CN113480650B - Preparation method and application of a fully human CD276-targeting CAR-T cell - Google Patents
Preparation method and application of a fully human CD276-targeting CAR-T cellDownload PDFInfo
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Abstract
Description
Technical Field
The invention belongs to the field of immunotherapy, and relates to a preparation method and application of a fully human-derived CD 276-targeted CAR-T cell.
Background
A Chimeric Antigen modified T cell (CAR-T) is a T cell which is subjected to genetic modification, and a CAR containing a tumor Antigen specific recognition single-chain antibody (scFv) and a T cell activation motif is introduced into a T cell of a patient by using a gene transduction technology, so that the CAR-transduced T cell can directly recognize a cancer cell surface Antigen to be activated, and further the cancer cell is killed. It is because CAR-T cells kill cancer cells without the need for antigen presentation, and greatly increase the efficiency of cancer cell killing. The professor Carl June, university of pennsylvania in 2012, cured the leukemia patient with Emily Whitehead using chimeric antigen receptor modified T cells targeting CD19, and the FDA in us in 2017 breakthroughs approval of two CAR-T cell drugs for B cell leukemia and lymphoma treatment, which became a milestone in the field of cell therapy.
CAR-T cells have a number of advantages over traditional autoimmune cell therapy: targeting (I): CAR-T cells can be specifically targeted to tumor cells by tumor antigen-specific single-chain antibody or ligand recognition; (II) high killing efficiency: nature reported in 2018 that in each case, 1 TET 2-deleted CAR-T cell killed all tumor cells to cure leukemia; (III) wide antigen recognition range: the CAR-T cell can find all tumor antigens recognized by the antibody, including protein antigens, carbohydrate antigens, lipid antigens and the like; (IV) no MHC restriction: CAR-T cells recognize tumor cells directly through armed scFv, overcoming the immune escape that occurs when tumors down-regulate MHC-I molecules.
CAR-T cell therapy has achieved tremendous success against hematological tumors, and currently, the FDA in the united states has approved CAR-T cell products from noval and gilded corporation in tandem for the treatment of acute B-lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL) priced at 47.5 ten thousand dollars (about 300 ten thousand minbi) and 37.3 thousand dollars (about 250 ten thousand minbi), respectively. Up to now, 407 clinical trials of CAR-T cell therapy tumor were registered on the clinical trials website, with 276 for hematologic tumors and 131 for solid tumors. In China, 26 CAR-T cell treatment tumor medicine clinical applications have been accepted by the national drug administration, only 1 target solid tumor (GPC3) exists, at present, 8 companies such as Legionella, Hengrunhengsheng, Keji biology and the like obtain medicine clinical test batches, and the CAR-T cell treatment standardization era in China is marked to come.
At present, CAR-T cells targeting CD19, CD22 and BCMA have been advanced greatly in treating tumors in the blood system, the curative effect is obvious, but the CAR-T cell treatment for solid tumors is slow in progress, and the lack of safe and effective specific tumor antigen targets is one of important blocking factors.
CD276, also known as B7H3, belongs to the B7 immune checkpoint superfamily, and is a type I transmembrane protein composed of an extracellular region and a shorter intracellular region containing two pairs of identical immunoglobulin variable and constant regions. The mRNA expression is wide, but the protein expression is very limited. Many studies reveal that CD276 is highly expressed in various tumors, including glioma, liver cancer, pancreatic cancer, lung cancer, melanoma, kidney cancer, breast cancer, prostate cancer, colorectal cancer, cervical cancer, ovarian cancer, etc., and the expression level is closely related to poor prognosis of patients. Therefore, the CAR-T cell prepared by the inventor by using the target as a target targets a wide range of solid tumors, and has a wide application range. While various I, II phase clinical tests aiming at tumors such as peritoneal cancer, glioma and central nervous tumor have been carried out on medicaments such as CD 276-targeted antibody medicaments, antibody-drug conjugates, radioactive material labeled antibodies, CD3-CD276 bispecific antibodies and the like, show encouraging preliminary results, have no adverse events and indicate that CD 276-targeted therapy is safe and reliable.
With the continuous and intensive research, the side effects of CAR-T therapy gradually appear, such as serious cytokine storm (CRS), disease recurrence and other problems. The majority of patients gradually recover from the development of CRS following symptomatic treatment, and it has also been reported that CRS can be reduced by lowering the CAR-T infusion dose or altering the CAR transmembrane domain. Disease recurrence remains a major problem for CAR-T therapy, with a 1-year recurrence rate of 41-45% in CTL019 clinical trials reported by novain; the applicant's completed CD 19-targeted murine CAR-T cell therapy also achieved 40% relapse in leukemia, and re-infusion of the same murine CAR-T cells was completely unable to expand, with 2 of the 3 relapsed patients being effectively expanded after replacement with humanized CD19 CAR-T cells. The reason is related to that the murine scFv has stronger immunogenicity, induces the body to generate human anti-mouse antibody reaction, and leads the murine CAR-T cells to be transiently alive in the human body. Although humanization of murine scFv reduced immunogenicity to some extent, fully human scFv would minimize CAR immunogenicity, reduce immune rejection, and improve CAR-T cell survival in vivo.
Disclosure of Invention
The invention aims to solve the defects of the background technology and provide a preparation method and application of a fully human CD276 targeting CAR-T cell, and the prepared fully human CD276 CAR-T cell has a killing effect on various solid tumor cells, has strong killing capability and a wide application range, and is safe and effective to human bodies.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an isolated antibody that specifically binds to CD276, comprising a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises: VH-CDR1, VH-CDR2, VH-CDR 3; the light chain variable region comprises: VL-CDR1, VL-CDR2, VL-CDR 3;
the amino acid sequence of the VH-CDR1 is shown as SEQ ID NO: 1;
the amino acid sequence of the VH-CDR2 is shown as SEQ ID NO. 2;
the amino acid sequence of VH-CDR3 is shown in SEQ ID NO. 3;
the amino acid sequence of the VL-CDR1 is shown in SEQ ID NO. 4;
the amino acid sequence of the VL-CDR2 is shown in SEQ ID NO. 5;
the amino acid sequence of VL-CDR3 is shown in SEQ ID NO 6.
Further, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 7, or an amino acid sequence having at least 85% identity thereto.
Further, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 8, or an amino acid sequence having at least 85% identity thereto.
Further, the antibody may be a full-length antibody or an antigen-binding domain of a full-length antibody, and in a specific embodiment of the present invention, the antigen-binding domain is a single chain antibody scFv, wherein the heavy chain variable region and the light chain variable region are linked by a glycine-and serine-rich linker peptide chain, and the order of the heavy chain variable region and the light chain variable region is interchangeable.
In a specific embodiment of the invention, the scFv sequence comprises the amino acid sequence shown as SEQ ID NO 13.
Further, the antibody is a fully human antibody.
The invention also provides nucleic acid molecules encoding the aforementioned antibodies.
Further, the nucleic acid molecule sequence comprises the sequences shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO. 14.
The invention also provides a recombinant vector comprising a nucleic acid molecule as described above.
Further, the recombinant vector includes a cloning vector or an expression vector.
Further, the vector includes DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector.
The invention also provides a recombinant host cell comprising a nucleic acid molecule as described above or a recombinant vector as described above.
The present invention also provides a method for preparing the recombinant vector as described above, comprising the steps of: cloning the nucleic acid molecule encoding the antibody into a vector to obtain a recombinant vector.
The present invention also provides a method for producing the recombinant host cell as described above, comprising the steps of: introducing a nucleic acid molecule encoding the antibody or the recombinant vector into a host cell to obtain the recombinant host cell.
The invention also provides a composition or kit comprising an antibody as hereinbefore described, a nucleic acid molecule as hereinbefore described, a recombinant vector as hereinbefore described, a recombinant host cell as hereinbefore described.
Further, the composition or kit may further comprise one or more pharmaceutically acceptable excipients, diluents or pharmaceutically acceptable carriers.
Further, the types of the composition include immunoconjugates, antibody drugs, cell drugs, nucleic acid drugs, chimeric antigen receptors.
The immunoconjugates are formed by conjugating an antibody of the invention to an effector molecule. The effector molecule may be any therapeutic molecule or marker molecule that facilitates detection. The effector molecule is not limited and may be any suitable effector molecule. For example, the effector molecule can be any one or more of a drug, a toxin, a label (e.g., any detectable label described herein), a small molecule, or another antibody.
For example, the toxin may be pseudomonas exotoxin a or a variant thereof.
Examples of drugs that may be suitable for use in the immunoconjugates of the invention include, but are not limited to, Pyrrolobenzodiazepine (PBD) dimers, tubulin binding agents such asdolastatin 10,monomethyl dolastatin 10, auristatin E, monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin F, HTI-286, tubulysin M, maytansinoid AP-3, cryptophycin, Boc-Val-Dil-Dap-OH, tubulysin IM-1, Boc-Val-Dil-Dap-Phe-OMe, tubulysin IM-2, Boc-Nme-Val-Val-Dil-Dap-OH, tubulysin IM-3 and colchicine DA; DNA alkylating agents (duocarmycin analogs), e.g., duocarmycin SA, duocarmycin CN, duocarmycin DMG, duocarmycin DMA, duocarmycin MA, duocarmycin TM, duocarmycin MB, duocarmycin GA; tomaymycin DM; SJG-136; (ii) illedin S; ilonffene, apaquinone, triptolide, staurosporine, camptothecin, methotrexate and other anti-cancer drugs, such as kinase inhibitors, Histone Deacetylase (HDAC) inhibitors, proteasome inhibitors and Matrix Metalloproteinase (MMP) inhibitors.
Marker molecules that can be suitable for use in the immunoconjugates of the invention include, for example, radioisotopes, fluorophores (e.g., Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).
Further, the chimeric antigen receptor includes the scFv described above.
Further, the chimeric antigen receptor also includes a transmembrane region, an intracellular signaling domain.
Further, the chimeric antigen receptor further comprises a hinge region.
Further, the chimeric antigen receptor also includes a co-stimulatory domain.
Further, the co-stimulatory domain comprises the signaling domains of the following co-stimulatory molecules: CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD-2, CD7, LIGHT, NKG2C, B7-H3.
Preferably, the co-stimulatory domain is the signaling domain of 4-1BB, which comprises the amino acid sequence set forth in SEQ ID NO: 29.
Further, the hinge and transmembrane regions include those of the following molecules: IgG1, IgG4, CD8 alpha, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, CD 34.
Preferably, the hinge and transmembrane regions are those of CD8 a.
Preferably, the hinge region of CD8 α comprises the amino acid sequence set forth in SEQ ID NO. 25.
Preferably, the transmembrane region of CD8 α comprises the amino acid sequence set forth in SEQ ID NO 27.
Further, the intracellular signaling domain comprises the intracellular signaling domains of the following molecules: fc γ R, Fc ∈ R, Fc α R, FcRn, CD3, CD3 ζ, CD3 γ, CD3 δ, CD3 ∈, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278, CD247 ζ, CD247 η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF- κ B, PLC- γ, iC3b, C3dg, C3d, Zap 70.
Preferably, the intracellular signaling domain is a CD3 ζ intracellular signaling domain comprising an amino acid sequence as set forth in SEQ ID NO. 31.
Further, the chimeric antigen receptor further comprises a signal peptide.
The signal peptide used in the CAR of the invention is preferably a signal peptide which is indicative of localization to the cell membrane, preferably a signal peptide of a membrane protein. Examples of the signal peptide include signal peptides such as an α chain and a β chain of a T cell receptor, CD3 ζ, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, an immunoglobulin heavy chain, and an immunoglobulin light chain. Specific examples of the amino acid sequence of the signal peptide include the amino acid sequence shown in SEQ ID NO. 23.
Further, the signal peptide comprises an amino acid sequence shown as SEQ ID NO. 23.
Further, the amino acid sequence of the chimeric antigen receptor comprises the amino acid sequence shown as SEQ ID No. 15 or SEQ ID No. 16.
The invention also provides a nucleotide molecule encoding the chimeric antigen receptor described above or a component thereof;
further, the components of the chimeric antigen receptor include a scFv, a transmembrane region, an intracellular signaling domain, a hinge region, a costimulatory domain, a signal peptide.
Further, the nucleotide molecule further comprises a coding sequence for a self-cleaving peptide; preferably, the coding sequence for the self-cleaving peptide is the coding sequence for T2A; preferably, the coding sequence for T2A is the coding sequence encoding the amino acid sequence shown in SEQ ID NO. 19; preferably, the coding sequence of T2A is shown in SEQ ID NO: 20.
Further, the nucleotide molecule further comprises a coding sequence for a tag molecule; the tag molecule is a molecule for characterizing whether the chimeric antigen receptor is expressed on the surface of a cell; preferably, the tag molecule comprises tEGFR; preferably, the amino acid sequence of tEGFR is shown as SEQ ID NO 21; preferably, the coding sequence of tEGFR is shown in SEQ ID NO: 22.
Further, the nucleotide molecule comprises a sequence shown by SEQ ID NO 26, SEQ ID NO 28,SEQ ID NO 30, SEQ ID NO 32,SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 33 and SEQ ID NO 24.
The invention also provides a vector comprising a nucleotide molecule as described above.
The invention also provides a recombinant virus comprising a nucleotide molecule as described above or a vector as described above.
The present invention also provides a recombinant cell expressing the chimeric antigen receptor described above.
Preferably, the recombinant cell comprises a nucleotide molecule as described above or a vector as described above.
Preferably, the recombinant cell comprises a modified immune cell.
Further, the immune cells include PBMCs, T cells, macrophages, dendritic cells, monocytes, NK cells, NKT cells.
Preferably, the immune cell is a T cell selected from the group consisting of: CD4+/CD8+ double positive T cells, CD4+ helper T cells, CD8+ T cells, tumor infiltrating cells, memory T cells, naive T cells, γ δ T cells, and α β T cells.
The present invention also provides a method for preparing the recombinant cell as described above, comprising the steps of: the nucleotide molecule or the vector is introduced into a cell to obtain a recombinant cell.
The invention also provides a derivative, which comprises the chimeric antigen receptor, the nucleotide molecule, the vector, the recombinant virus and the recombinant cell.
Further, the type of the derivative includes a composition or a kit.
The invention also provides an application, which comprises any one of the following items:
1) use of an antibody as hereinbefore described, a nucleic acid molecule as hereinbefore described, a recombinant vector as hereinbefore described, a recombinant host cell as hereinbefore described, a composition as hereinbefore described in the manufacture of a medicament for the treatment of a disease or condition associated with the expression of CD 276;
2) use of a nucleic acid molecule as hereinbefore described in the preparation of a recombinant vector as hereinbefore described, a recombinant host cell as hereinbefore described, a composition as hereinbefore described or a kit of parts as hereinbefore described;
3) use of a recombinant vector as hereinbefore described for the preparation of a recombinant host cell as hereinbefore described, a composition or a kit as hereinbefore described;
4) use of a recombinant host cell as hereinbefore described in the preparation of a composition or kit as hereinbefore described;
5) use of a chimeric antigen receptor as described above, a nucleotide molecule as described above, a vector as described above, a recombinant virus as described above, a recombinant cell as described above, a derivative as described above for the manufacture of a medicament for the treatment of a disease or condition associated with the expression of CD 19;
6) use of a chimeric antigen receptor as described above for the preparation of a nucleotide molecule as described above, a vector as described above, a recombinant virus as described above, a recombinant cell as described above, a derivative as described above;
7) use of a nucleic acid molecule as described above for the preparation of a vector as described above, a recombinant virus as described above, a recombinant cell as described above, a derivative as described above;
8) use of the vector as described above for the preparation of a recombinant virus as described above, a recombinant cell as described above, or a derivative as described above;
9) use of the recombinant virus as described above for the preparation of the recombinant cell as described above, the derivative as described above;
10) use of a recombinant cell as hereinbefore described for the preparation of a derivative as hereinbefore described;
11) the application of the antibody in the preparation of a kit for detecting CD276 protein or an antigen fragment thereof;
12) use of an antibody according toclaim 1 for the preparation of a kit for the diagnosis of a disease or disorder associated with the expression of CD 276.
The invention also provides a method of treating a disease or disorder associated with the expression of CD276, comprising administering to a subject the antibody described above, the nucleic acid molecule described above, the recombinant vector described above, the recombinant host cell described above, the composition described above, the nucleotide molecule described above, the vector described above, the recombinant cell described above, the derivative described above.
The present invention also provides a method for detecting a CD276 protein or an antigenic fragment thereof, said method comprising administering an antibody as described above, a composition as described above or a kit as described above.
Further, the method comprises the steps of:
1) obtaining a sample containing a CD276 protein or antigenic fragment thereof;
2) contacting the sample collected in step 1) with the antibody or composition as described above;
3) detecting the presence of the antibody-antigen complex.
The present invention also provides a method of diagnosing whether a subject has a disease or disorder associated with expression of CD276, comprising detecting the amount of CD276 in a sample from the subject using the aforementioned antibody or antigen-binding fragment thereof.
Preferably, the method further comprises: comparing the amount of the CD276 in the sample from the subject to its amount in a known standard or reference sample, and determining whether the level of CD276 in the sample from the subject falls within the level of CD276 associated with the tumor.
Further, the disease or disorder associated with expression of CD276 may include any solid and non-solid tumor. Solid and non-solid tumors are characterized by the expression or overexpression of CD 276.
Further, examples of diseases or disorders associated with the expression of CD276 include, but are not limited to, acute lymphocytic cancer, acute myelogenous leukemia, rhabdomyosarcoma, bladder cancer, bone cancer, any of brain cancer (e.g., medulloblastoma, neuroblastoma, and glioblastoma), breast cancer, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, gallbladder cancer, pleural cancer, nasal cancer, oral cancer, vulval cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, ewing's sarcoma, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal tract carcinoid, head and neck squamous cell carcinoma, hodgkin's lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-hodgkin's lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, Acute Lymphocytic Leukemia (ALL) and burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omentum and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer.
Preferably, the disease or disorder associated with expression of CD276 is a solid tumor, including but not limited to glioma, liver cancer, pancreatic cancer, lung cancer, melanoma, renal cancer, breast cancer, prostate cancer, colorectal cancer, cervical cancer, ovarian cancer, and the like.
In a particular embodiment of the invention, the disease or disorder associated with the expression of CD276 is ovarian cancer, lung cancer.
As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. The antibody may be an intact immunoglobulin derived from a natural source or from a recombinant source, and may be an immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of the invention may exist in a variety of forms, including polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies; antibody formats of the invention also include full-length antibodies, antibody fragments, such as Fab, Fab ', F (ab') 2, Fv, scFv, di-scFv, tri-scFv, Fd, and other antibody fragments that retain antigen-binding function; it may also be a dimer structure Diabody or a trimer structure Triabody. Typically, the fragment should include an antigen-binding fragment, which typically includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it need not necessarily include both. For example, the so-called Fd antibody fragments consist only of the VH and CH1 domains, but still retain some of the antigen binding function of the intact antibody. The term "antibody" as an immunoglobulin or fragment thereof or derivative thereof includes any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The VH or VL region may be further subdivided into: hypervariable regions, termed Complementarity Determining Regions (CDRs), and interspersed with more conserved regions, termed framework regions (FWRs). The variable regions of the heavy and light chains comprise binding domains that interact with an antigen. The CDRs in the heavy chain are abbreviated VH-CDRs, e.g., VH-CDR1, VH-CDR2, VH-CDR3, and the CDRs in the light chain are abbreviated VL-CDRs, e.g., VL-CDR1, VL-CDR2, VL-CDR 3. The CDRs of the antibodies and antigen binding fragments disclosed herein are defined or identified by Kabat numbering.
The terms "single chain variable fragment," "single chain antibody," or "scFv" as used herein refer to an antibody formed by recombinant DNA techniques in which an immunoglobulin heavy chain variable region and a light chain variable region are joined by an amino acid peptide fragment (linker). Various methods of generating single chain antibodies are known, including those described in U.S. patent nos. 4,694,778; bird (1988) Science242: 423-; huston et al (1988) Proc.Natl.Acad.Sci.USA 85: 5879-; ward et al (1989) Nature 334: 54454; skerra et al (1988) Science242: 1038-. The variable regions of the heavy and light chains comprise binding domains that interact with an antigen. The CDRs in the heavy chain are abbreviated VH-CDRs, e.g., VH-CDR1, VH-CDR2, VH-CDR3, and the CDRs in the light chain are abbreviated VL-CDRs, e.g., VL-CDR1, VL-CDR2, VL-CDR 3. The CDRs of the antibodies and antigen binding fragments disclosed herein are defined or identified by Kabat numbering.
The term "vector" as used herein refers to a molecular tool for the transport, transduction, and expression of an included exogenous gene of interest (e.g., a polynucleotide of the present invention) in a target cell, which provides a suitable nucleotide sequence, i.e., a promoter, for initiating transcription in the target cell. Vectors which include the isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, e.g., polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, sendai viral vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.
As used herein, "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector includes sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art that incorporate recombinant polynucleotides, such as plasmids (e.g., naked or contained in liposomes) and viruses (e.g., sendai virus, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
"cloning vector" as used herein refers to a DNA molecule such as a plasmid, cosmid, or phage that is capable of autonomous replication in a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences are inserted in a defined manner without loss of the essential biological function of the vector, and a marker gene suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
The host cell may be any prokaryotic or eukaryotic cell that contains a cloning or expression vector, including those that have been genetically engineered to contain a cloned gene in the chromosome or genome of the host cell. Suitable mammalian host cells include myeloma cells, such as SP2/0 cells and NS0 cells, as well as Chinese Hamster Ovary (CHO) cells, hybridoma cell lines, and other mammalian host cells useful for expressing antibodies. Specific transgenic animals with modified immune systems can also be used to produce antibodies.
"identity" as used herein refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions in each sequence that are aligned for comparison purposes. When a position in the compared sequences is occupied by the same base, then the molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and/or programs can be used to calculate identity between two sequences, including that available as part of the GCG sequence analysis package (University of wisconsin, Madison, Wis.), and FASTA or BLAST that can be used, for example, in default settings. For example, one skilled in the art can expect polypeptides that are at least 70%, 85%, 90%, 95%, 98%, or 99% identical and preferably exhibit substantially the same function as the particular polypeptides described herein, as well as polynucleotides encoding the same.
As used herein, "isolated" refers to being altered or removed from a natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide, partially or completely separated from its coexisting materials of its natural state, is "isolated. An isolated nucleic acid or protein may be present in a substantially purified form, or may be present in a non-natural environment, such as a host cell.
As used herein, unless otherwise specified, a nucleotide sequence that "encodes" a nucleic acid molecule of a protein or an amino acid sequence of a protein includes all nucleotide sequences that are degenerate versions of each other (degenergency) and that encode the same amino acid sequence. The nucleotide sequence may also include one or more introns.
As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, rats, mice, amphibians, reptiles, and the like. The terms "patient" or "subject" are used interchangeably unless otherwise indicated. In the present invention, a preferred subject is a human.
As used herein, the term "treating" refers to administering to a subject an effective amount of a cell having a polynucleotide sequence of a target gene altered ex vivo according to the methods described herein, such that the subject has a reduction in at least one symptom of the disease or an improvement, e.g., a beneficial or desired clinical outcome, of the disease. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treatment may refer to an extended survival compared to the expected survival in the absence of treatment. Thus, those skilled in the art recognize that treatment may improve the disease condition, but may not be a complete cure for the disease. As used herein, the term "treatment" includes prophylaxis. Alternatively, treatment is "effective" in cases where progression of the disease is reduced or halted. "treatment" may also mean prolonging survival as compared to expected survival in the absence of treatment. Patients in need of treatment include those who have been diagnosed with a condition associated with expression of a polynucleotide sequence, and who may develop such a condition due to genetic susceptibility or other factors.
The invention has the beneficial effects that: the invention provides a preparation method of a fully human CD276 CAR-T cell, and the prepared CD 276-targeted CAR-T cell has killing effect on various solid tumor cells, has high killing activity, is safe and effective, and can be used for immune cell therapy of kidney cancer, lung cancer, liver cancer, glioma, ovarian cancer, breast cancer and the like.
Drawings
FIG. 1 is a graph showing the results of a color reaction using ELISA to detect CD276-02 recognition and binding to a CD276 target antigen;
FIG. 2 is a graph showing the results of a color reaction for detecting the ability of the purified scFv protein to recognize a target antigen by ELISA;
FIG. 3 shows graphs showing scFv protein purification results;
FIG. 4 shows the results of flow cytometry analysis of the ability of scFv to recognize and bind to a target antigen;
FIG. 5 shows a statistical plot of mean fluorescence intensity of cell surface scFv binding to target antigen;
FIG. 6 shows a graph of the results of detecting the expression of CD276-CAR in CAR-T using flow cytometry;
FIG. 7 shows CAR-T cell growth profiles;
figure 8 shows a graph of the results of the killing ability of CAR-T of the invention against SKOV3 cells, wherein a: 2: 1; b: 1: 1; c: 1: 2;
figure 9 shows a graph of the results of the killing ability of CAR-T of the invention against a549 cells, where a: 2: 1; b: 1: 1; c: 1: 2;
FIG. 10 is a graph showing the results of the ability of the CAR-T of the present invention to clear a mouse ovarian cancer graft tumor, wherein A: an experimental flow chart; b: mouse fluorescence imaging; c: and (6) carrying out statistical graph.
Detailed Description
The invention will be described in detail below with reference to the accompanying drawings and examples. It should be noted that the drawings and their embodiments of the present invention are for illustrative purposes only and are not to be construed as limiting the invention. The embodiments and features of the embodiments in the present application may be combined with each other without contradiction.
Example 1 screening for scFv targeting CD276
1. Experimental conditions set
Experimental group CD276 antigen + CD276-Phage
2. Experimental methods
The specific binding antibody sequence is enriched through four rounds of screening, and the conditions of phage adding amount, antigen adding amount, reaction time and the like are changed in each round.
The final result was obtained by counting the number of phages having infectious ability contained in the 0.1M HCl (PH 2.0) eluate per 100 μ l of the control group of the experimental group, and the enrichment was judged.
3. Analysis of screening results
Enrichment appears in the third round of screening, and the ratio of the number of the phases (antigen-antibody specific binding) eluted by the experimental group to the number of the phases (non-specific binding between the antigen and the antibody, or no affinity) eluted by the control group is close to 10 times; after the fourth round of changing the experimental conditions, the experimental group and the control group still maintain 10-fold difference, which indicates that the screened phase should have scFv with the affinity with the target protein of CD 276.
4. scFv sequence analysis
Selecting 24 monoclonals for sequencing, wherein 13 scFv sequences are completely expressed, and different sequences are enriched
Clone 02: VH IGHV3-23 × 01/IGHV3-23D × 01, IGHJ4 × 02/IGHJ4 × 0303;
VK:IGKV1-39*01/IGKV1D-39*01、IKJ1*01；
clone 03: VH IGHV 3-33X 06, IGHJ 6X 03; VL, IGKV2-14 × 01, IGLJ2 × 01/IGLJ3 × 01;
the scFv sequence of clone 02 is shown in SEQ ID NO 13.
The scFv sequence of clone 03 was sequenced as shown in SEQ ID NO: 34.
Example 1 detection of the ability of CD 276-Targeted scFv to bind to target antigens
First,experiment 1
1. Detection condition setting
Experimental groups: antigen + phase
Positive control group: BCMA antigen and phase scFv-BCMA
Negative control group 1: other biotin-free antigens (PRPS1) + phase
Negative control group 2: antigen-free + phase
2. Experimental protocol
Preparation of monoclonal phase: CD276-02 and CD276-03, and whether the affinity of the CD276-02 and the CD276-03 with the target antigen is preliminarily judged through ELISA color reaction and OD value.
3. Experimental procedure
Adding equal amount of antigen coating into each group of experimental wells and control wells, adding equal amount of phase, incubating, washing for multiple times to remove unbound phase, adding phase detection antibody and secondary antibody, developing TMB, and measuring OD with enzyme-labeling instrument450nmAnd (6) reading.
4. Analysis of results
As shown in FIG. 1, Table 1 and Table 2, the results of color development and OD development by ELISA450nmThe reading shows that clone CD276-02 can recognize and bind to the CD276 target antigen, and CD276-03 cannot recognize and bind to the CD276 target antigen.
TABLE 1 CD276-03 statistics
TABLE 2 CD276-02 statistics
Second,experiment 2
1. scFv antibody expression purification
The pET-22b is used for constructing a CD276-scFv antibody expression vector, two purified scFv proteins are obtained through induced expression and purification, and a purification result graph is shown in figure 3.
CD276-02 scFv antibody: 0.456. mu.g/. mu.l.
2. Experimental protocol
Adding equal amount of antigen coating into each group of experimental wells and control wells, adding purified scFv antibody, incubating, washing for multiple times, adding His antibody and secondary antibody, developing TMB, and measuring OD with enzyme labeling instrument450nmAnd (6) reading.
3. Analysis of ELISA results
As shown in FIG. 2 and Table 2, CD276-02 still has good affinity when the purified scFv antibody is diluted 1000-fold.
TABLE 2 statistical results of affinity
| CD276 antigen | scFv-02 | 10x(10μl) | 100x | 1000x |
| + | + | 3.968 | 2.357 | 0.306 |
| + | - | 0.128 | 0.129 | 0.158 |
| - | + | 0.264 | 0.19 | 0.192 |
| - | - | 0.09 | 0.088 | 0.104 |
Third, experiment 3
1. Experimental protocol
CD276-02 scFv was constructed into eukaryotic expression vectors containing GPI anchor sequences, transfected into 293T cells, and the binding of scFv expressed on the cell membrane surface to the target antigen was detected by flow-testing CD276-Fc (R & D systems,1027-B3-100) and PE-Anti-Human IgG Fc (Thermo, 12-4998-82).
2. Analysis of results
As a result of flow detection, it was found that CD276-02 scFv could recognize and bind to CD276 target antigen, and that CD276-02 scFv has strong binding ability, and its binding ability to antigen was not as good as that of the positive control 8H9 clone scFv (FIG. 4), and that CD276-02 scFv was strong as the average fluorescence intensity of cell surface scFv binding to target antigen CD276, indicating that the scFv bound to the target antigen in a large amount, i.e., a large number of fluorophores (FIG. 5).
Example 3 preparation of fully human CD276 CAR-T cells
One, step
(1) Preparation of PBMC cells
Taking peripheral blood of a healthy person, centrifuging, reserving autologous plasma for later use, diluting the residual blood cells with equal volume of normal saline, adding the diluted residual blood cells into the upper layer of the lymphocyte separation liquid, centrifuging, sucking the cells of the middle leucocyte layer, adding the normal saline for washing, centrifuging, and discarding the supernatant to obtain the leucocyte plasma.
(2) Construction of shuttle plasmid MSCV-M13B702 containing CAR Structure
a. Synthesizing a CAR-encoding nucleotide sequence targeting human CD276, wherein the CAR-encoding nucleotide sequence is shown as SEQ ID NO.17, wherein the nucleotide sequence of the heavy chain VH of the scFv targeting human CD276 is shown as SEQ ID NO.9, the nucleotide sequence of the light chain VL is shown as SEQ ID NO.10, the nucleotide sequence of the G4S short peptide is shown as SEQ ID NO.12, and the nucleotide sequence of the scFv is shown as SEQ ID NO. 14.
b. Carrying out double enzyme digestion on a retrovirus vector MSCV and the CAR coding nucleotide sequence of the target human CD276 synthesized in the step 1) through Nco I and Mlu I, recovering fragments, connecting the recovered target fragments with T4 ligase, and then transforming Stbl3 competent cells;
c. and selecting a single clone to extract plasmids, carrying out enzyme digestion identification, and then sending sequencing confirmation to confirm that the correct plasmid is MSCV-M13B 702.
(3) Packaging viruses
Mixing shuttle plasmid MSCV-M13B 7026 μ g containing CAR structure and helper plasmid pCL-Ampho 4 μ g in 300 μ l of opti-MEM culture medium, adding 30 μ l PEI reagent dropwise in another 300 μ l of opti-MEM culture medium, shaking and mixing uniformly, standing at room temperature for 5 min, adding the mixture containing PEI reagent dropwise into the plasmid mixture, shaking and mixing uniformly, standing at room temperature for 15 min, adding PEI and plasmid mixture dropwise into a pre-paved 293T cell culture dish, shaking and mixing uniformly, collecting supernatant after 48-72 h, filtering through a 0.45 μ M needle filter, and storing in an ultra-low temperature refrigerator for later use.
(4) CAR-T cell preparation
a. Isolation of PBMC cells
Collecting peripheral blood of a healthy volunteer, centrifuging at room temperature of 1300g for 10 minutes, removing a plasma part, and diluting and uniformly mixing residual blood cells with physiological saline with the same volume; slowly adding the blood cell suspension into the upper layer of the lymphocyte separation liquid, and centrifuging for 25 minutes at the room temperature of 600 g; sucking the intermediate leucocyte layer lymphocytes, adding physiological saline for washing, performing lysis erythrocyte treatment if necessary, centrifuging at room temperature of 400g for 10 minutes, and removing supernatant to obtain PBMC cells.
b. PBMC cell culture activation
Firstly, coating a 24-well plate with 1 mu g/ml anti-human CD3(OKT3) and anti-human CD28(CD28.2), and incubating overnight at 4 ℃; PBMC cells were then resuspended to1X 10 with X-Vivo medium containing 5% fetal bovine serum, 200U/ml IL-2, 10ng/ml IL-7 and 5ng/ml IL-156Per ml, 1ml of cell suspension was inoculated per well and activated in culture.
c. Infection of activated PBMC cells with CD276-CAR Virus
Adding CD276-CAR virus solution into 10 μ M HEPES and 6-8 μ g/ml polybrene, mixing, resuspending activated PBMC cells with the virus solution, adding into RetroNectin coated 24-well plate, centrifuging at 1500g and 30 deg.C for 2 hr, removing supernatant, adding X-Vivo culture medium containing 5% fetal calf serum, 200U/ml IL-2, 10ng/ml IL-7 and 5ng/ml IL-15, and culturing.
(5) Detection of infection efficiency in CAR-T cells
Infection efficiency was analyzed by flow cytometry to detect CD276-CAR expression in CAR-T.
(6) CAR-T cell proliferation potency assay
The number of CAR-T cells cultured for different days was determined to plot the growth curve.
Second, result in
Results as shown in figure 6, CAR-T of the invention can efficiently express CD 276-targeted CARs with high infection efficiency.
Results figure 7 shows that the CAR-T cells of the invention proliferate rapidly.
Example 3 in vitro functional validation of fully human CD276 CAR-T cells
One, step
To the E-Plate assay Plate 50ul of cytokine-free T cell complete medium (without cytokine) was added and the background impedance value was determined. Adding 1 x 10 to E-Plate assay Plate4Tumor cells (tumor cells/100 ul), and observing the ratio of target to target (E/T) 2: 1. 1:1, 1: 2CAR-T cells were added and 200ul of the system was trimmed with medium and placed on a test bench (test bench)Placed in an incubator in advance), real-time dynamic cell proliferation detection is performed.
Second, result in
Results as shown in figures 8 and 9, CAR-T cells of the invention can kill tumor cells efficiently in vitro.
Example 4 in vivo functional validation of fully human CD276 CAR-T cells
One, step
5 x 10 that will carry a fluorescent signal5SKOV3-luc-GFP was intraperitoneally injected into NCG mice, the mice were monitored for abdominal tumor formation by photography weekly using live imaging of the mice, fully human fhCD276-02CAR-T cells were intraperitoneally injected after the abdominal tumors had formed, and humanized CD276 CAR-T cells were used in the control group. Mice were then observed weekly for peritoneal tumor regression by in vivo imaging.
Second, result in
Results as shown in figure 10, CAR-T cells of the invention can clear mouse peritoneal ovarian cancer transplantable tumors.
It should be noted that the above-mentioned embodiments are merely preferred examples of the present invention, and the present invention is not limited thereto. It will be understood by those skilled in the art that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Sequence listing
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ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc caggggtgtt 300
ggccggggct ttgactactg gggccagggg accacggtca ccgtctcctc a 351
<210> 10
<211> 324
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc atctatttaa attggtatcg gcagcaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttactt ctgtcaacag acttacagta cccctccgtg gacgttcggc 300
caagggacca aagtggatat caaa 324
<210> 11
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 12
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
agtggcggtg gctctggcgg tggtgggtcg ggtggcggcg gatca 45
<210> 13
<211> 240
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Glu Val Gln Leu Phe Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Val Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110
Val Thr Val Ser Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser
130 135 140
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
145 150 155 160
Ile Ser Ile Tyr Leu Asn Trp Tyr Arg Gln Gln Pro Gly Lys Ala Pro
165 170 175
Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
180 185 190
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
195 200 205
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Thr Tyr
210 215 220
Ser Thr Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys
225 230 235 240
<210> 14
<211> 720
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gaggtgcagc tgttccagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc caggggtgtt 300
ggccggggct ttgactactg gggccagggg accacggtca ccgtctcctc aagtggcggt 360
ggctctggcg gtggtgggtc gggtggcggc ggatcagaca tccagttgac ccagtctcca 420
tcctccctgt ctgcatctgt aggagacaga gtcaccatca cttgccgggc aagtcagagc 480
attagcatct atttaaattg gtatcggcag caaccaggga aagcccctaa gctcctgatc 540
tatgctgcat ccagtttgca aagtggggtc ccatcaaggt tcagtggcag tggatctggg 600
acagatttca ctctcaccat cagcagtctg caacctgaag attttgcaac ttacttctgt 660
caacagactt acagtacccc tccgtggacg ttcggccaag ggaccaaagt ggatatcaaa 720
<210> 15
<211> 488
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Val Gln Leu Phe Gln Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
85 90 95
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Gly Val Gly Arg Gly Phe Asp Tyr Trp Gly Gln
115 120 125
Gly Thr Thr Val Thr Val Ser Ser Ser Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser
145 150 155 160
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
165 170 175
Ser Gln Ser Ile Ser Ile Tyr Leu Asn Trp Tyr Arg Gln Gln Pro Gly
180 185 190
Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly
195 200 205
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
210 215 220
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln
225 230 235 240
Gln Thr Tyr Ser Thr Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val
245 250 255
Asp Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
260 265 270
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
275 280 285
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
290 295 300
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
305 310 315 320
Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser Val Val Lys Arg Gly
325 330 335
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
340 345 350
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
355 360 365
Glu Glu Gly Gly Cys Glu Leu Arg Arg Val Lys Phe Ser Arg Ser Ala
370 375 380
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
385 390 395 400
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
405 410 415
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg
485
<210> 16
<211> 469
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Glu Val Gln Leu Phe Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Val Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110
Val Thr Val Ser Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser
130 135 140
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
145 150 155 160
Ile Ser Ile Tyr Leu Asn Trp Tyr Arg Gln Gln Pro Gly Lys Ala Pro
165 170 175
Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
180 185 190
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
195 200 205
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Thr Tyr
210 215 220
Ser Thr Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys
225 230 235 240
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
245 250 255
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
260 265 270
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
275 280 285
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
290 295 300
Ile Thr Leu Tyr Cys Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys
305 310 315 320
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
325 330 335
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
340 345 350
Gly Cys Glu Leu Arg Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
355 360 365
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
370 375 380
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
385 390 395 400
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
405 410 415
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
420 425 430
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
435 440 445
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
450 455 460
Ala Leu Pro Pro Arg
465
<210> 17
<211> 1470
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
atggaatttg gcctgagctg gctgtttctg gtggcgattc tgaaaggcgt gcagtgcgag 60
gtgcagctgt tccagtctgg gggaggcttg gtacagcctg gggggtccct gagactctcc 120
tgtgcagcct ctggattcac ctttagcagc tatgccatga gctgggtccg ccaggctcca 180
gggaaggggc tggagtgggt ctcagctatt agtggtagtg gtggtagcac atactacgca 240
gactccgtga agggccggtt caccatctcc agagacaatt ccaagaacac gctgtatctg 300
caaatgaaca gcctgagagc cgaggacacg gccgtatatt actgtgccag gggtgttggc 360
cggggctttg actactgggg ccaggggacc acggtcaccg tctcctcaag tggcggtggc 420
tctggcggtg gtgggtcggg tggcggcgga tcagacatcc agttgaccca gtctccatcc 480
tccctgtctg catctgtagg agacagagtc accatcactt gccgggcaag tcagagcatt 540
agcatctatt taaattggta tcggcagcaa ccagggaaag cccctaagct cctgatctat 600
gctgcatcca gtttgcaaag tggggtccca tcaaggttca gtggcagtgg atctgggaca 660
gatttcactc tcaccatcag cagtctgcaa cctgaagatt ttgcaactta cttctgtcaa 720
cagacttaca gtacccctcc gtggacgttc ggccaaggga ccaaagtgga tatcaaaacg 780
cgtaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 840
ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg 900
ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 960
ctcctgtcac tggttatcac cctttactgc aggttcagtg tcgtgaagag aggccggaag 1020
aagctgctgt acatcttcaa gcagcctttc atgaggcccg tgcagactac ccaggaggaa 1080
gatggatgca gctgtagatt ccctgaagag gaggaaggag gctgtgagct gagaagagtg 1140
aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1200
gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260
cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1320
cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1380
ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1440
gcccttcaca tgcaggccct gccccctcgc 1470
<210> 18
<211> 1413
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gaggtgcagc tgttccagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc caggggtgtt 300
ggccggggct ttgactactg gggccagggg accacggtca ccgtctcctc aagtggcggt 360
ggctctggcg gtggtgggtc gggtggcggc ggatcagaca tccagttgac ccagtctcca 420
tcctccctgt ctgcatctgt aggagacaga gtcaccatca cttgccgggc aagtcagagc 480
attagcatct atttaaattg gtatcggcag caaccaggga aagcccctaa gctcctgatc 540
tatgctgcat ccagtttgca aagtggggtc ccatcaaggt tcagtggcag tggatctggg 600
acagatttca ctctcaccat cagcagtctg caacctgaag attttgcaac ttacttctgt 660
caacagactt acagtacccc tccgtggacg ttcggccaag ggaccaaagt ggatatcaaa 720
acgcgtacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 780
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 840
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 900
cttctcctgt cactggttat caccctttac tgcaggttca gtgtcgtgaa gagaggccgg 960
aagaagctgc tgtacatctt caagcagcct ttcatgaggc ccgtgcagac tacccaggag 1020
gaagatggat gcagctgtag attccctgaa gaggaggaag gaggctgtga gctgagaaga 1080
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1140
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1200
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1260
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1320
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1380
gacgcccttc acatgcaggc cctgccccct cgc 1413
<210> 19
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu
1 5 10 15
Glu Asn Pro Gly Pro
20
<210> 20
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ggatctggag agggcagagg cagcctgctg acatgtggcg acgtggaaga gaaccctggc 60
ccc 63
<210> 21
<211> 352
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile
1 5 10 15
Ser Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser
20 25 30
Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser
35 40 45
Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser
50 55 60
Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys
65 70 75 80
Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu
85 90 95
Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly
100 105 110
Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn
115 120 125
Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp
130 135 140
Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn
145 150 155 160
Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser
165 170 175
Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala
180 185 190
Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val
195 200 205
Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn
210 215 220
Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile
225 230 235 240
Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr
245 250 255
Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly
260 265 270
Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn
275 280 285
Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys
290 295 300
His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys
305 310 315 320
Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly
325 330 335
Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
340 345 350
<210> 22
<211> 1059
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tcgcaaagtg 60
tgtaacggaa taggtattgg tgaatttaaa gactcactct ccataaatgc tacgaatatt 120
aaacacttca aaaactgcac ctccatcagt ggcgatctcc acatcctgcc ggtggcattt 180
aggggtgact ccttcacaca tactcctcct ctggatccac aggaactgga tattctgaaa 240
accgtaaagg aaatcacagg gtttttgctg attcaggctt ggcctgaaaa caggacggac 300
ctccatgcct ttgagaacct agaaatcata cgcggcagga ccaagcaaca tggtcagttt 360
tctcttgcag tcgtcagcct gaacataaca tccttgggat tacgctccct caaggagata 420
agtgatggag atgtgataat ttcaggaaac aaaaatttgt gctatgcaaa tacaataaac 480
tggaaaaaac tgtttgggac ctccggtcag aaaaccaaaa ttataagcaa cagaggtgaa 540
aacagctgca aggccacagg ccaggtctgc catgccttgt gctcccccga gggctgctgg 600
ggcccggagc ccagggactg cgtctcttgc cggaatgtca gccgaggcag ggaatgcgtg 660
gacaagtgca accttctgga gggtgagcca agggagtttg tggagaactc tgagtgcata 720
cagtgccacc cagagtgcct gcctcaggcc atgaacatca cctgcacagg acggggacca 780
gacaactgta tccagtgtgc ccactacatt gacggccccc actgcgtcaa gacctgcccg 840
gcaggagtca tgggagaaaa caacaccctg gtctggaagt acgcagacgc cggccatgtg 900
tgccacctgt gccatccaaa ctgcacctac ggatgcactg ggccaggtct tgaaggctgt 960
ccaacgaatg ggcctaagat cccgtccatc gccactggga tggtgggggc cctcctcttg 1020
ctgctggtgg tggccctggg gatcggcctc ttcatgtaa 1059
<210> 23
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys
<210> 24
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
atggaatttg gcctgagctg gctgtttctg gtggcgattc tgaaaggcgt gcagtgc 57
<210> 25
<211> 47
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
35 40 45
<210> 26
<211> 141
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgatatcta c 141
<210> 27
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
1 5 10 15
Val Ile Thr Leu Tyr Cys
20
<210> 28
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 60
tactgc 66
<210> 29
<211> 48
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
1 5 10 15
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
20 25 30
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
35 40 45
<210> 30
<211> 144
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
aggttcagtg tcgtgaagag aggccggaag aagctgctgt acatcttcaa gcagcctttc 60
atgaggcccg tgcagactac ccaggaggaa gatggatgca gctgtagatt ccctgaagag 120
gaggaaggag gctgtgagct gaga 144
<210> 31
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 32
<211> 336
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 33
<211> 2598
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
atggaatttg gcctgagctg gctgtttctg gtggcgattc tgaaaggcgt gcagtgcgag 60
gtgcagctgt tccagtctgg gggaggcttg gtacagcctg gggggtccct gagactctcc 120
tgtgcagcct ctggattcac ctttagcagc tatgccatga gctgggtccg ccaggctcca 180
gggaaggggc tggagtgggt ctcagctatt agtggtagtg gtggtagcac atactacgca 240
gactccgtga agggccggtt caccatctcc agagacaatt ccaagaacac gctgtatctg 300
caaatgaaca gcctgagagc cgaggacacg gccgtatatt actgtgccag gggtgttggc 360
cggggctttg actactgggg ccaggggacc acggtcaccg tctcctcaag tggcggtggc 420
tctggcggtg gtgggtcggg tggcggcgga tcagacatcc agttgaccca gtctccatcc 480
tccctgtctg catctgtagg agacagagtc accatcactt gccgggcaag tcagagcatt 540
agcatctatt taaattggta tcggcagcaa ccagggaaag cccctaagct cctgatctat 600
gctgcatcca gtttgcaaag tggggtccca tcaaggttca gtggcagtgg atctgggaca 660
gatttcactc tcaccatcag cagtctgcaa cctgaagatt ttgcaactta cttctgtcaa 720
cagacttaca gtacccctcc gtggacgttc ggccaaggga ccaaagtgga tatcaaaacg 780
cgtaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 840
ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg 900
ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 960
ctcctgtcac tggttatcac cctttactgc aggttcagtg tcgtgaagag aggccggaag 1020
aagctgctgt acatcttcaa gcagcctttc atgaggcccg tgcagactac ccaggaggaa 1080
gatggatgca gctgtagatt ccctgaagag gaggaaggag gctgtgagct gagaagagtg 1140
aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1200
gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260
cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1320
cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1380
ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1440
gcccttcaca tgcaggccct gccccctcgc gcatgcggat ctggagaggg cagaggcagc 1500
ctgctgacat gtggcgacgt ggaagagaac cctggcccca tgtggctgca gagcctgctg 1560
ctcttgggca ctgtggcctg cagcatctct cgcaaagtgt gtaacggaat aggtattggt 1620
gaatttaaag actcactctc cataaatgct acgaatatta aacacttcaa aaactgcacc 1680
tccatcagtg gcgatctcca catcctgccg gtggcattta ggggtgactc cttcacacat 1740
actcctcctc tggatccaca ggaactggat attctgaaaa ccgtaaagga aatcacaggg 1800
tttttgctga ttcaggcttg gcctgaaaac aggacggacc tccatgcctt tgagaaccta 1860
gaaatcatac gcggcaggac caagcaacat ggtcagtttt ctcttgcagt cgtcagcctg 1920
aacataacat ccttgggatt acgctccctc aaggagataa gtgatggaga tgtgataatt 1980
tcaggaaaca aaaatttgtg ctatgcaaat acaataaact ggaaaaaact gtttgggacc 2040
tccggtcaga aaaccaaaat tataagcaac agaggtgaaa acagctgcaa ggccacaggc 2100
caggtctgcc atgccttgtg ctcccccgag ggctgctggg gcccggagcc cagggactgc 2160
gtctcttgcc ggaatgtcag ccgaggcagg gaatgcgtgg acaagtgcaa ccttctggag 2220
ggtgagccaa gggagtttgt ggagaactct gagtgcatac agtgccaccc agagtgcctg 2280
cctcaggcca tgaacatcac ctgcacagga cggggaccag acaactgtat ccagtgtgcc 2340
cactacattg acggccccca ctgcgtcaag acctgcccgg caggagtcat gggagaaaac 2400
aacaccctgg tctggaagta cgcagacgcc ggccatgtgt gccacctgtg ccatccaaac 2460
tgcacctacg gatgcactgg gccaggtctt gaaggctgtc caacgaatgg gcctaagatc 2520
ccgtccatcg ccactgggat ggtgggggcc ctcctcttgc tgctggtggt ggccctgggg 2580
atcggcctct tcatgtaa 2598
<210> 34
<211> 240
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Asn Ser His
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Val Ile Trp Tyr Asp Gly Ser Asn Ala Tyr Tyr Ala Asp Ser Ala
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Val Tyr
65 70 75 80
Leu Gln Leu Asn Ser Leu Gly Val Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys Ser Asp Arg Pro His Pro Met Gly Phe Tyr Tyr Tyr Met Asp
100 105 110
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala Ser Val
130 135 140
Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser
145 150 155 160
Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro
165 170 175
Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val Ser Asn Arg Pro Ser
180 185 190
Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
195 200 205
Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
210 215 220
Ser Ser Tyr Thr Ser Ser Ser Thr Leu Val Phe Gly Gly Gly Thr Lys
225 230 235 240
Claims (47)
1. An isolated antibody that specifically binds to CD276, comprising a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises: VH-CDR1, VH-CDR2, VH-CDR 3; the light chain variable region comprises: VL-CDR1, VL-CDR2, VL-CDR 3;
the amino acid sequence of the VH-CDR1 is shown as SEQ ID NO: 1;
the amino acid sequence of the VH-CDR2 is shown as SEQ ID NO. 2;
the amino acid sequence of VH-CDR3 is shown in SEQ ID NO. 3;
the amino acid sequence of the VL-CDR1 is shown in SEQ ID NO. 4;
the amino acid sequence of the VL-CDR2 is shown in SEQ ID NO. 5;
the amino acid sequence of VL-CDR3 is shown in SEQ ID NO 6.
2. The antibody of claim 1, wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID No. 7; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.
3. The antibody of claim 1, wherein the antibody is an scFv.
4. The antibody of claim 1, wherein said antibody is a fully human antibody.
5. A nucleic acid molecule encoding the antibody of any one of claims 1-4, a recombinant vector comprising the nucleic acid molecule, or a recombinant host cell comprising the nucleic acid molecule or the recombinant vector.
6. A method of making, comprising: a method for producing the recombinant vector according to claim 5 or a method for producing the recombinant host cell according to claim 5;
the preparation method of the recombinant vector comprises the following steps: cloning a nucleic acid molecule encoding the antibody of any one of claims 1-4 into a vector to obtain a recombinant vector;
the preparation method of the recombinant host cell comprises the following steps: introducing a nucleic acid molecule encoding the antibody of any one of claims 1-4 or the recombinant vector of claim 5 into a host cell to obtain the recombinant host cell.
7. A composition or kit comprising the antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the recombinant vector of claim 5, or the recombinant host cell of claim 5.
8. The composition or kit of claim 7, wherein the composition comprises an immunoconjugate, an antibody drug, a cellular drug, a nucleic acid drug, a chimeric antigen receptor.
9. The composition or kit of claim 8, wherein the chimeric antigen receptor comprises the scFv of claim 3.
10. The composition or kit of claim 9, wherein the chimeric antigen receptor further comprises a transmembrane region, an intracellular signaling domain.
11. The composition or kit of claim 10, wherein the chimeric antigen receptor further comprises a hinge region.
12. The composition or kit of claim 11, wherein the chimeric antigen receptor further comprises a co-stimulatory domain.
13. The composition or kit of claim 12, wherein the chimeric antigen receptor further comprises a signal peptide.
14. The composition or kit of claim 12, wherein the co-stimulatory domain comprises an intracellular signaling domain of: CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD-2, CD7, LIGHT, NKG2C, or B7-H3.
15. The composition or kit of claim 14, wherein the co-stimulatory domain is the intracellular signaling domain of 4-1BB, comprising the amino acid sequence set forth in SEQ ID No. 29.
16. The composition or kit of claim 11, wherein the hinge and transmembrane regions comprise hinge and transmembrane regions of the following molecules: IgG1, IgG4, CD8 a, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, or CD 34.
17. The composition or kit of claim 16, wherein the hinge and transmembrane regions are those of CD8 a.
18. The composition or kit of claim 17, wherein the hinge region of CD8 a comprises the amino acid sequence set forth in SEQ ID No. 25.
19. The composition or kit of claim 17, wherein the transmembrane region of CD8 a comprises the amino acid sequence of SEQ ID No. 27.
20. The composition or kit of claim 10, wherein the intracellular signaling domain comprises the intracellular signaling domains of the following molecules: fc γ R, Fc ∈ R, Fc α R, FcRn, CD3, CD3 ζ, CD3 γ, CD3 δ, CD3 ∈, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278, CD247 ζ, CD247 η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF- κ B, PLC- γ, iC3b, C3dg, C3d, or Zap 70.
21. The composition or kit of claim 20, wherein the intracellular signaling domain is a CD3 ζ intracellular signaling domain comprising an amino acid sequence as set forth in SEQ id No. 31.
22. The composition or kit of claim 13, wherein the signal peptide comprises a signal peptide of a membrane protein.
23. The composition or kit of claim 22, wherein the signal peptide comprises a signal peptide from an alpha chain and a beta chain of a T cell receptor, CD3 ζ, CD28, CD3 ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, an immunoglobulin heavy chain, or an immunoglobulin light chain.
24. The composition or kit of claim 23, wherein the signal peptide comprises the amino acid sequence set forth in SEQ id No. 23.
25. The composition or kit of any one of claims 8-24, wherein the amino acid sequence of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ id No. 15 or SEQ id No. 16.
26. A nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 8-24.
27. The nucleic acid molecule of claim 26, wherein said nucleic acid molecule further encodes a self-cleaving peptide.
28. The nucleic acid molecule of claim 27, wherein the coding sequence for the self-cleaving peptide is a coding sequence for T2A.
29. The nucleic acid molecule of claim 28, wherein the coding sequence for T2A is a coding sequence encoding the amino acid sequence set forth in SEQ ID No. 19.
30. The nucleic acid molecule of claim 29, wherein the coding sequence for T2A is set forth in SEQ ID No. 20.
31. The nucleic acid molecule of claim 26, wherein said nucleic acid molecule further encodes a tag molecule; the tag molecule is a molecule that is used to characterize whether the chimeric antigen receptor is expressed on the surface of a cell.
32. The nucleic acid molecule of claim 31, wherein said tag molecule comprises tEGFR.
33. The nucleic acid molecule of claim 32, wherein the amino acid sequence of tEGFR is set forth in SEQ ID NO 21.
34. The nucleic acid molecule of claim 33, wherein the coding sequence for tEGFR is set forth in SEQ ID NO 22.
35. The nucleic acid molecule of claim 26, wherein said nucleic acid molecule comprises the sequence set forth in SEQ ID No.17, SEQ ID No. 18, or SEQ ID No. 33.
36. A vector or a recombinant virus comprising the same, wherein the vector comprises the nucleic acid molecule of any one of claims 26-35; the recombinant virus comprises the vector or the nucleic acid molecule of any one of claims 26-35.
37. A recombinant cell expressing the chimeric antigen receptor of any one of claims 8-25.
38. The recombinant cell of claim 37, wherein the recombinant cell comprises the nucleic acid molecule of any one of claims 26-35, or the vector or recombinant virus of claim 36.
39. The recombinant cell of claim 37, wherein the recombinant cell comprises a modified immune cell.
40. The recombinant cell of claim 39, wherein the immune cell is a T cell.
41. A method of making the recombinant cell of any one of claims 37-40, comprising the steps of: introducing the nucleic acid molecule of any one of claims 26-35 or the vector or recombinant virus of claim 36 into a cell to obtain a recombinant cell.
42. A derivative comprising the chimeric antigen receptor of any one of claims 8-25, the nucleic acid molecule of any one of claims 26-35, the vector or recombinant virus of claim 36, or the recombinant cell of any one of claims 37-40.
43. The derivative of claim 42, wherein the derivative comprises a composition or a kit.
44. A method of detecting CD276 protein or an antigenic fragment thereof for non-diagnostic purposes, said method comprising administering the antibody of any one of claims 1-4, the immunoconjugate of claim 8.
45. The method of claim 44, comprising the steps of: 1) Obtaining a sample containing a CD276 protein or antigenic fragment thereof; 2) contacting the sample collected in step 1) with the antibody of any one of claims 1-4 or the immunoconjugate of claim 8; 3) detecting the presence of the antibody-antigen complex.
46. An application, the application comprising any one of:
1) use of the antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the recombinant vector of claim 5, the recombinant host cell of claim 5, the composition of any one of claims 7-25 for the manufacture of a medicament for the treatment of a solid tumor;
2) use of the nucleic acid molecule of claim 5 in the preparation of the recombinant vector of claim 5, the recombinant host cell of claim 5, the composition or kit of any one of claims 7-25;
3) use of the recombinant vector of claim 5 in the preparation of the recombinant host cell of claim 5, the composition or kit of any one of claims 7-25;
4) use of the recombinant host cell of claim 5 in the preparation of a composition or kit of any one of claims 7-25;
5) use of the nucleic acid molecule of any one of claims 26 to 35, the vector or recombinant virus of claim 36, the recombinant cell of any one of claims 37 to 40, the derivative of claim 42 or 43 for the manufacture of a medicament for the treatment of a solid tumor;
6) use of the nucleic acid molecule of any one of claims 26 to 35 in the preparation of the vector or recombinant virus of claim 36, the recombinant cell of any one of claims 37 to 40, the derivative of claim 42 or 43;
7) use of the vector of claim 36 for the preparation of the recombinant virus of claim 36, the recombinant cell of any one of claims 37-40, the derivative of claim 42 or 43;
8) use of the recombinant virus of claim 36 in the preparation of the recombinant cell of any one of claims 37-40, the derivative of claim 42 or 43;
9) use of the recombinant cell of any one of claims 37-40 for the preparation of the derivative of claim 42 or 43;
10) use of an antibody of any one of claims 1-4 in the preparation of a kit for detecting a CD276 protein or an antigenic fragment thereof;
11) use of an antibody according to any one of claims 1 to 4 for the preparation of a kit for the diagnosis of solid tumors.
47. The use of claim 46, wherein the solid tumor comprises cancer of the bladder, glioma, bone, brain, liver, pancreas, lung, melanoma, kidney, breast, prostate, cervix, ovary, anus, anal canal, anorectum, eye, intrahepatic bile duct, joint, gall bladder, pleura, nose, mouth, vulva, colon, esophagus, fibrosarcoma, head and neck, malignant mesothelioma, skin, soft tissue, thyroid, ureter, testicular.
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| PCT/CN2021/115806WO2023272924A1 (en) | 2021-06-30 | 2021-08-31 | Novel fully human antibody for human b7h3, chimeric antigen receptor and uses thereof |
| US18/401,115US20240245771A1 (en) | 2021-06-30 | 2023-12-29 | Fully human antibody for human b7h3, chimeric antigen receptor and uses thereof |
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| CN113621068B (en)* | 2021-10-11 | 2022-01-07 | 上海恒润达生生物科技股份有限公司 | Antibody or antigen binding fragment thereof specifically binding to CD276, and preparation method and application thereof |
| CN117362434A (en)* | 2022-06-30 | 2024-01-09 | 南京北恒生物科技有限公司 | Antibodies targeting B7-H3 and uses thereof |
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