutical composition. In one or more embodiments, the combination of the invention is a composition comprising a T cell and a high affinity PD-1 fusion protein. The composition may comprise a pharmaceutically acceptable carrier.
The high-affinity PD-1 fusion protein can specifically identify the cell surface and/or the soluble PD-L1 and PD-L2 in the tumor microenvironment, so that the PD-1 signal path for inhibiting the function of the T cells is blocked, and the cytotoxicity and proliferation capacity of the T cells are enhanced.
In one or more embodiments, the combinations of the invention include T cells that specifically recognize NY-ESO-1 and a high affinity PD-1 fusion protein. In one or more embodiments, the T cell that specifically recognizes NY-ESO-1 and the high affinity PD-1 fusion protein can be administered separately, sequentially or simultaneously. In one or more embodiments, the combination of the invention is a composition comprising T cells that specifically recognize NY-ESO-1 and a high affinity PD-1 fusion protein.
According to the invention, through combining the T cells specifically recognizing the NY-ESO-1 and the high-affinity PD-1 fusion protein, the combination of two immunotherapies is realized for treating the NY-ESO-1+ tumor, so that an excellent treatment effect which cannot be achieved by single-drug treatment is achieved, and the growth of tumor cells is obviously limited.
The T cell specifically recognizing NY-ESO-1 means that the T cell contains a nucleic acid encoding a high affinity TCR specifically recognizing NY-ESO-1 and expresses a high affinity TCR specifically recognizing NY-ESO-1. The nucleic acid includes a nucleic acid fragment encoding an alpha chain of the TCR and an acid fragment encoding a beta chain of the TCR, and the high affinity TCR includes an alpha chain and a beta chain.
Preferably, the high affinity TCR has the property of binding to the SLLMWITQC-HLA A2 complex; and the TCR has a binding affinity for the SLLMWITQC-HLA A2 complex that is at least 2-fold greater than the binding affinity of a wild-type TCR for the SLLMWITQC-HLA A2 complex; more preferably the binding affinity of the high affinity TCR to the SLLMWITQC-HLA A A2 complex is between 10. Mu.M and 0.1. Mu.M.
The high affinity TCR has 3 CDR regions in its alpha chain variable domain
CDR1α：TSINN
CDR2α：IRSNERE
CDR3 a: ATDANGKII, a mutation; and/or 3 CDR regions in its beta chain variable domain
CDR1β：SGHDY
CDR2β：FNNNVP
CDR3 β: ASSLGSNEQY, a mutation; furthermore, the affinity and/or binding half-life of the inventive TCRs for the SLLMWITQC-HLA A2 complex described above after mutation is at least 2-fold that of the wild-type TCR.
In a specific embodiment, the high affinity TCRs of the present invention are constructed, generated and screened as described in WO2018/099402 A1. The entire contents of WO2018/099402A1 are incorporated herein by reference.
The term "T cell" as used herein is an immune system cell that matures in the thymus and produces T Cell Receptors (TCRs). The T cells in the invention can be CD4+T cells and/or CD8+T cells, and the two cells can be used singly or in a mixture. Cd8+ T cells are preferred.
The term "T Cell Receptor (TCR)" as used herein consists of two chains (αβ or γδ) that pair on the surface of a T cell to form a heterodimeric receptor. αβ TCRs are expressed on most T cells in the body and are known to be involved in recognition of MHC-restricted antigens. The molecular genetics, structure and biochemistry of αβ TCRs have now been thoroughly studied. Each alpha and beta chain consists of two domains: a constant domain (C) that anchors proteins in the cell membrane and associates with a constant subunit of the CD3 signaling device, and a variable domain (V) that confers antigen recognition through six loops, known as Complementarity Determining Regions (CDRs). Each V domain has three CDRs. The interaction of these CDRs with a peptide (peptide-MHC) bound to a protein encoded by a major histocompatibility complex (Davis and Bjorkman (1988), nature 334,395-402; davis et al (1998), immunology annual. 16, 523-544; murphy (2012), xix, page 868).
The term "wild-type TCR" as used herein refers to a TCR comprising a naturally occurring or unmodified V domain (e.g., isolated from an initial or maternal T cell clone) and having specific recognition for an antigen. The wild type TCR described in the present invention preferably has the property of binding to the SLLMWITQC-HLA A2 complex; more preferably, the amino acid sequence of the alpha chain of the wild-type TCR is shown as SEQ ID NO. 7, and the corresponding nucleic acid sequence is shown as SEQ ID NO. 8; the amino acid sequence of the beta chain of the wild TCR is shown as SEQ ID NO. 9, and the corresponding nucleic acid sequence is shown as SEQ ID NO. 10.
The term "high affinity TCR" as used herein refers to engineering a normally low affinity wild-type TCR by directed evolution means to give TCR mutants with increased affinity for a particular peptide-MHC. Such engineering means include, but are not limited to, yeast display (Holler et al (2003) Nature. Immunology, 4,55-62; holler et al (2000) Proc. Natl. Acad. Sci. U.S., 97,5387-92), phage display (Li et al (2005) Nature. Biotechnology, 23,349-54), and the like.
As used herein, "T cells that specifically recognize NY-ESO-1" refers to T cells that contain a nucleic acid encoding a high affinity TCR that specifically recognizes NY-ESO-1, and/or express a high affinity TCR that specifically recognizes NY-ESO-1. The T cells can specifically recognize NY-ESO-1 and NY-ESO-1 positive tumors. Preferably, in a T cell specifically recognizing NY-ESO-1, the alpha chain variable domain sequence of the high affinity TCR specifically recognizing NY-ESO-1 is SEQ ID NO:6 and the beta chain variable domain sequence of the high affinity TCR specifically recognizing NY-ESO-1 is SEQ ID NO:5.
The nucleic acid molecules described herein 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 the fusion protein of the invention may be identical to or degenerate from the nucleic acid sequences shown in the figures of the present invention. By "degenerate variant" is meant, as used herein, a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO. 7, but differing from the sequence of SEQ ID NO. 8, of the alpha chain having a TCR.
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 entirely possible to obtain DNA sequences encoding the invention by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells as known in the art.
The high-affinity PD-1 fusion protein disclosed by the invention refers to a polypeptide formed by fusing a high-affinity PD-1 molecule and/or a human IgG Fc segment; preferably a soluble, dimeric molecule; preferably, the amino acid sequence of the fusion protein is SEQ ID NO.2 or SEQ ID NO. 3. The term "high affinity PD-1 fusion protein" is used interchangeably herein with "high affinity PD-1 fusion protein".
The term "high affinity PD-1 molecule" as used herein means that it has at least 100-fold, preferably at least 150-fold, more preferably at least 200-fold, more preferably at least 500-fold higher affinity for PD-L1 than the wild-type PD-1 molecule, and that the amino acid sequence of the molecule has at least 90%, at least 95% or at least 99% similarity to the wild-type PD-1 amino acid sequence, and/or does not affect its binding capacity to PD-L1 after fusion to a human IgG Fc fragment. The affinity of the wild-type PD1 molecule to the PDL1 molecule was 2.815E-06M as measured by the same method.
The term "Fc segment of IgG" as used herein refers to the constant region of an immunoglobulin heavy chain, e.g., the Fc segment of IgG comprises a combination of two or more domains of heavy chains CH1, CH2, CH3, CH4 and a hinge region, and in one or more embodiments, the Fc region of an immunoglobulin used comprises at least one immunoglobulin hinge region, one CH2 domain and one CH3 domain, preferably lacks a CH1 domain, more preferably lacks a CH1, CH4 domain.
The term "dimer" as used herein refers to two fusion protein polypeptide chains that are stably linked together by covalent or non-covalent interactions. In particular, a stable dimer structure is formed by covalent bonds formed by disulfide bonds in the Fc region of immunoglobulins.
The high affinity PD-1 fusion proteins of the invention can be produced by the T cells or can be produced separately. Preferably, it is produced separately.
Furthermore, based on the teachings of the present invention, one skilled in the art will appreciate that the combinations of the present invention also include combinations of T cells and high affinity PD-1 molecules, wherein the T cells and the high affinity PD-1 molecules are as described above. The high affinity PD-1 molecules of the invention were generated as described in CN 107987153A. The entire contents of CN 107987153A are incorporated herein by reference.
The term "production" as used herein refers to the expression of proteins known in the art. For example, a vector encoding a high affinity PD-1 fusion protein can be transferred into T cells by lentiviral infection to express the protein of interest; vectors carrying the high affinity PD-1 fusion protein encoding can also be transferred into engineered or commercial protein expression systems to express the protein of interest, such as commercial 293FTM eukaryotic expression systems.
The high affinity TCR molecules and the high affinity PD-1 fusion proteins of the combinations described herein can be expressed from one vector or from two vectors. Preferably, two vectors are used for expression.
The term "vector" as used herein may be a eukaryotic expression and/or recombinant virus; the eukaryotic expression plasmid may be, for example, pcDNA3.1, pcDNA3.4, pUSE, pVAX1, transposon plasmid; the recombinant virus may be a recombinant retrovirus, a recombinant lentivirus. In one embodiment of the invention the vectors are pFUSE and recombinant lentiviruses.
The combination of the invention is used for treating tumors. Preferably for the treatment of tumors that highly express NY-ESO-1.
The term "tumor" as used herein 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 invasion. 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 compositions and methods of the present invention. "tumor" includes, but is not limited to, melanoma, multiple myeloma, lung cancer, breast cancer, leukemia, glioma, sarcoma, glioma, skin cancer, oral cancer, colon cancer, stomach cancer, ovarian cancer, prostate cancer, uterine cancer, endometrial cancer, cervical cancer, bladder cancer, pancreatic cancer, bone cancer, liver cancer, gall bladder cancer, kidney cancer, and testicular cancer.
The combination according to the invention may be a pharmaceutical composition.
The pharmaceutical compositions of the present invention generally further comprise a pharmaceutically acceptable carrier. 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 pharmaceutical compositions of the present invention 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.
The pharmaceutical compositions of the present invention may be used alone or in combination with one or more other therapeutic agents. Thus, in a further aspect the invention provides a further combination comprising a combination of the invention and one or more other therapeutic agents, and a composition comprising said combination of the invention and the use of such further combination, composition in therapy; including but not limited to, combined use with any one or at least two of surgery, chemotherapy, or radiation therapy.
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.
These pharmaceutical compositions may also 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, 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.
When the pharmaceutical composition of the present invention is used for actual treatment, the pharmaceutical composition 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 combinations of the invention may allow separate, sequential or simultaneous administration of the T cells, in particular T cells specifically recognizing NY-ESO-1 with high affinity, and PD-1 fusion proteins. The combinations of the invention may be provided in the form of pharmaceutical compositions, or in the form of T cells expressing TCR and PD-1 fusion proteins which specifically recognize NY-ESO-1 with high affinity.
Typically, a therapeutically effective amount of a combination of the invention will be administered. The term "therapeutically effective amount" when administered to an animal, preferably a mammal, more preferably a human in need of such treatment, refers to an amount sufficient to achieve therapeutic purposes as defined below. The therapeutically effective amount will vary depending on the subject and nature of the disease being treated, the severity of the tumor and the manner of administration, and can be routinely determined by one of ordinary skill in the art.
The term "treatment" as used herein encompasses any treatment of a disease in an animal, preferably a human, and includes: (i) inhibiting a disease; (ii) alleviating a disease; or (iii) alleviating symptoms of a condition caused by a disease, such as a tumor.
The term "preventing" as used herein encompasses the prevention of a disease in an animal, preferably a human, and includes the prevention of the occurrence of a disease, such as a tumor, in a subject that has not been diagnosed as having the disease.
The term "subject" as used herein refers to living organisms such as humans, dogs, cats and other mammals. In one or more embodiments, the subject is a human.
The invention has the advantages that:
1. The combination of the invention can realize the combination of TCR-T adoptive cell therapy and immunodetection point therapy, thereby solving the problem that the high-affinity TCR-T adoptive immunotherapy can not overcome the inhibition effect mediated by PD-1 signaling pathway;
2. The combination of the invention achieves an excellent treatment effect which cannot be achieved by single drug treatment; and
3. The combination of the present invention significantly limits the growth of tumor cells.
The invention will be further illustrated with reference to specific examples. 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 the specific conditions in the examples below, is generally followed by molecular cloning under conventional conditions such as Sambrook et al: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
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.
Examples
EXAMPLE 1 lentiviral vector construction and viral packaging of high affinity TCR against SLLMWITQC-HLA A A2
First, a first nucleic acid fragment (nucleic acid sequence shown as SEQ ID NO: 8) encoding a wild-type TCR alpha chain, a second nucleic acid sequence (nucleic acid sequence shown as SEQ ID NO: 10) encoding a beta chain of a wild-type NY-ESO-1-TCR, and a connecting sequence 2A of the two nucleic acid fragments are synthesized; cloning into a universal lentiviral packaging vector through restriction endonuclease, transforming into stbl3 escherichia coli, and obtaining a recombinant containing the complete wild-type TCR through sequencing; a lentiviral expression vector for expressing a high affinity TCR is cloned by means of point mutation on the basis of the wild-type recombinant. Phage display technology is one means of generating libraries of TCR high affinity variants to screen for high affinity variants. The TCR phage display and screening method described by Li et al (2005) Nature Biotech 23 (3): 349-354) can be used to obtain high affinity TCRs. In particular, for a detailed procedure for the construction, generation and screening of high affinity TCRs of the present invention, see WO2018/099402A1. The alpha chain variable domain amino acid sequence of the high affinity TCR used in this example is SEQ ID NO. 6 and the beta chain variable domain amino acid sequence is SEQ ID NO. 5. Its affinity KD value with SLLMWITQC-HLA A A2 complex is 9.69E-07M. The affinity KD value of the corresponding wild-type TCR and SLLMWITQC-HLA A2 complex was measured as 4.3E-05M by the same method.
The resulting lentiviral packaging vector expressing the high affinity TCR was transferred to 293T cells via PEI with lentiviral backbone vector PMDLG PRRE, pRSV-Rev and pmd2.G (adedge) for lentiviral packaging. The method comprises the following specific steps: 1. 293T was cultured in DMEM medium containing 10% FBS; 2. the 293T cells are spread in a 15cm culture dish one day before transfection, so that the cell confluency is ensured to be about 70% during transfection; 3. 4 plasmids were diluted with opti-MEM and mixed, and at the same time, equal amounts of PEI were diluted and allowed to stand for 5 minutes, respectively. PEI diluent was added dropwise to plasmid diluent, allowed to stand at room temperature for 20 minutes, and then the mixture was added to a cell culture dish and after 6 to 8 hours, replaced with DMEM containing 10% FBS. 4. Viral supernatants were collected at 48 and 72 hours, respectively. 5. Concentrating virus with ultrafiltration tube of 50kD, subpackaging, and preserving at-80deg.C.
Example 2 preparation of T cells specifically recognizing NY-ESO-1
First, peripheral blood of healthy volunteers was collected, and then the peripheral blood was separated using Ficoll separating liquid (Lymphoprep TM) to obtain Peripheral Blood Mononuclear Cells (PBMCs). After counting, the cells were resuspended in serum-free 1640, plated in a 6-well plate for two hours, the culture medium was collected, and the cell pellet obtained by centrifugation was PBL, which was a mixture of CD4 + T cells and CD8 + T cells as the main component, and CD4 + T cells and CD8 + T cells both expressed CD3 molecules (CD 3 molecules were linked to TCR via salt bridge, involved in T cell signaling), so CD3 was selected for labeling this cell population in this example.
Then, after PBL cells were stimulated with anti-CD3/CD28beads (Gibco TM), the cells were infected with the virus at MOI=5, and cultured in an expanded manner according to the growth state of the cells. In order to ensure a good cell status, a certain cell density should be maintained during the expansion culture, typically 1-3 x 10 6 cells/ml.
Finally, a small fraction of cells were removed and after removal of the magnetic beads, the efficiency of high affinity TCR transduction of PBL cells was examined by flow cytometry. The basic process is as follows: the cells were centrifuged at 300g for 5 minutes, and after removing the culture medium, the cells were washed twice with PBS containing 2% FBS. After 200. Mu.l of wash solution was resuspended, the solution was split into two tubes, one tube was added with 1. Mu.l of APC-labeled anti-CD 3 antibody (bioleged) and PE-labeled anti-TCR V.beta.8 antibody (bioleged), and the other tube was added with 1. Mu.l of PE-labeled anti-CD 3 antibody (bioleged) and tetramer of the APC-labeled SLLMWITQC-HLA-A2 complex. After being left on ice for 30 minutes and washed twice with wash solution, 200. Mu.l of wash solution was resuspended and flow-detected. The results are shown in FIG. 8. The results showed that more than 90% of the cells obtained by expansion were CD3 positive cells, i.e.T cells, and that 85% of the T cells expressed high affinity TCR against NY-ESO-1 tumor antigen, regardless of whether the antibodies were raised against the TCR beta chain V region or the SLLMWITQC-HLA-A2 complex tetramer.
EXAMPLE 3 expression and purification of high affinity PD-1 fusion proteins
Based on the wild type PD-1 fusion protein sequence stored in the experiment, the sequence for encoding the high-affinity PD-1 fusion protein is obtained by utilizing a PCR technology in a point mutation mode; then cloned into the pFUSE-hIgG1e1-Fc2 (InvivoGen) vector using both EcoR I and Nhe I cleavage sites. Bacterial solutions carrying the right pFUSE (InvivoGen) plasmid encoding the high affinity PD-1 fusion protein were inoculated at 1:100 into 200ml LB medium (containing 100ug/ml ampicillin) and incubated overnight at 37℃after which the pellet was collected by centrifugation at 4500g for 30min. Plasmid was extracted according to the standard procedure for large plasmid extraction (see PureLink TM HiPure PLASMID FILTER Maxiprep Kit standard technical procedure, life), OD 260/280 was used to determine plasmid concentration, plasmid concentration was adjusted to 1mg/ml, and the plasmid was stored at-20℃after packaging.
The expi293F TM cell line was recovered and stably cultured at 37℃and 125rpm with 5% CO 2 for 2-3 passages. The day before transfection, the cell density was adjusted to 2 x 10 6 cells/ml and cultured overnight. On the day of transfection (taking 30ml transfection system as an example), the density of overnight cultured cells was first adjusted to 2.5 x 10 6 cells/ml in 25.5ml (cell viability was required to be greater than 95%); next, 30. Mu.g of the plasmid and 81. Mu. l ExpiFectamine TM 293Reagent were diluted into opti-MEM medium (1.5 ml each) and left to stand for 5min, and the transfection Reagent mixture was slowly added dropwise to the plasmid mixture and left to stand for 20min. Finally, the mixture of plasmid and transfection reagent was added dropwise to the cell culture, incubated at 37℃and 125rpm under 5% CO 2 for 16-18h, 150. Mu. L ENHANCER 1 and 1.5ml of enhancer 2 were added, incubation was continued, supernatant was collected by centrifugation at 12000g at 4℃and filtered at 0.45. Mu.m.
Proteins were purified by a linear gradient elution with 500mM imidazole phosphate buffer (pH 7.2) through a nickel column (GE HEALTHCARE) at a sample flow rate of 2ml/min and the collected eluted fractions were analyzed by SDS-PAGE. According to the analysis result, the target PD-1 fusion protein component is collected, concentrated and then further purified by a molecular sieve Superdex 200 (GE HEALTHCARE), and the target component is collected and subjected to SDS-PAGE analysis to identify the protein purity.
The ability of high affinity PD-1 fusion proteins to recognize PD-L1 was determined by an Octet system (forteBio) based on a biofilm interference technique. The high affinity PD-1 fusion protein is first loaded onto the NTA probe (poll). The PD-L1 antigen was then diluted to different concentrations in a 2-fold gradient, with HBS dilution buffer (70mM NaCl,750mM Na 2HPO4·2H2 O,25mM HEPS) as baseline, to bind to the high affinity PD-1 fusion protein. Data was collected using Data Acquisition software, and DATA ANALYSIS software analyzed the Data and calculated kinetic parameters. After each round of assay, the chip was regenerated with 10mM Gly-HCl pH 1.75. The affinity of the high affinity PD-1 fusion protein for binding to the antigen of PD-L1 is shown in Table 1.
TABLE1 affinity assay of high affinity PD-1 fusion proteins for PD-L1 molecules (Octet system)
Example 4 high affinity PD-1 fusion proteins are capable of promoting cytotoxicity of T cells specifically recognizing NY-ESO-1
This example determines the ability of a pharmaceutical composition to clear tumor cells by measuring LDH release by a non-radioactive cytotoxicity assay. The assay is a colorimetric surrogate assay for the 51 Cr-release cytotoxicity assay, quantitatively determining Lactate Dehydrogenase (LDH) released after cell lysis. LDH released in the medium was detected using a 30 minute coupled enzyme reaction in which LDH converts one tetrazolium salt (INT) to red formazan (formanzan). The amount of red product produced is proportional to the number of cells lysed. The 490nm absorbance data can be collected using a standard 96-well plate reader.
Methods for detecting cell function using LDH release assays (Promega) are well known to those skilled in the art. The target cell lines selected for the LDH experiments of this example were MDA-MB-231 (breast cancer cell line, ATCC), A375 (melanoma cell line, ATCC), IM9 (multiple bone marrow cell line, ATCC), NCI-H1299 (HLA-A 2) (lung cancer cell line, shanghai cell bank, transfected into HLA-A2 gene) and NCI-H1299 (lung cancer cell line, shanghai cell bank). Wherein MDA-MB-231, A375, IM9 and NCI-H1299 (HLA-A 2) are positive tumor cell lines and NCI-H1299 is a negative tumor cell line.
LDH plates were first prepared. On experiment day 1, the individual components were added in the order of the target cells followed by the pharmaceutical composition. Culturing and adjusting each target cell line to 2X 10 5 cells/ml, and adding 100 μl of target cells to each well; the culture medium adjusts the concentration of the high affinity PD-1 fusion protein to 40nM, and each well is added with 20 μl and the final concentration is 4nM, and incubated for 30 minutes at room temperature; the medium was conditioned to 6.25X10 5 cells/ml of T cells specifically recognizing NY-ESO-1, and 80. Mu.l were added per well; three complex holes are provided per hole. Control wells required for LDH experiments were also set. After 24h incubation, the supernatant was taken to detect the release of LDH (Promega). The results are shown in FIG. 9, where the ability of the drug composition group to clear tumors was significantly enhanced over the T cell group specifically recognizing NY-ESO-1 alone. When the MDA-MB-231 cells are killed, the killing capacity of the specific T cells is improved by 17.4 percent and 28.2 percent by L5B7Fc and L45-123Fc respectively; when killing A375 cells, the L5B7Fc and the L45-123Fc respectively improve the killing capacity of specific T cells by 29.7 percent and 29.6 percent; when killing IM9 cells, the L5B7Fc and the L45-123Fc respectively improve the killing capacity of specific T cells by 17.5 percent and 87.7 percent; L5B7Fc and L45-123Fc increased killing capacity of specific T cells by 26.9% and 23.9%, respectively, when NCI-H1299 (HLA-A 2) cells were killed. Effector cells have no killing effect on negative tumor cell lines.
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
<120> Combination of T cells and high affinity PD-1 fusion proteins
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Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
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Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
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Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala
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Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
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Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
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Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
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Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
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Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
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Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala
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Pro Tyr Ile Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
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Glu Arg Arg Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
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Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
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Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
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Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
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Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
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Asn Trp Tyr Arg Glu Ser Pro Ser Gly Gln Thr Asp Lys Leu Ala Ala
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Phe Pro Glu Asp Arg Ser Gln Leu Gly Gln Asp Cys Arg Phe Arg Val
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Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
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Arg Arg Asn Asp Ser Gly Thr Tyr Met Cys Val Ala Ile Ser Leu Ala
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Pro Lys Ile Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
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Glu Arg Arg Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
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Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
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<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu Gly
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Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn Leu
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Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile Leu
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Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val Thr
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Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser Arg
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Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp Ala Asn Gly Lys
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Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro
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<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
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Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr
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Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
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Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln
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Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr
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Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn Leu
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Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro
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<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu Gly
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Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn Leu
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<210> 8
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agtcaacagg gagaagagga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 60
atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 120
agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 180
ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 240
gcagcagaca ctgcttctta cttctgtgct acggacgcaa acggcaagat catctttgga 300
aaagggacac gacttcatat tctccccaat atccagaacc ctgaccctgc cgtgtaccag 360
ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 420
acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 480
atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 540
gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 600
gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 660
tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 720
ctgctcatga cgctgcggct gtggtccagc 750
<210> 9
<211> 291
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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1 5 10 15
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35 40 45
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50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln
65 70 75 80
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85 90 95
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Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro
115 120 125
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Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn
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Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys
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Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu
180 185 190
Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys
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Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp
210 215 220
Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg
225 230 235 240
Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser
245 250 255
Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala
260 265 270
Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp
275 280 285
Ser Arg Gly
290
<210> 10
<211> 873
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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gatgctggag ttatccagtc accccggcac gaggtgacag agatgggaca agaagtgact 60
ctgagatgta aaccaatttc aggacacgac taccttttct ggtacagaca gaccatgatg 120
cggggactgg agttgctcat ttactttaac aacaacgttc cgatagatga ttcagggatg 180
cccgaggatc gattctcagc taagatgcct aatgcatcat tctccactct gaagatccag 240
ccctcagaac ccagggactc agctgtgtac ttctgtgcca gcagtttagg gagcaacgag 300
cagtacttcg ggccgggcac caggctcacg gtcacagagg acctgaaaaa cgtgttccca 360
cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420
ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 480
gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 540
ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 600
aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660
tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 720
gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgc caccatcctc 780
tatgagatct tgctagggaa ggccaccttg tatgccgtgc tggtcagtgc cctcgtgctg 840
atggccatgg tcaagagaaa ggattccaga ggc 873
<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
Leu Trp Asn Arg Val Ser Pro Ala 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 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> 117
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 12
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> 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
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> 14
<211> 117
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 14
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> 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 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> 16
<211> 117
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 16
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> 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> 117
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 18
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> 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 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> 20
<211> 117
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 20
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> 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 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> 22
<211> 117
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 22
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> 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 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> 24
<211> 117
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 24
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> 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 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> 26
<211> 117
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 26
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> 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 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> 28
<211> 117
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 28
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> 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 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> 30
<211> 117
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 30
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> 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 Glu Ser Pro Ser Gly Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Leu Gly Gln Asp Cys 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 Ile 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> 229
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 32
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys
225
<210> 33
<211> 345
<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 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 Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
115 120 125
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
130 135 140
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
145 150 155 160
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
165 170 175
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
180 185 190
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
195 200 205
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
210 215 220
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
225 230 235 240
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
245 250 255
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
260 265 270
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
275 280 285
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
290 295 300
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
305 310 315 320
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
325 330 335
Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 34
<211> 345
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 34
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 Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
115 120 125
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
130 135 140
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
145 150 155 160
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
165 170 175
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
180 185 190
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
195 200 205
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
210 215 220
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
225 230 235 240
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
245 250 255
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
260 265 270
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
275 280 285
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
290 295 300
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
305 310 315 320
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
325 330 335
Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345
<210> 35
<211> 345
<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 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 Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro
115 120 125
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
130 135 140
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
145 150 155 160
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
165 170 175
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
180 185 190
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
195 200 205
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
210 215 220
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
225 230 235 240
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
245 250 255
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
260 265 270
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
275 280 285
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
290 295 300
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
305 310 315 320
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
325 330 335
Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 345

Claims (22)

1. A combination comprising a T cell and a high affinity PD-1 fusion protein comprising a high affinity PD-1 molecule and an Fc fragment of an IgG molecule, characterized in that,
The PD-1 molecule has an amino acid sequence selected from the group consisting of SEQ ID NOs 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31;
the T cells contain a nucleic acid encoding a high affinity TCR that specifically recognizes NY-ESO-1 and express a high affinity TCR that specifically recognizes NY-ESO-1;
The alpha chain variable domain sequence of the high affinity TCR specifically recognizing NY-ESO-1 is SEQ ID NO: 6, and the beta chain variable domain sequence of the high affinity TCR specifically recognizing NY-ESO-1 is SEQ ID NO: 5.
2. The combination of claim 1, wherein the PD-1 molecule has an amino acid sequence selected from the group consisting of SEQ ID NOs 15, 16, 28, 30, and 31.
3. The combination of claim 1, wherein the high affinity PD-1 molecule is located N-terminal to the Fc fragment of an IgG molecule in the high affinity PD-1 fusion protein.
4. The combination of claim 1, wherein the Fc fragment of IgG is selected from the group consisting of human IgG1, igG2, igG3 and IgG 4.
5. The combination of claim 4, wherein the Fc fragment of IgG is selected from the group consisting of the Fc fragment of IgG 4.
6. The combination of claim 1, wherein the Fc fragment comprises a hinge region, a CH2 and a CH3 domain.
7. The combination of claim 6, wherein the Fc fragment does not comprise CH1 and CH4 domains.
8. The combination of claim 7, wherein the Fc fragment has the amino acid sequence of SEQ ID No. 32.
9. The combination of claim 1, wherein the amino acid sequence of the high affinity PD-1 fusion protein is selected from the group consisting of SEQ ID NOs 2,3, 33, 34, and 35.
10. The combination of claim 9, wherein; the amino acid sequence of the high affinity PD-1 fusion protein is SEQ ID NO 2 or 3.
11. The combination of claim 1, wherein the fusion protein comprises two high affinity PD1 molecules.
12. The combination of claim 11, wherein two high affinity PD1 molecules in the fusion protein are linked by disulfide bonds to form a dimer structure.
13. The combination of claim 12, wherein the disulfide bond is that of the hinge region of the Fc fragment of an IgG molecule.
14. A combination as claimed in claim 1 wherein the C-or N-terminus of the α and/or β chain of the TCR is conjugated to a conjugate.
15. The combination of claim 14, wherein the conjugate that binds to the TCR is a detectable label, a therapeutic agent, a PK modifying moiety, or a combination of any of these.
16. A combination according to claim 15, wherein the therapeutic agent that binds to the TCR is an anti-CD 3 antibody linked to the C-or N-terminus of the alpha or beta chain of the TCR.
17. The combination of claim 1, wherein the combination is a pharmaceutical composition.
18. The combination of claim 17, wherein the composition further comprises a pharmaceutically acceptable carrier.
19. The combination of any one of claims 1-18, wherein the high affinity PD-1 fusion protein is expressed on T cells that express the high affinity TCR that specifically recognizes NY-ESO-1.
20. The combination of claim 19, wherein the high affinity TCR that specifically recognizes NY-ESO-1 and the high affinity PD-1 fusion protein are expressed by T cells via the same vector or different vectors.
21. Use of a combination according to any one of claims 1 to 20 for the preparation of a medicament for the treatment of tumors.
22. The use of claim 21, wherein the tumor is a tumor that highly expresses NY-ESO-1.
CN201910224044.5A 2019-03-22 2019-03-22 Combination of T cells and high affinity PD-1 fusion proteins Active CN111714618B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
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Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
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Publications (2)

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