CN116964097A

CN116964097A - CD27 extracellular domain CAR targeting CD70 positive tumors

Info

Publication number: CN116964097A
Application number: CN202280018183.2A
Authority: CN
Inventors: K·雷兹瓦尼; E·施帕尔; R·巴萨尔; S·阿查亚; N·尤普赖特瑞; D·马林科斯达; E·恩斯雷
Original assignee: University of Texas System
Current assignee: University of Texas System
Priority date: 2021-01-25
Filing date: 2022-01-24
Publication date: 2023-10-27

Abstract

Embodiments of the present disclosure encompass methods and compositions utilizing an anti-CD 70 Chimeric Antigen Receptor (CAR) that lacks an antibody or antibody fragment as part of the receptor, including as part of the antigen binding region of the CAR. In particular embodiments, the CAR molecule utilizes receptor CD27 for ligand CD70, rather than a CAR employing antibodies that bind CD 70. In particular embodiments, the CAR comprises a truncated form of CD27, rather than full length CD27. In particular cases, the CAR lacks a CD27 transmembrane domain.

Description

CD27 extracellular domain CAR targeting CD70 positive tumors

The present application claims priority from U.S. provisional application No. 63/141,016, filed on 25 months 1 and 2021, and U.S. provisional application No. 63/270,414, filed on 21 months 10 and 2021, both of which are hereby incorporated by reference in their entireties.

Sequence listing

The present application comprises a sequence listing submitted in ASCII format, hereby incorporated by reference in its entirety. The ASCII copy was created at 2022, 1/18, and was named "MDAC.P1261WO_ST25SL.txt" with a size of 209,719 bytes.

Background

Embodiments of the present disclosure include at least the fields of cell biology, molecular biology, immunology, and medicine (including cancer medicine).

Background

Gene reprogramming of Natural Killer (NK) cells for adoptive cancer immunotherapy has clinically relevant applications and benefits such as 1) congenital anti-tumor monitoring without prior sensitization, 2) alloefficacy without graft-versus-host reactivity, and 3) direct cell-mediated cytotoxicity and cytolysis of target tumors. Development and acquisition of self-tolerance, alloreactivity and effector functions of human NK cells are adaptive processes of licensing, calibration and setup. At the molecular level, specific activating and inhibitory receptors direct NK cell function by aggregating, balancing and integrating extracellular signals into different effector functions. The functional activity of NK cells and responsiveness to external stimuli follow a continuously educational "rheostat" model and can therefore be reprogrammed. Genetic modification of NK cells to redirect their effector functions is an effective way to exploit their cytotoxic ability to kill tumor cells.

CD70 is a ligand for the cytokine receptor CD27, an attractive "pan-cancer antigen" because it is expressed on many solid tumors, including renal, bladder, lung, triple negative breast, renal cell, pancreatic and melanoma, in addition to malignant hematological diseases such as Acute Myeloid Leukemia (AML) and lymphomas, and it is only transiently present on activated T and B lymphocytes and dendritic cells. CD70 is particularly advantageous as a target for immunotherapy of AML because, unlike other AML targets, it is not expressed on normal hematopoietic stem cells and is therefore unlikely to lead to long-term cytopenia and the need for hematopoietic stem cell transplantation by the recipient following CAR treatment, in contrast to the current clinical majority of CAR-T therapies for AML. The present disclosure provides a solution to the long felt need in the field of cancer therapy for CD70 positive cancers.

Disclosure of Invention

The present disclosure relates to methods and compositions for treating or preventing cancer having CD70 expressing cells. The present disclosure relates to specific anti-CD 70 Chimeric Antigen Receptors (CARs) and their use for targeting any type of CD70 positive cancer. In certain embodiments, the antigen binding domain that targets CD70, including specific binding to CD70, is not an antibody. In certain embodiments, the current anti-CD 70 CAR lacks any type of antibody, including scFv that binds CD 70. In certain embodiments, the extracellular domain of the current anti-CD 70 CAR does not comprise an scFv or any other type of antibody.

In a particular embodiment, the antigen binding domain that targets CD70 is a natural receptor for CD70, such as receptor CD27. In certain cases, some or all of CD27 is employed in the CAR molecule. In aspects of the disclosure, the antigen binding domain present in an anti-CD 70 CAR molecule comprises part or all of the extracellular domain of CD27, and in particular instances, the CAR molecule utilizes or does not utilize the transmembrane domain of CD27. In some cases, although a CD27 extracellular domain is used in the CAR, a CD28 transmembrane domain is used instead of a CD27 transmembrane domain. Specific intracellular domains may be used in the CAR, such as CD3 ζ alone or a combination of CD3 ζ and DAP10 or DAP12, in some cases the intracellular domains do not comprise the intracellular domains of 4-1 BB.

Embodiments of the disclosure include any polynucleotide encoding part or all of the anti-CD 70 CAR. Particular embodiments encompass polynucleotides encoding an anti-CD 70 CAR comprising an antigen binding domain, a transmembrane domain, and at least one intracellular domain (including one or more costimulatory domains), wherein the CAR comprises an antigen binding domain that is free of antibodies. Vectors comprising expression constructs encoding the anti-CD 70 CAR and optionally other genes are contemplated. The vector may or may not be a virus. Cells comprising the vector are also contemplated, including any type of immune cell, such as at least NK cells or T cells. In some cases, the cells are off-the-shelf cells, including NK cells, and these cells may or may not have been manipulated to express one or more heterologous genes, such as one or more exogenous cytokines.

Contemplated herein is a method of treating or preventing any type of cancer comprising administering a cell expressing a particular anti-CD 70 CAR in a therapeutically effective amount to ameliorate or prevent the cancer, or reduce the risk of the cancer, reduce the severity of the cancer, prevent metastasis or the risk thereof, or delay the onset of the cancer.

Embodiments of the present disclosure include methods of targeting CD70 expressing cells, in particular embodiments, the cells are cancerous, while in other methods, CD70 expressing cells are immunoregulatory cells, such as tregs.

The present disclosure includes methods of genetically engineering human NK cells, including from umbilical Cord Blood (CB), to target cd70+ve tumors using a CD 27-ectodomain CAR. Contemplated herein are novel CAR constructs that fuse the extracellular binding portion of CD27 (CD 27eCAR or CD27 EC) with an activating signaling intracellular domain (endodomain) that incorporates CD3 zeta, alone or in combination with a co-stimulatory or adapter signaling domain (such as 4-1BB, CD28, DAP10 or DAP 12) and a cytokine gene (such as IL-15, IL-12, IL-18 or IL-21) to support NK cell survival and proliferation. In a series of in vitro studies, the activity of CD27eCAR/IL-15 transduced CB-NK cells against AML and lymphoma targets was demonstrated.

Embodiments of the present disclosure include a polynucleotide encoding an anti-CD 70 Chimeric Antigen Receptor (CAR) comprising a signal peptide, an anti-CD 70 antigen binding domain, a transmembrane domain, and at least one intracellular domain, wherein the anti-CD 70 antigen binding domain does not comprise an antibody, and optionally wherein the transmembrane domain of the CAR is not a CD27 transmembrane domain. In specific embodiments, the anti-CD 70 antigen binding domain is contained in the extracellular domain of CD27 and/or can be codon optimized. In a specific aspect, the antigen binding domain comprises, consists of, or consists essentially of SEQ ID NO. 2. In some cases, the antigen binding domain and the transmembrane domain comprise, consist of, or consist essentially of SEQ ID NO. 1. The sequence encoding the transmembrane domain may be codon optimized and/or the transmembrane domain may be from CD28, the alpha chain of a T cell receptor, the beta chain of a T cell receptor, the zeta chain of a T cell receptor, cd3ζ, cd3ε, cd3γ, cd3δ, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS/CD278, GITR/CD357, NKG2D, DAP or DAP12. In a particular case, the CD28 transmembrane domain comprises, consists of, or consists essentially of SEQ ID NO 3. The CAR may comprise two or more, or three or more, intracellular domains. The intracellular domain may comprise any ITAM-containing signaling domain. The intracellular domain may comprise cd3ζ. The intracellular domain may be an intracellular co-stimulatory domain, such as an intracellular co-stimulatory domain selected from the group consisting of CD27, CD28, 4-1BB, DAP12, NKG2D, OX-40 (CD 134), DAP10, CD40L, 2B4, DNAM, CS1, CD48, NKp30, NKp44, NKp46, NKp80 and combinations thereof.

In some embodiments, the signal peptide is from the CD27 or granulocyte-macrophage colony-stimulating factor receptor (GMSCF-R), and the sequence of the CD27 signal peptide may comprise, consist of, or consist essentially of SEQ ID NO. 6. The signal peptide may be from CD27, the anti-CD 70 antigen binding domain may be from CD27, the transmembrane domain may be from CD27, and the intracellular domain from CD27. The signal peptide may comprise, consist of or consist essentially of SEQ ID NO. 6. The CAR may comprise, consist of, or consist essentially of SEQ ID NO. 4. In some cases, the transmembrane domain comprises, consists of, or consists essentially of SEQ ID NO 3 or SEQ ID NO 7. In certain aspects, the intracellular domain comprises one or more of SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, or SEQ ID NO. 12. The CAR may comprise, consist of, or consist essentially of SEQ ID No. 5. The CAR may comprise, consist of, or consist essentially of SEQ ID No. 1.

In particular embodiments, the polynucleotide encodes a CAR, and the CAR comprises one or more of the following:

(a) CD27 Signal Peptide (SP), CD27 extracellular domain (EC), CD27 transmembrane domain (TMD), DAP12 intracellular domain (ICD), and cd3ζ;

(b) GMSCF-R SP, codon optimized CD27 EC, codon optimized CD27 TMD, DAP12 ICD;

(c) CD27 SP, CD27 EC, CD28 TMD, DAP12 ICD and cd3ζ;

(d) GMSCF-R SP, codon optimized CD27 EC, CD28 TMD, DAP12 ICD and cd3ζ;

(e) CD27 SP, CD27 EC, CD27 TMD, natural killer group 2 member D (NKG 2D) ICD and cd3ζ;

(f) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, NKG2D ICD and cd3ζ;

(g) CD27 SP, CD27 EC, CD28 TMD, natural killer group 2 member D (NKG 2D) ICD and cd3ζ;

(h) GMSCF-R SP, codon optimized CD27 EC, CD28 TMD, NKG2D ICD and cd3ζ;

(i) CD27 SP, CD27 EC, CD27 TMD, 4-1BB ICD and CD3 ζ;

(j) GMSCF-R SP, codon optimized CD27 EC, codon optimized CD27 TMD, 4-1BB ICD and cd3ζ;

(k) CD27 SP, CD27 EC, CD28 TMD, 4-1BB ICD and CD3 ζ;

(l) GMSCF-R SP, codon optimized CD27 EC, CD28 TMD, 4-1BB ICD and cd3ζ;

(m) CD27 SP, CD27 EC, CD27 TMD, DAP10 ICD, and cd3ζ;

(n) GMSCF-R SP, codon optimized CD27 EC, codon optimized CD27 TMD, DAP10 ICD and cd3ζ;

(o) CD27 SP, CD27 EC, CD28 TMD, DAP10 ICD, and cd3ζ;

(p) GMSCF-R SP, codon optimized CD27 EC, CD28 TMD, DAP10ICD and cd3ζ;

(q) CD27 SP, CD27 Full Length (FL), CD27 TMD, CD27 ICD, and CD3 ζ;

(R) GMSCF-R SP, codon optimized CD27 FL, codon optimized CD27 TMD, codon optimized CD27 ICD and cd3ζ;

(s) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, CD28 ICD and CD3 ζ;

(t) GMSCF-R SP, codon optimized CD27 FL, codon optimized CD27 TMD, codon optimized CD27 ICD, CD28 ICD and cd3ζ;

(u) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, 4-1BB ICD and CD3 ζ;

(v) GMSCF-R SP, codon optimized CD27 FL, codon optimized CD27 TMD, codon optimized CD27 ICD, 4-1BB ICD and cd3ζ;

(w) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, DAP10ICD, and cd3ζ;

(x) GMSCF-R SP, CD27 FL, CD27 TMD, CD27 ICD, DAP10ICD and cd3ζ;

(y) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, DAP12 ICD, and cd3ζ;

(z) GMSCF-R SP, codon optimized CD27 FL, codon optimized CD27 TMD, codon optimized CD27 ICD, DAP12 ICD and cd3ζ;

(aa) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, NKG2D ICD and cd3ζ;

(bb) GMSCF-R SP, codon optimized CD27 FL, codon optimized CD27 TMD, codon optimized CD27 ICD, NKG2D ICD and CD3 ζ;

(cc) CD27 SP, CD27 EC, CD27 TMD and CD3 ζ;

(dd) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD and cd3ζ;

(ee) CD27 SP, CD27 EC, CD28 TMD and CD3 ζ;

(ff) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD28 TMD and cd3ζ;

(gg) CD27 SP, CD27 EC, CD27 TMD, CD28 ICD and cd3ζ;

(hh) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, CD28 ICD and cd3ζ;

(ii) CD27 SP, CD27 EC, CD28 TMD, CD28 ICD and cd3ζ; or (b)

(jj) GMSCF-R SP, codon optimized CD27 EC, CD28 TMD and CD3 ζ.

Embodiments of the present disclosure include cells comprising any of the polynucleotides encompassed herein. The cells may or may not be immune cells. Examples of immune cells include Natural Killer (NK) cells, T cells, γδ T cells, invariant NKT (iNKT) cells, B cells, macrophages, MSCs or dendritic cells. When the cells are NK cells, they may be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, hematopoietic Stem Cells (HSCs), from cell lines, or mixtures thereof. The NK cell line may be an NK-92 cell line or another NK cell line derived from tumor or healthy NK cells, or a progenitor cell. In particular embodiments, the NK cells are derived from umbilical cord blood mononuclear cells. The NK cells may be CD56+ NK cells. In some cases, NK cells express one or more exogenously supplied cytokines, such as IL-15, IL-2, IL-12, IL-18, IL-21, IL-7, or a combination thereof.

Embodiments of the present disclosure include populations of immune cells of the present disclosure, the cells being present in a suitable medium. In a particular case, the immune cells are NK cells. The immune cells may or may not be obtained from a cryopreserved repository.

In a particular embodiment, a method of killing CD70 positive cells in an individual is provided comprising the step of administering to the individual a therapeutically effective amount of cells comprising any of the polynucleotides covered herein. The cell may be an NK cell, a T cell, a γδ T cell, an Invariant NKT (iNKT) cell, a B cell, a macrophage, a γδ T cell or a dendritic cell. NK cells may be derived from cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, HSCs, or from cell lines. NK cells can be derived from umbilical cord blood mononuclear cells. In some cases, the CD70 positive cells are not cancer cells, but may be regulatory T cells. The individual may have or be suspected of having any CD70 expressing cancer. The individual may have or be suspected of having, for example, acute myeloid leukemia, lymphoma, lung cancer, kidney cancer, bladder cancer, melanoma, glioblastoma, breast cancer, head and neck cancer, mesothelioma, multiple myeloma, pancreatic cancer, or a combination thereof. The cells may be allogeneic or autologous with respect to the individual. The individual may be a human. In some embodiments, the cells are administered to the individual one or more times. The duration between each administration of the cells to the individual may be 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, or one or more years.

In certain embodiments of the methods contemplated herein, the methods further comprise the step of providing an effective amount of additional therapy (such as surgery, radiation, gene therapy, immunotherapy, and/or hormonal therapy) to the individual. In some cases, the additional therapy includes one or more antibodies.

In particular embodiments, the cells are administered to the individual intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, transdermally, subcutaneously, topically, by perfusion in a tumor microenvironment, or a combination thereof. In particular embodiments, the method further comprises the step of identifying CD70 positive cells in the individual.

The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages will be described hereinafter that form the subject of the claims herein. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present design. Those skilled in the art will also recognize that such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The novel features which are believed to be characteristic of the design disclosed herein, both as to organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.

Drawings

For a more complete understanding of this disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings.

FIGS. 1A-1B illustrate the expression of CD70 on AML patient samples using Tstone diagrams.

Figures 2A-2E illustrate various anti-CD 70 CAR constructs.

The following table provides information about truncated (tr) and full length (fl) CD27 constructs and their respective reference numbers used in the data contained herein. S.no refers to the sequence number.

For the present table, information about truncated (tr) and full length (fl) CD27 constructs and their respective identification numbers used in the figures are as follows:

GSP: GMCSF receptor signal peptides

co: codon optimized

CD28 tmd: CD28 transmembrane domain

CD27 tmd: CD27 transmembrane domain

3z: CD3 zeta signal

CD28 ic: CD28 intracellular domain

Natural killer group 2 member D (NKG 2D) ic: NKG2D intracellular domain

Dap12 icd: DAP12 intracellular domain

Dap10 icd: DAP12 intracellular domain

41bb icd:41bb intracellular Domain

FIG. 3 shows transfection efficiency of truncated (tr) and full length (fl) CD27 constructs in 293T cells.

FIG. 4 shows transduction efficiencies of truncated (tr) and full length (fl) CD27 constructs in NK cells of cord blood origin.

FIGS. 5A-5C show the killing activity of CD70 positive Raji cells (FIG. 5A) and Karpas cells (FIG. 5B) by cord blood natural killer (CB-NK) cells transduced with various trCD27 constructs using 4 hours annexin-V assay (FIG. 5C) for CD70 expression on tumor targets. Fig. 5C shows that CAR-transduced CBNK cells have better killing effect against various trCD70 transduced CB-NK cells of Raji (black bars) and Karpas (gray bars) cells compared to NT (non-transduced), IL15 (construct with IL15 cytokine gene only) or CD70-IL15 (construct with CD70 scFv and IL 15).

FIG. 6 shows CD107a degranulation of CB-NK cells transduced with various trCD27 constructs against Raji and Karpas cells (markers of the cytotoxic activity of NK cells). trCD27 CAR transduced CBNK cells and cancer cells (Raji and Karpas) were co-cultured in the presence of CD107a antibody at a ratio of 1:1 for 6hr. After incubation, CD107a expression was assessed by flow cytometry. Among the three groups of columns, the left (green) column is a control using CB-NK cells not co-cultured with cancer cells, the middle (black) column is trCD 70-transduced CB-NK cells co-cultured with Raji cells, and the right (gray) column is trCD 70-transduced CB-NK cells co-cultured with Karpas cells. Most trCD27 transduced CB-NK cells show enhanced cytotoxic activity against Raji and Karpas cells when compared to NT, IL15 or CD70-IL15 CAR transduced CB-NK cells.

FIGS. 7A-7B show the killing activity of CB-NK cells transduced with various trCD27 constructs against Raji cells (FIG. 7A) and Karpas cells (FIG. 7B) measured using in vivo imaging of intucyte. trCD27 CAR transduced CBNK cells and Raji or Karpas cells were co-cultured in a ratio of 1:1, and real-time cytotoxicity of NK cells against Raji and Karpas cells was measured every hour over 12 hours. trCD27 transduced CB-NK cells showed enhanced cytotoxic activity against Raji and Karpas cells when compared to NT, IL15 or CD70-IL15 CAR transduced CB-NK cells. Cancer cells cultured in the absence of CBNK cells (cancer cells alone) served as controls.

FIGS. 8A-8B show the in vivo antitumor activity of CB-NK cells transduced with various trCD27 constructs against Raji cells with high CD70 expression. Fig. 8A shows bioluminescence imaging of Raji tumors following infusion of CB-NK cells transduced with various trCD27 CARs. Fig. 8B shows survival curves after infusion of CB-NK cells transduced with various trCD27 CARs.

FIGS. 9A-9B show the in vivo antitumor activity of CB-NK cells transduced with various trCD27 constructs against THP-1 cells with high CD70 expression. Fig. 9A shows bioluminescence imaging of THP-1 tumors following infusion with various trCD27 CAR transduced CB-NK cells. Fig. 9B shows survival curves after infusion of CB-NK cells transduced with various trCD27 CARs.

Detailed Description

In keeping with the long-standing patent law convention, the words "a" and "an" when used in this specification (including the claims) with the word "comprising" mean "one or more". Some embodiments of the present disclosure may consist of or consist essentially of one or more elements, method steps, and/or methods of the present disclosure. It is contemplated that any of the methods or compositions described herein may be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises", and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. "consisting of" means including and limited to any content following the phrase "consisting of". Thus, the phrase "consisting of" means that the listed elements are required or mandatory and that no other elements are present. "consisting essentially of" is meant to include any element listed after that phrase and is limited to other elements that do not interfere with or contribute to the activity or effect specified in the disclosure of the listed elements. Thus, the phrase "substantially..the term" consisting of "means that the listed elements are necessary or mandatory, but that no other elements are optional, and may or may not be present, depending on whether they affect the activity or effect of the listed elements.

Throughout this specification, reference to "one embodiment," "an embodiment," "particular embodiment," "related embodiment," "particular embodiment," "additional embodiment" or "another embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places in the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

As used herein, the terms "or" and/or "are used to describe a plurality of components that are combined or mutually exclusive. For example, "x, y, and/or z" may refer to "x" alone, "y" alone, "z," x, y, and z, "" x and y, "or" z, "" x or (y and z) "or" x or y or z. It is specifically contemplated that x, y or z may be specifically excluded from embodiments.

Throughout this application, the term "about" is used in accordance with its ordinary and customary meaning in the art of cell and molecular biology to indicate that a value includes the standard deviation of the error of the device or method used to determine the value.

The term "engineered" as used herein refers to artificially created entities, including cells, nucleic acids, polypeptides, vectors, and the like. In at least some cases, the engineered entity is synthetic and includes elements that do not naturally occur or are configured in the manner in which it is used in the present disclosure. In particular embodiments, the vector is engineered by recombinant nucleic acid technology, and the cells are engineered by transfection or transduction of the engineered vector.

As used herein, "prevent" and similar words such as "prevent", "preventing" and the like mean methods for preventing, inhibiting or reducing the likelihood of occurrence or recurrence of a disease or disorder (e.g., cancer). It also refers to delaying the onset or recurrence of a disease or disorder, or delaying the onset or recurrence of symptoms of a disease or disorder. As used herein, "preventing" and like terms also include reducing the intensity, effect, symptoms, and/or burden of a disease or disorder prior to the onset or recurrence of the disease or disorder.

The term "sample" as used herein generally refers to a biological sample. The sample may be taken from a tissue or cell of the individual. In some examples, the sample may include or be derived from a tissue biopsy, blood (e.g., whole blood), plasma, extracellular fluid, dried blood spots, cultured cells, discarded tissue. The sample may have been separated from the source prior to collection. Non-limiting examples include blood (including but not limited to cord blood), serum, plasma, cerebrospinal fluid, pleural fluid, amniotic fluid, lymph, saliva, urine, feces, tears, sweat, bone marrow or mucosal secretions, as well as other bodily fluids isolated from a primary source prior to collection. In some examples, the sample is separated from its original source (cells, tissue, bodily fluids such as blood, environmental samples, etc.) during sample preparation. The sample may or may not be purified or otherwise enriched from its original source. In some cases, the initial source is homogenized prior to further processing. The sample may be filtered or centrifuged to remove buffy coat, lipids or particulate matter. The nucleic acid of the sample may also be purified or enriched, or the sample may be treated with an RNase. The sample may comprise intact, disrupted or partially degraded tissue or cells.

As used herein, the term "subject" generally refers to an individual having a biological sample being processed or analyzed and, in particular instances, having or suspected of having cancer. The subject may be any organism or animal subject, including mammals, such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), domestic animals (e.g., cattle, sheep, goats, pigs, turkeys, and chickens), domestic pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals, as subjects of the method or material. The subject may be a patient, e.g., suffering from or suspected of suffering from a disease (which may be referred to as a medical condition), such as a benign or malignant tumor or cancer. The subject may be receiving or has received treatment. The subject may be asymptomatic. The subject may be a healthy individual desiring to prevent cancer. The term "individual" may be used interchangeably, at least in some instances. As used herein, a "subject" or "individual" may or may not be disposed in a medical facility, and may be treated as an outpatient to the medical facility. The individual may receive one or more medical compositions via the internet. Individuals may include humans or non-human animals of any age, and thus include adults and adolescents (i.e., children) and infants, and include intrauterine individuals. The term does not mean that medical treatment is required, and thus, an individual may voluntarily or involuntarily be part of a clinical or basic scientific study supporting experiment.

As used herein, "treatment" or "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reduction of one or more measurable markers of the disease or disorder (e.g., cancer) being treated. Treatment may optionally include alleviating or ameliorating symptoms of the disease or condition, or delaying the progression of the disease or condition. "treating" does not necessarily mean complete eradication or cure of a disease or disorder or associated symptoms thereof.

**********

The disclosure includes targeting CD 70-expressing cancers with anti-CD 70 CARs, and in some cases, CARs are expressed on NK cells derived from cord blood. Described herein are a number of novel CAR molecules produced by fusing the extracellular binding portion of CD27 (CD 27 eCAR) with an activating signaling intracellular domain, alone or in combination with a costimulatory or adapter signaling domain, such as 4-1BB, CD28, DAP10, or DAP12, and at least in some cases a cytokine gene, such as IL 15, IL-12, IL-18, or IL-21, to incorporate CD3 zeta. In some embodiments, the CAR is a fusion of the extracellular domain of CD27 (a natural ligand of CD 70) that is specific for the human CD70 antigen. In addition, in some cases, codon optimization is utilized to increase the surface expression of CD27 on the surface of transduced cells. In some cases, vectors comprising expression constructs encoding anti-CD 70 CARs also carry cytokine genes, such as to produce human interleukin 15 (IL-15), IL-12, IL-18, or IL-21, to aid in the survival and maintenance of cells (including, for example, NK cells) carrying the vector.

I. Chimeric Antigen Receptor (CAR)

The immune cells of the present disclosure can be genetically engineered to express one or more antigen binding receptors, such as an engineered CAR, that target one or more antigens (e.g., CD 70). For example, the immune cell can be an immune cell modified to express a CAR having antigen specificity for CD 70. Other CARs may be expressed by the same cell as the cell expressing the CD70 antigen receptor, and they may be directed against different antigens. In some aspects, immune cells are engineered to express a CD 70-specific CAR by knocking in the CAR using, for example, CRISPR/Cas technology.

Suitable methods for modifying cells are known in the art. See, for example, sambrook and Ausubel, supra. For example, cells can be transduced to express CARs with antigen specificity for cancer antigens using transduction techniques described in heimskerk et al, 2008 and Johnson et al, 2009.

In some embodiments, the cells comprise one or more nucleic acids introduced by genetic engineering that encode one or more antigen-targeted receptors (at least one of which may be directed against CD 70), as well as genetically engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., is not normally present in the cell or in a sample obtained from the cell, such as a sample obtained from another organism or cell, e.g., is not normally present in the cell being engineered and/or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as a nucleic acid that does not exist in nature (e.g., chimeric).

The present disclosure encompasses a CD 70-specific CAR comprising: a) An extracellular domain comprising an antigen binding region that targets CD70 (including specifically binding to CD 70); b) A transmembrane domain; and c) one or more intracellular domains, including an intracellular signaling domain. In some aspects, an antigen-specific binding or recognition component is linked to the transmembrane domain and one or more intracellular signaling domains. In some embodiments, the CAR comprises a transmembrane domain fused to an extracellular domain of the CAR, the extracellular domain comprising an antigen binding region or domain. In one embodiment, the transmembrane domain naturally associated with one domain in the CAR is used, although in other cases it is not used. In some cases, the transmembrane domains are selected or modified by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

The present disclosure relates to anti-CD 70 CARs comprising an antigen binding domain that does not use an antibody or antibody fragment. In certain embodiments, in some cases, the CAR utilizes part or all of CD27 in the CAR (including using the extracellular domain of CD27 as the antigen binding domain of the CAR), rather than using an anti-CD 70 CAR that utilizes an antibody or antibody fragment as the antigen binding domain.

For reference, the homo sapiens CD27 molecule (CD 27) on chromosome 12 is obtained from the National Center for Biotechnology Information (NCBI)Accession number ng_031995.1 (which is incorporated herein by reference in its entirety). The nucleotide sequence encoding the complete CD27 protein includes the following sequences:

ATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTC CAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCATCCTTGTGATCTTCTCTGGAATGTTCCTTGTTTTCACCCTGGCCGGGGCCCTGTTCCTCCATCAACGAAGGAAATATAGATCAAACAAAGGAGAAAGTCCTGTGGAGCCTGCAGAGCCTTGTCGTTACAGCTGCCCCAGGGAGGAGGAGGGCAGCACCATCCCCATCCAGGAGGATTACCGAAAACCGGAGCCTGCCTGCTCCCCCTGA(SEQ ID NO:9)

any polynucleotide encompassed by the present disclosure may utilize SEQ ID NO 9 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO 9.

An example of an intact wild-type CD27 protein sequence is NCBIAccession number P26842 (also identical to the amino acid sequence in accession number ng_ 031995.1):

(SEQ ID NO:4)

in particular embodiments, some or all of SEQ ID NO. 4 is used in the CAR. Although in certain embodiments, a full length wild-type (or codon-optimized) CD27 protein is used in the CAR, in other embodiments, a truncated form of the wild-type CD27 protein is used as the antigen binding domain in the CAR. In particular embodiments, part or all of the extracellular domain of CD27 is used, such that the truncated CD27 used as the antigen binding domain is a truncated form of CD27 truncated at the C-terminus of the native protein.

The nucleotide sequence encoding a truncated form of the wild-type CD27 protein includes the following sequences:

GCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGG AACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGC(SEQ ID NO:63)

any polynucleotide encompassed by the present disclosure may utilize SEQ ID NO. 63 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO. 63.

Polypeptide sequences encoding truncated forms of the wild-type CD27 protein include the following sequences:

ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR(SEQ ID NO:5)

any polypeptide encompassed by the present disclosure may utilize SEQ ID NO. 5 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO. 5.

In the particular case where full length CD27 protein is used in the CAR molecule, no additional transmembrane domain is used. In the particular case where full length CD27 protein is used in the CAR molecule, the CAR molecule comprises its own intracellular signaling domain, which may or may not be used in the CAR. In the particular case where the full length CD27 protein is used in the CAR molecule, although it contains its own signal peptide, additional signal peptide may or may not be used. In particular embodiments, cd3ζ (and/or DAP10, DAP12, 2B4, CD28, NKG2D, 41BB or any ITAM-containing signaling domain) is used in the CAR.

In one example, the region of CD27 that serves as the antigen binding domain in the CAR molecule comprises, consists of, or consists essentially of amino acids 1-211 of SEQ ID NO. 4 (including CD27 TMD). In another example, the region of CD27 that serves as the antigen binding domain in the CAR molecule comprises, consists of, or consists essentially of amino acids 1-191 of SEQ ID NO. 4 (lacking CD27 TMD). In a specific example, the region of CD27 used in the CAR molecule comprises SEQ ID NO:4, 1-50, 1-51, 1-52, 1-53, 1-54, 1-55, 1-56, 1-57, 1-58, 1-59, 1-60, 1-61, 1-62, 1-63, 1-64, 1-65, 1-66, 1-67, 1-68, 1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, 1-81, 1-82, 1-83, 1-84, 1-85, 1-86, 1-87, 1-88, 1-89, 1-90, 1-91 1-92, 1-93, 1-94, 1-95, 1-96, 1-97, 1-98, 1-99, 1-100, 1-101, 1-102, 1-103, 1-104, 1-105, 1-106, 1-107, 1-108, 1-109, 1-110, 1-111, 1-112, 1-113, 1-114, 1-115, 1-116, 1-117, 1-118, 1-119, 1-120, 1-121, 1-122, 1-123, 1-124, 1-125, 1-126, 1-127, 1-128, 1-129, 1-130, and, 1-131, 1-132, 1-133, 1-134, 1-135, 1-136, 1-137, 1-138, 1-139, 1-140, 1-141, 1-142, 1-143, 1-144, 1-145, 1-146, 1-147, 1-148, 1-149, 1-150, 1-151, 1-152, 1-153, 1-154, 1-155, 1-156, 1-157, 1-158, 1-159, 1-160, 1-161, 1-162, 1-163, 1-164, 1-165, 1-166, 1-167, 1-168, 1-169, 1-170, 1-171, 1-172, 1-173 1-174, 1-175, 1-176, 1-177, 1-178, 1-179, 1-180, 1-181, 1-182, 1-183, 1-184, 1-185, 1-186, 1-187, 1-188, 1-189, 1-190, 1-191, 1-192, 1-193, 1-194, 1-195, 1-196, 1-197, 1-198, 1-199, 1-200, 1-201, 1-202, 1-203, 1-204, 1-205, 1-206, 1-207, 1-208, 1-209, 1-210, 1-211, 1-212, 1-213, 1-214, 1-215, 1-216, 1-217, 1-218, 1-219, 1-220, 1-221, 1-222, 1-223, 1-224, 1-225, 1-226, 1-227, 1-228, 1-229, 1-230, 1-231, 1-232, 1-233, 1-234, 1-235, 1-236, 1-237, 1-238, 1-239, 1-240, 1-241, 1-242, 1-243, 1-244, 1-245, 1-246, 1-247, 1-248, 1-249, 1-250, 1-251, 1-252, 1-253, 1-254, 1-255, 1-256, 1-57, 1-258, 1-259, or all (1-260) amino acids, consisting of or consisting essentially of them; in particular embodiments, such amino acids within these ranges are contiguous. In some embodiments, a region of SEQ ID NO. 4 having a truncation at the N-terminus is utilized, such as truncating 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids from the N-terminus. In certain instances, truncations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids are present at the N-terminus, and truncations are present at the C-terminus, as described in the list above.

In certain embodiments, any region of the CAR molecule is codon optimized. In some embodiments, the antigen binding domain comprises any instance of the CD27 sequences encompassed herein, but is codon optimized. In certain instances, the antigen binding domain comprises a C-terminally truncated CD27 (e.g., an extracellular domain) and is also codon optimized. In certain aspects, the antigen binding domain comprises, consists of, or consists essentially of amino acids 1-191 of CD27, and is also codon optimized. In other aspects, the antigen binding domain and the transmembrane domain comprise, consist of, or consist essentially of amino acids 1-211 of CD27, and are also codon optimized.

In some cases, specific components of the CAR molecule are utilized. In particular embodiments, the CAR comprises a particular transmembrane domain. Examples include transmembrane domains from CD28 or CD 27. In some embodiments, the CD27 transmembrane domain or CD28 transmembrane domain is not used in the CAR, while in alternative embodiments, the CD27 transmembrane domain is used in the CAR. In particular embodiments, the signal peptide is used in a CAR, examples include the CD27 or GMCSF-R signal peptide, or both may be used. Examples of intracellular signaling domains of a CAR include at least CD27, CD28, DAP10, DAP12, NKG2D, 4-1BB, or a combination thereof.

In some cases, a CD27 signal peptide (MARPHPWWLCVLGTLVGLS (SEQ ID NO: 6); ATGGCACG GCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGG GGCTCTCA (SEQ ID NO: 61)) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:6 or 61 is used in the CAR. In some cases, a GMCSF-R signal peptide (MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 14); ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCA CACCCAGCATTCCTCCTGATCCCA (SEQ ID NO: 62)) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:14 or 62 is used in the CAR.

The CAR may or may not comprise CD27 TM (ILVIFSGMFLVFTLAGALFLH (SEQ ID NO: 7); ATCCTTGT GATCTTCTCTGGAATGTTCCTTGTTTTCACCCTGGCCGGGGC CCTGTTCCTCCAT (SEQ ID NO: 66)), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:7 or 66.

The CAR may or may not comprise an ICD of CD27, which is QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPA CSP (SEQ ID NO: 8), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 8.

In some embodiments, CD3 ζICD is RVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKPRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATKDTYDALHMQA LPPRG (SEQ ID NO: 10), CGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGAC (SEQ ID NO: 67), and the sequences may be used in CARs, or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:10 or 67.

In some embodiments, CD28 ICD (RSKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS; SEQ ID NO: 11) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:11 is used in the CAR molecule.

In some embodiments, 4-1BB ICD (KRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEEGGCEL (SEQ ID NO: 12); AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAA CCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGC TGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGA ACTG (SEQ ID NO: 68)) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:12 or 68 is used in the CAR molecule.

In some embodiments, DAP10 ICD (LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 72); CTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGG CAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID NO: 69)) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:69 or 72 is used in the CAR molecule.

In some embodiments, DAP12 ICD (YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSD LNTQRPYYK (SEQ ID NO: 74); TACTTCCTGGGCCGGCTGGT CCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAA (SEQ ID NO: 71)) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:71 or 74 is used in the CAR molecule.

In some embodiments, NKG2D ICD (SANERCKSKVVPCRQKQWRTSFDSKKLDLNYNHFESMEWSHR SRRGRIWGM (SEQ ID NO: 73); AGCGCGAACGAACGCTGC AAAAGCAAAGTGGTGCCGTGCCGCCAGAAACAGTGGCGCACCAGCTTTGATAGCAAAAAACTGGATCTGAACTATAACCATTTTGAAAGCATGGAATGGAGCCATCGCAGCCGCCGCGGCCGCATTTGGGGCATG (SEQ ID NO: 70)) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:70 or 73 is used in the CAR molecule.

In a particular embodiment, codon optimized CD27 is utilized, one example being as follows:

ATG GCG AGA CCA CAC CCT TGG TGG CTG TGC GTA CTC GGC ACA CTT GTA GGT CTG TCC GCT ACA CCG GCT CCG AAG TCC TGC CCG GAG CGG CAT TAT TGG GCA CAG GGC AAG TTG TGT TGT CAA ATG TGT GAG CCG GGA ACC TTT CTC GTG AAG GAT TGC GAT CAG CAT CGG AAG GCC GCG CAG TGC GAC CCA TGT ATA CCA GGG GTC TCA TTT TCC CCA GAT CAC CAT ACG AGG CCG CAC TGT GAG TCT TGC AGG CAT TGT AAT TCC GGC TTG TTG GTC CGC AAC TGT ACT ATT ACT GCG AAT GCA GAG TGT GCT TGT AGA AAC GGA TGG CAG TGC AGG GAC AAA GAA TGT ACG GAG TGT GAT CCA CTG CCT AAC CCC AGT CTT ACA GCA AGA TCT TCA CAG GCC CTC AGC CCG CAT CCT CAA CCA ACA CAT CTT CCT TAC GTG TCA GAA ATG TTG GAG GCG CGA ACC GCA GGC CAT ATG CAG ACC CTG GCG GAC TTT CGG CAG CTG CCA GCA CGC ACA CTT AGT ACA CAC TGG CCA CCA CAA CGC AGC TTG TGC TCT TCC GAT TTC ATC CGC ATA CTG GTC ATC TTT TCT GGA ATG TTC CTT GTG TTC ACC CTG GCA GGA GCC CTG TTC CTT CAC CAG AGA CGC AAG TAC AGG TCA AAC AAG GGT GAG AGC CCC GTT GAA CCC GCA GAG CCG TGT AGA TAC TCA TGT CCT AGA GAA GAA GAG GGC TCT ACT ATC CCT ATT CAG GAA GAT TAT AGA AAA CCC GAA CCC GCG TGC AGC CCC(SEQ ID NO:13)。

any polynucleotide encompassed by the present disclosure may utilize SEQ ID NO. 13 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO. 13.

In a particular embodiment, a codon-optimized extracellular domain of CD27 is utilized, an example of which is as follows:

GCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGC(SEQ ID NO:64)

any polynucleotide encompassed by the present disclosure may utilize SEQ ID NO. 64 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO. 64.

Figures 2A-2E provide specific examples of CAR constructs comprising specific components. These examples are specific examples wherein the CAR construct may comprise full length CD27 (FL; all 260 amino acids). In the alternative, when full length CD27 is not utilized, the extracellular domain of CD27 (CD 27 EC), which is residues 20-191 of CD27, may be utilized. In particular embodiments, when truncated CD27 is utilized in the CAR, the CD27 intracellular co-stimulatory domain (ICD) may not be used in the CAR and/or the CD27 transmembrane domain (TMD) may not be used in the CAR.

In fig. 2A, example (1) of an anti-CD 70 CAR comprises CD27 Signal Peptide (SP), full length CD27 (FL), CD27 TMD, CD27ICD, and CD3 ζ. Example (2) of fig. 2 includes granulocyte-macrophage colony-stimulating factor receptor (GMSCF-R) signal peptide, codon-optimized (co) full-length CD27, codon-optimized CD27 transmembrane domain, codon-optimized CD27ICD, and cd3ζ. Example (3) of fig. 2A includes CD27SP, full length CD27, CD27 TMD, CD27ICD, CD28 ICD, and CD3 ζ. Example (4) of fig. 2A includes GMSCF-R SP, codon optimized full length CD27 TMD, codon optimized CD27ICD, CD28 ICD and CD3 ζ. Example (4) of FIG. 2A includes CD27SP, full length CD27, CD27 TMD, CD27ICD, 4-1BB ICD, and CD3 ζ. Example (6) includes GMSCF-R SP, codon optimized full length CD27, codon optimized CD27 TMD, codon optimized CD27ICD, 4-1BB ICD and CD3 ζ.

Examples (7) in fig. 2B include CD27SP, full length CD27, CD27 TMD, CD27ICD, DAP10 ICD, and cd3ζ. Example (8) of FIG. 2B includes GMSCF-R SP, codon optimized full length CD27, codon optimized CD27 TMD, codon optimized CD27ICD, DAP10 ICD, and CD3 ζ. Example (9) in fig. 2B includes CD27SP, full length CD27, CD27 TMD, CD27ICD, DAP12 ICD, and cd3ζ. Example (10) of fig. 2B includes GMSCF-R SP, codon optimized full length CD27, codon optimized CD27 TMD, codon optimized CD27ICD, DAP12 ICD, and CD3 ζ. Example (11) of fig. 2B includes CD27SP, full length CD27, CD27 TMD, CD27ICD, NKG2D ICD, and cd3ζ. Examples (12) of FIG. 2B include GMSCF-R SP, codon optimized full length CD27, codon optimized CD27 TMD, codon optimized CD27ICD, NKG2D ICD and CD3 ζ.

Example (13) in fig. 2C includes CD27SP, CD27 extracellular domain (EC), CD27TMD, and CD3 ζ. Examples (14) in FIG. 2C include GMSCF-R SP, codon optimized CD27 EC, codon optimized TMD and CD27. Examples (15) in fig. 2C include CD27SP, CD27 EC, CD28TMD, and CD3 ζ. Examples (16) in FIG. 2C include GMSCF-R SP, codon optimized CD27 EC, codon optimized CD28TMD, and CD3 ζ. Examples (17) in fig. 2C include CD27SP, CD27 EC, CD27TMD, CD28ICD, and CD3 ζ. Example (18) of FIG. 2C includes GMSCF-R SP, codon optimized CD27 EC, codon optimized CD27TMD, CD28ICD and CD3 ζ. Examples (19) of fig. 2C include CD27SP, CD27 EC, CD28TMD, CD28ICD, and cd3ζ. Examples (20) of FIG. 2C include GMSCF-R SP, codon optimized CD27 EC, CD28TMD, CD28ICD and CD3 ζ.

Example (21) of FIG. 2D includes CD27SP, CD27 EC, CD27TMD, 4-1BB ICD, and CD3 ζ. Example (22) of FIG. 2D includes GMSCF-R SP, codon optimized CD27 EC, codon optimized CD27TMD, 4-1BB ICD, and CD3 ζ. Examples (23) of FIG. 2D include CD27SP, CD27 EC, CD28TMD, 4-1BB ICD, and CD3 ζ. Examples (24) of FIG. 2D include GMSCF-R SP, codon optimized CD27 EC, CD28TMD, 4-1BB ICD, and CD3 ζ. Examples (25) of fig. 2D include CD27SP, CD27 EC, CD27TMD, DAP10ICD, and cd3ζ. Examples (26) of FIG. 2D include GMSCF-R SP, codon optimized CD27 EC, codon optimized CD27TMD, DAP10ICD and CD3 ζ. Example (27) of fig. 2D includes CD27SP, CD27 EC, CD28 co-stimulatory domain, DAP10ICD, and cd3ζ. Examples (28) of FIG. 2D include GMSCF-R SP, codon optimized CD27 EC, CD28TMD, DAP10ICD, and CD3 ζ.

Examples (29) of fig. 2E include CD27SP, CD27EC, CD27 TMD, DAP12ICD, and cd3ζ. Examples (30) of FIG. 2E include GMSCF-R SP, codon optimized CD27EC, codon optimized CD27 TMD, DAP12ICD and CD3 ζ. Examples (31) of FIG. 2E include CD27SP, CD27EC, CD28 TMD, DAP12ICD, and CD3 ζ. Examples (32) of FIG. 2E include GMSCF-R SP, codon optimized CD27EC, CD28 TMD, DAP12ICD, and CD3 ζ. Examples (33) of fig. 2E include CD27SP, CD27EC, CD27 TMD, NKG2D ICD, and CD3 ζ. Examples (34) of FIG. 2E include GMSCF-R SP, codon optimized CD27EC, CD28 TMD, NKG2D ICD and CD3 ζ. Examples (35) of fig. 2E include CD27SP, CD27EC, CD28 TMD, NKG2D ICD, and CD3 ζ. Examples (36) of FIG. 2E include GMSCF-R SP, codon optimized CD27EC, CD28 TMD, NKG2D ICD and CD3 ζ.

One example of an anti-CD 70 CAR of the present disclosure comprises SEQ ID No. 1 comprising a CD27 extracellular domain (not underlined in SEQ ID No. 1 below) operably linked to a CD28 transmembrane domain (underlined in SEQ ID No. 1 below) and a CD27 signal peptide (bold in SEQ ID No. 1 below):

MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWV(SEQ ID NO:1)

the CD27 extracellular domain of SEQ ID NO. 1 comprises SEQ ID NO. 2 as follows:

MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR。

The CD28 transmembrane domain of SEQ ID NO. 1 comprises SEQ ID NO. 3 as follows:

FWVLVVVGGVLACYSLLVTVAFIIFWV(TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG(SEQ ID NO:65))。

the CD27 signal peptide of SEQ ID NO. 1 comprises SEQ ID NO. 6 as follows:

MARPHPWWLCVLGTLVGLS(ATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCA(SEQ IDNO:61))。

the present disclosure provides cells (including immune cells, such as NK cells) containing a vector encoding at least one CAR, and the CAR may be, for example, first generation, second generation, or third generation or subsequent generation. The CAR may or may not have dual specificity for two or more different antigens, one of which is CD70. In particular embodiments, the CAR is not a bivalent tandem CAR. The CAR may comprise one or more co-stimulatory domains. For example, each co-stimulatory domain may comprise a co-stimulatory domain, e.g., any one or more of the following: TNFR superfamily members, CD28, CD137 (4-1 BB), CD134 (OX 40), DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1 (CD 11a/CD 18), lck, TNFR-I, TNFR-II, fas, CD, CD40, any ITAM-containing signaling domain, or a combination thereof. In particular embodiments, the CAR comprises cd3ζ. In certain embodiments, the CAR lacks one or more specific co-stimulatory domains; for example, the CAR may lack 4-1BB and/or lack CD28.

In particular embodiments, the CAR polypeptide in the cell comprises an extracellular spacer domain that links the antigen binding domain and the transmembrane domain. The extracellular spacer domain may include, but is not limited to, an Fc fragment of an antibody or fragment or derivative thereof, a hinge region of an antibody or fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include, but are not limited to, CD 8-alpha hinge, CD28, artificial spacer made of polypeptides such as Gly3, or CH1 domain, CH3 domain of IgG (such as human IgG1 or IgG 4). In particular cases, the extracellular spacer domain can comprise (i) a hinge region of IgG4, a CH2 region, and a CH3 region, (ii) a hinge region of IgG4, (iii) a hinge region of IgG4 and a CH2 region, (iv) a hinge region of Cd8- α, (v) a hinge region of IgG1, a CH2 region, and a CH3 region, (vi) a hinge region of IgG1, or (vii) a hinge region of IgG1 and a CH2 region, or (viii) a hinge region of Cd28, or a combination thereof.

In particular embodiments, the hinge is from IgG1, and in certain aspects, the CAR polypeptide comprises or is encoded by a particular IgG1 hinge amino acid sequence.

In particular embodiments, the hinge is from CD28, and in certain aspects, the CAR polypeptide comprises or is encoded by a particular CD28 hinge amino acid sequence.

In some embodiments, the transmembrane domain is derived from a natural or synthetic source. When the source is natural, in some aspects, the domain is derived from any membrane-bound protein or transmembrane protein. The transmembrane regions include those derived from (i.e., comprising at least one or more of the following transmembrane regions) the alpha, beta or zeta chain, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS/CD278, GITR/CD357, NKG2D, and DAP molecules (such as DAP10 or DAP 12) of a T cell receptor. Alternatively, in some embodiments, the transmembrane region is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues, such as leucine and valine. In some aspects, it was found that triplets of phenylalanine, tryptophan, and valine can be present at each end of the synthetic transmembrane domain.

In certain embodiments, the CD 70-specific CAR may be co-expressed with one or more cytokines to improve persistence in the presence of a small amount of tumor-associated antigen. For example, the CAR can be co-expressed with one or more cytokines (such as IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, IL-7, GMCSF, or a combination thereof).

In specific examples, the anti-CD 70 CAR can have the following nucleotide sequence:

CD27tr28tdmCD3zIL15：

ATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG(SEQ ID NO:15)

the corresponding amino acid sequence of CD27tr28tdmCD3zIL is as follows:

MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWV(SEQ ID NO:38)

CD27Tr28tmd41BBicd3zIL15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:16)

the corresponding amino acid sequence of CD27Tr28tmd41BBicd3zIL is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:39)

GSPco27Tr28tmd41BBicCD3zIL15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:17)

the corresponding amino acid sequence of GSPco27Tr28tmd41 bbicd3zIL is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:40)

CD27Tr28tmdDAP10icd3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGCTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:18)

the corresponding amino acid sequence of CD27Tr28 tmdap 10icd3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVLCARPRRSPAQEDGKVYINMPGRGKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:41)

GSPco27Tr28tmdDAP10IL15:

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGCTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:19)

the corresponding amino acid sequence of GSPco27Tr28 tmdap 10IL15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVLCARPRRSPAQEDGKVYINMPGRGKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:42)

CD27Tr28tmdDAP12icd3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAAAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:20)

the corresponding amino acid sequence of CD27Tr28 tmdap 12icd3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:43)

GSPco27Tr28tmddap12icd15:

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAAAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:21)

the corresponding amino acid sequence of GSPco27Tr28tmddap12icd15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:44)

CD27Tr28tmdNKG2Dic3z15:

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGCGCGAACGAACGCTGCAAAAGCAAAGTGGTGCCGTGCCGCCAGAAACAGTGGCGCACCAGCTTTGATAGCAAAAAACTGGATCTGAACTATAACCATTTTGAAAGCATGGAATGGAGCCATCGCAGCCGCCGCGGCCGCATTTGGGGCATGAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:22)

the corresponding amino acid sequence of CD27Tr28tmdNKG2Dic3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVSANERCKSKVVPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:45)

GSPco27Tr28tmdNKG2Dicd3z15：ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGCGCGAACGAACGCTGCAAAAGCAAAGTGGTGCCGTGCCGCCAGAAACAGTGGCGCACCAGCTTTGATAGCAAAAAACTGGATCTGAACTATAACCATTTTGAAAGCATGGAATGGAGCCATCGCAGCCGCCGCGGCCGCATTTGGGGCATGAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:23)

the corresponding amino acid sequence of GSPco27Tr28tmdNKG2 dcd 3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVSANERCKSKVVPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:46)

CD27Tr41BBicd3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCATCCTTGTGATCTTCTCTGGAATGTTCCTTGTTTTCACCCTGGCCGGGGCCCTGTTCCTCCATAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:24)

the corresponding amino acid sequence of CD27Tr41BBicd3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:47)

GSPco27Tr41BBicd3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGCATACTGGTCATCTTTTCTGGAATGTTCCTTGTGTTCACCCTGGCAGGAGCCCTGTTCCTTCACAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:25)

the corresponding amino acid sequence of GSPco27Tr41BBicd3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:48)

CD27TrCD3ZIL15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCATCCTTGTGATCTTCTCTGGAATGTTCCTTGTTTTCACCCTGGCCGGGGCCCTGTTCCTCCATAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:26)

the corresponding amino acid sequence of CD27TrCD3ZIL is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:49)

GSPco27Tr3z15:

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGCATACTGGTCATCTTTTCTGGAATGTTCCTTGTGTTCACCCTGGCAGGAGCCCTGTTCCTTCACCGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:27)

the corresponding amino acid sequence of GSPco27Tr3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:50)

CD27TrCD28icd3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCATCCTTGTGATCTTCTCTGGAATGTTCCTTGTTTTCACCCTGGCCGGGGCCCTGTTCCTCCATAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:28)

the corresponding amino acid sequence of CD27TrCD28icd3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:51)

GSPco27Tr28CD3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGCATACTGGTCATCTTTTCTGGAATGTTCCTTGTGTTCACCCTGGCAGGAGCCCTGTTCCTTCACAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:29)

the corresponding amino acid sequence of GSPco27Tr28CD3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:52)

CD27TrCD28tmdicd3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:30)

the corresponding amino acid sequence of CD27TrCD28 tmdcd 3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:53)

GSPco27Tr28tmdicCD3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:31)

the corresponding amino acid sequence of GSPco27Tr28 tmdccd 3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI SLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:54)

CD27TrDAP10icd3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCATACTGGTCATCTTTTCTGGAATGTTCCTTGTGTTCACCCTGGCAGGAGCCCTGTTCCTTCACCTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:32)

the corresponding amino acid sequence of CD27TrDAP10icd3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHLCARPRRSPAQEDGKVYINMPGRGKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:55)

GSPco27FLdap10icd3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGCATACTGGTCATCTTTTCTGGAATGTTCCTTGTGTTCACCCTGGCAGGAGCCCTGTTCCTTCACCTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:33)

The corresponding amino acid sequence of GSPco27FLdap10icd3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHLCARPRRSPAQEDGKVYINMPGRGKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:56)

CD27TrDAP12icd3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCATCCTTGTGATCTTCTCTGGAATGTTCCTTGTTTTCACCCTGGCCGGGGCCCTGTTCCTCCATTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAAAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:34)

the corresponding amino acid sequence of CD27TrDAP12icd3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:57)

GSPco27FLdap12icd3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGCATACTGGTCATCTTTTCTGGAATGTTCCTTGTGTTCACCCTGGCAGGAGCCCTGTTCCTTCACTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAAAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:35)

the corresponding amino acid sequence of GSPco27FLdap12icd3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:58)

CD27TrNKG2Dic3z15:

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCATCCTTGTGATCTTCTCTGGAATGTTCCTTGTTTTCACCCTGGCCGGGGCCCTGTTCCTCCATAGCGCGAACGAACGCTGCAAAAGCAAAGTGGTGCCGTGCCGCCAGAAACAGTGGCGCACCAGCTTTGATAGCAAAAAACTGGATCTGAACTATAACCATTTTGAAAGCATGGAATGGAGCCATCGCAGCCGCCGCGGCCGCATTTGGGGCATGAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:36)

the corresponding amino acid sequence of CD27TrNKG2Dic3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHSANERCKSKVVPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:59)

GSPco27TrNKG2Dicd3z15：

ATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATGCTCGAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGCATACTGGTCATCTTTTCTGGAATGTTCCTTGTGTTCACCCTGGCAGGAGCCCTGTTCCTTCACAGCGCGAACGAACGCTGCAAAAGCAAAGTGGTGCCGTGCCGCCAGAAACAGTGGCGCACCAGCTTTGATAGCAAAAAACTGGATCTGAACTATAACCATTTTGAAAGCATGGAATGGAGCCATCGCAGCCGCCGCGGCCGCATTTGGGGCATGAAACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:37)

the corresponding amino acid sequence of GSPco27TrNKG2 dcd 3z15 is as follows:

MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSS DFIRILVIFSGMFLVFTLAGALFLHSANERCKSKVVPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:60)

the sequence encoding the open reading frame of the chimeric receptor may be obtained from a genomic DNA source, a cDNA source, or may be synthesized (e.g., by PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, as introns are found to stabilize mRNA. Furthermore, it may be more advantageous to stabilize mRNA using endogenous or exogenous non-coding regions.

It is contemplated that the chimeric construct may be introduced into any type of immune cell as naked DNA or in a suitable vector. Methods for stably transfecting cells by electroporation using naked DNA are known in the art. See, for example, U.S. patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor contained in a plasmid expression vector in the appropriate direction of expression.

Alternatively, the chimeric construct may be introduced into the immune cell using a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector). Suitable vectors for use in accordance with the methods of the present disclosure are non-replicating in immune cells. A large number of viral-based vectors (e.g., HIV, SV40, EBV, HSV or BPV-based vectors) are known, wherein the number of copies of the virus maintained in the cell is low enough to maintain viability of the cell. Non-viral vectors include plasmids, transposons, nanoparticles, liposomes, lipids, metals, or combinations thereof.

II. cytokines

In some embodiments, the cell expressing the anti-CD 70 CAR is engineered to express one or more heterologous cytokines and/or is engineered to up-regulate normal expression of one or more heterologous cytokines. One or more cytokines may or may not be transduced or transfected on the same vector as other genes.

One or more cytokines may be co-expressed from the vector as a polypeptide separate from the antigen receptor. For example, interleukin-15 (IL-15) is tissue limiting and only in pathological conditions can any level of interleukin be observed in serum or systemically. IL-15 has several attributes required for adoptive therapy. IL-15 is a homeostatic cytokine that induces natural killer cell development and cell proliferation, promotes eradication of established tumors by alleviating functional inhibition of tumor resident cells, and inhibits activation-induced cell death (AICD). In addition to IL-15, other cytokines are also contemplated. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells for human applications. NK cells expressing IL-15 are able to sustain cytokine signaling, which is useful for their survival after infusion.

In particular embodiments, the cells express one or more exogenously supplied cytokines. As one example, the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, GMCSF, or a combination thereof. As another example, the cytokine is IL-21. As another example, the cytokine is IL-12. Cytokines can be provided exogenously to NK cells because they are expressed by intracellular expression vectors. In another case, the endogenous cytokine in the cell is up-regulated after manipulation of expression regulation of the endogenous cytokine (e.g., genetic recombination at one or more promoter sites of the cytokine). In the case where the cytokine is provided to the cell on an expression construct, the cytokine may be encoded by the same vector along with the suicide gene and/or the anti-CD 70 CAR. In some embodiments, the disclosure relates to the co-utilization of CAR with IL-15. In some embodiments, the disclosure relates to the co-utilization of CAR with IL-21. In some embodiments, the disclosure relates to the co-utilization of a CAR with IL-12.

III suicide Gene

In certain embodiments, suicide genes are used in combination with anti-CD 70 cell therapies to control their use and allow termination of cell therapies at a desired event and/or time. Suicide genes are used in transduced cells in order to trigger the death of the transduced cells when needed. The cells of the present disclosure that have been modified to carry the vectors encompassed by the present disclosure may comprise one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that upon administration of a prodrug or other agent effects a conversion of the gene product to a compound that kills its host cell. In other embodiments, the suicide gene encodes a gene product that is targeted, when desired, by an agent (such as an antibody) that targets the suicide gene product.

In some cases, a cell therapy may require the use of one or more suicide genes of any type when an individual receiving the cell therapy and/or having received the cell therapy exhibits one or more symptoms of one or more adverse events, such as cytokine release syndrome, neurotoxicity, allergic response/anaphylaxis, and/or mid-target/tumor-free toxicity (on-target/off tumor toxicitiy), as examples, or is considered to be at risk of having one or more symptoms, including impending symptoms. The use of suicide genes may be part of the planned treatment regimen or only used when confirmation of need for use is desired. In some cases, cell therapy is terminated by the use of one or more agents that target suicide genes or gene products therefrom, as treatment is no longer required.

The use of suicide genes may be elicited when an individual experiences at least one adverse event, and the adverse event may be identified by any means, including upon routine monitoring, which may be continuous or discontinuous from the beginning of cell therapy. One or more adverse events may be detected during inspection and/or testing. In the case of an individual suffering from a cytokine release syndrome (which is also referred to as a cytokine storm), the individual may, for example, have elevated one or more inflammatory cytokines (by way of example only: interferon-gamma, granulocyte macrophage colony stimulating factor, IL-10, IL-6 and TNF-alpha), fever, fatigue, hypotension, hypoxia, tachycardia, nausea, capillary leakage, heart/kidney/liver dysfunction or a combination thereof. In the case of an individual having neurotoxicity, the individual may have confusion, delirium, hypoplasia and/or seizures. In some cases, the individual is tested for markers associated with the onset and/or severity of cytokine release syndrome, such as C-reactive protein, IL-6, TNF- α, and/or ferritin.

Examples of suicide genes include engineered non-secretable (including membrane-bound) Tumor Necrosis Factor (TNF) -alpha mutant polypeptides (see PCT/US19/62009, which is incorporated herein by reference in its entirety), and they may be affected by the delivery of antibodies that bind TNF-alpha mutants. Examples of suicide gene/prodrug combinations that can be used are herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir, acyclovir or FIAU; oxidoreductases and cycloheximides; cytosine deaminase and 5-fluorocytosine; thymidine kinase, thymidine kinase (Tdk: tmk), and AZT; deoxycytidine kinase and cytarabine. An E.coli purine nucleoside phosphorylase, a so-called suicide gene which converts the prodrug 6-methylpurine deoxyribose glycoside to the toxic purine 6-methylpurine, can be used. As examples, other suicide genes include CD20, CD52, inducible caspase 9, purine Nucleoside Phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase, methionine- α, γ -lyase (MET), and Thymidine Phosphorylase (TP).

In certain embodiments, the vector encoding a CD 70-targeted CAR, or any vector in an NK cell encompassed herein, comprises one or more suicide genes. The suicide gene may or may not be on the same vector as the CD 70-targeted CAR. Where the suicide gene is present on the same vector as the CD 70-targeted CAR, the suicide gene and CAR may be separated by, for example, an IRES or 2A element.

IV pharmaceutical composition

The pharmaceutical compositions of the present disclosure comprise an effective amount of cells expressing an anti-CD 70CAR dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when properly administered to an animal (e.g., a human). The preparation of pharmaceutical compositions comprising cells expressing an anti-CD 70CAR is known to those of skill in the art in light of the present disclosure, as exemplified by Remington, the Science and Practice of Pharmacy, 21 st edition, lippincott Williams and Wilkins,2005 (incorporated herein by reference). Furthermore, for animal (e.g., human) administration, it should be understood that the formulation should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA office of biological standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof known to those of ordinary skill in the art (see, e.g., remington's Pharmaceutical Sciences, 18 th edition, mack Printing Company,1990, pages 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated.

The pharmaceutical composition may comprise different types of carriers, depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for the route of administration, such as injection. The compositions disclosed herein may be administered intravenously, intradermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, transmucosally, orally, topically, locally, inhaled (e.g., aerosol inhaled), injected, infused, continuously infused, locally infused, directly infused into target cells, via catheter, via lavage, in a cream, in a lipid composition (e.g., liposomes), or by other methods known to one of ordinary skill in the art or any combination of the foregoing (see, e.g., remington's Pharmaceutical Sciences, 18 th edition, mack Printing Company,1990, incorporated herein by reference).

Cells expressing the anti-CD 70 CAR can be formulated into compositions in free alkaline, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, for example salts with the free amino groups of the proteinaceous composition, or salts with inorganic acids, for example hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric or mandelic acid. Salts with free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or an organic base such as isopropylamine, trimethylamine, histidine or procaine. After formulation, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are readily administered in a variety of dosage forms, such as formulated for parenteral administration, such as injection solutions, or aerosols for delivery to the lungs, or formulated for digestive tract administration, such as drug release capsules and the like.

In addition, in accordance with the present disclosure, the compositions of the present disclosure suitable for administration are provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be absorbable, including liquid, semi-solid (i.e., paste) or solid carriers. Except insofar as any conventional medium, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effect of the composition contained therein, its use in an administrable composition for practicing the methods of the invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. The composition may also contain various antioxidants to retard oxidation of one or more components. In addition, the action of microorganisms may be prevented by preservatives such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methyl parahydroxybenzoate, propyl parahydroxybenzoate), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

In accordance with the present disclosure, the compositions are combined with the carrier in any convenient and practical manner (i.e., by solution, suspension, emulsification, mixing, encapsulation, absorption, etc.). Such procedures are routine to those skilled in the art.

In a specific embodiment of the present disclosure, the composition is fully combined or mixed with a semi-solid or solid carrier. The mixing may be performed in any convenient manner such as milling. Stabilizers may also be added during mixing to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as glucose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, and the like.

In other embodiments, the disclosure can relate to the use of a pharmaceutical lipid vehicle composition comprising cells expressing an anti-CD 70 CAR and optionally an aqueous solvent. As used herein, the term "lipid" will be defined to include any of a variety of substances that are characteristically insoluble in water and extractable with organic solvents. This general class of compounds is well known to those skilled in the art, and the term "lipid" as used herein is not limited to any particular structure. Examples include compounds containing long chain aliphatic hydrocarbons and derivatives thereof. Lipids may be naturally occurring or synthetic (i.e., designed or produced by humans). However, lipids are typically biological substances. Biological lipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids and polymerizable lipids having ether and ester linked fatty acids, and combinations thereof. Of course, the compositions and methods of the present invention also encompass compounds understood by those of skill in the art as lipids in addition to the compounds specifically described herein.

Those of ordinary skill in the art will be familiar with the technical scope that may be used to disperse the composition in a lipid vehicle. For example, cells expressing the anti-CD 70 CAR may be dispersed in a solution containing the lipid, solubilized with the lipid, emulsified with the lipid, mixed with the lipid, combined with the lipid, covalently bound to the lipid, contained as a suspension in the lipid, contained in or complexed with the micelle or liposome, or otherwise associated with the lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

The actual dosage of the compositions of the present disclosure to be administered to an animal patient may be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, idiopathic disease (idiopath) of the patient, and route of administration. Depending on the dose and route of administration, the preferred number of doses and/or effective amounts administered may vary depending on the subject's response. In any event, the physician in charge of administration will determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) of the individual subject.

In certain embodiments, the pharmaceutical composition may comprise, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may, for example, comprise from about 2% to about 75%, or from about 25% to about 60%, by weight, and any range derivable therein. Naturally, the amount of one or more active compounds in each therapeutically useful composition can be prepared such that the appropriate dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be considered by those skilled in the art of preparing such pharmaceutical formulations and, therefore, may require multiple dosages and treatment regimens.

In other non-limiting examples, the dosage may also include about 1 μg/kg/body weight, about 5 μg/kg/body weight, about 10 μg/kg/body weight, about 50 μg/kg/body weight, about 100 μg/kg/body weight, about 200 μg/kg/body weight, about 350 μg/kg/body weight, about 500 μg/kg/body weight, about 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 50 mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about 350 mg/kg/body weight, about 500 mg/kg/body weight, or about 1000 mg/kg/body weight or more per administration, as well as any range derivable therein. In non-limiting examples of ranges derivable from the values set forth herein, ranges from about 5 mg/kg/body weight to about 100 mg/kg/body weight, from about 5 mg/kg/body weight to about 500 mg/kg/body weight, etc., can be administered according to the values recited above.

A. Nutritional compositions and formulations

In a particular embodiment of the invention, the cells expressing anti-CD 70 CARs are formulated for administration by the gut route. The gut route includes all possible routes of administration wherein the composition is in direct contact with the gut. In particular, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. Thus, these compositions may be formulated with inert diluents or absorbable carriers, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be mixed directly with the meal.

In certain embodiments, the active compounds may be admixed with excipients and used in the form of ingestible tablets, troches, lozenges, capsules, elixirs, suspensions, syrups, wafers (wafer) and the like (Mathiowitz et al, 1997; hwang et al, 1998; U.S. Pat. Nos. 5,641,515, 5,580,579 and 5,792,451, each of which is specifically incorporated herein by reference in its entirety). Tablets, troches, pills, capsules and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch, gelatin or a combination thereof; excipients, for example dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; disintegrants, such as corn starch, potato starch, alginic acid or a combination thereof; lubricants, such as magnesium stearate; sweeteners such as sucrose, lactose, saccharin or combinations thereof; flavoring agents, such as peppermint, oil of wintergreen, cherry flavoring, orange flavoring, and the like. When the dosage unit form is a capsule, it may contain, in addition to materials of the type described above, a liquid carrier. Various other substances may be present as coatings or otherwise alter the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar or both. When the dosage form is a capsule, it may contain, in addition to materials of the type described above, a carrier, such as a liquid carrier. Gelatin capsules, tablets or pills may be coated with an enteric coating. The enteric coating prevents the composition from denaturing in the stomach or upper intestine where the pH is acidic. See, for example, U.S. patent No. 5,629,001. When the small intestine is reached, the alkaline pH therein dissolves the coating and allows the composition to be released and absorbed by specialized cells, such as epithelial intestinal cells and Peyer's patch M cells. Syrups for elixirs may contain the active compounds sucrose (as a sweetening agent), methyl and propylparabens (as preservatives), a dye and a flavoring such as cherry or orange flavor. Of course, any material used to prepare any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, the active compounds can be incorporated into sustained release preparations and formulations.

For oral administration, the compositions of the present disclosure may optionally be admixed with one or more excipients in the form of a mouthwash, dentifrice, lozenge, oral spray or sublingual oral administration formulation. For example, mouthwashes can be prepared by incorporating the desired amount of the active ingredient in a suitable solvent such as a sodium borate solution (dobell solution). Alternatively, the active ingredient may be incorporated into an oral solution (such as a solution containing sodium borate, glycerol, and potassium bicarbonate), or dispersed in a dentifrice, or added in a therapeutically effective amount to a composition which may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively, the composition may be molded into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.

Other formulations suitable for other modes of gut administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually containing a drug for insertion into the rectum. After insertion, the suppository softens, melts or dissolves in the luminal fluid. In general, for suppositories, conventional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing the active ingredient, for example, in the range of about 0.5% to about 10%, preferably about 1% to about 2%.

B. Parenteral compositions and formulations

In other embodiments, the compositions may be administered by parenteral route. As used herein, the term "parenteral" includes routes that bypass the digestive tract. In particular, the pharmaceutical compositions disclosed herein may be administered, for example, but not limited to, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneously, or intraperitoneally. U.S. Pat. nos. 6,613,308, 5,466,468, 5,543,158, 5,641,515, and 5,399,363 (each of which is specifically incorporated herein by reference in its entirety).

Solutions of the active compound as the free base or pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and oils. Under normal conditions of storage and use, these formulations contain preservatives to prevent microbial growth. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, this form must be sterile and must be fluid to the extent that easy injection exists. It must be stable under the conditions of production and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (i.e., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like) can be used to prevent the action of microorganisms. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents which delay absorption, for example, aluminum monostearate and gelatin.

For example, for parenteral administration in aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those of skill in the art in light of the present disclosure. For example, a dose may be dissolved in isotonic NaCl solution, added to a subcutaneous infusion or injected at the proposed infusion site (see, e.g., remington's Pharmaceutical Sciences, "15 th edition, pages 1035-1038 and 1570-1580). The dosage will necessarily vary somewhat depending on the patient being treated. In any event, the person responsible for administration will determine the appropriate dosage for the individual subject. Furthermore, for human administration, the formulation should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA office of biological standards.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powdered composition is combined with a liquid carrier, such as water or a saline solution, with or without the presence of a stabilizing agent.

C. Various pharmaceutical compositions and formulations

In other specific embodiments of the present disclosure, the active compound cells expressing the anti-CD 70 CAR can be formulated for administration by a variety of different routes, e.g., topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.), and/or inhalation.

Pharmaceutical compositions for topical administration may include active compounds formulated for pharmaceutical applications such as ointments, pastes, creams or powders. Ointments include all oily, adsorptive, emulsion and water-soluble base compositions for topical application, with creams and lotions being those compositions containing only an emulsion base. Topically applied drugs may contain penetration enhancers to facilitate absorption of the active ingredient through the skin. Suitable permeation enhancers include glycerol, alcohols, alkyl methyl sulfoxides, pyrrolidones and laurocapram (laurocapram). Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum, and any other suitable absorption, emulsion or water-soluble ointment base. Topical formulations may also include emulsifying agents, gelling agents, and antimicrobial preservatives, as are necessary to preserve the active ingredients and provide a homogeneous mixture. Transdermal administration of the present invention may also include the use of "patches". For example, the patch may provide one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.

In certain embodiments, the pharmaceutical composition may be delivered by eye drops, intranasal sprays, inhalants, and/or other aerosol delivery vehicles. Methods of delivering compositions directly to the lungs by nasal aerosol spraying have been described, for example, in U.S. Pat. nos. 5,756,353 and 5,804,212 (each of which is specifically incorporated herein by reference in its entirety). Also, the use of intranasal microparticle resins (Takenaga et al, 1998) and lysophosphatidylglycerol compounds (U.S. patent No. 5,725,871, specifically incorporated herein by reference in its entirety) for drug delivery is also well known in the pharmaceutical arts. Also, transmucosal drug delivery in the form of polytetrafluoroethylene support matrices is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid or liquid particles dispersed in a liquefied or pressurized gas propellant. A typical aerosol formulation for inhalation of the present invention consists of a suspension of the active ingredient in a liquid propellant or a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary depending on the pressure requirements of the propellant. The administration of the aerosol will vary depending on the age, weight and severity of the symptoms and response of the subject.

V. combination therapy

In certain embodiments, the compositions and methods of embodiments of the invention relate to cancer therapies other than compositions comprising cells expressing an anti-CD 70 CAR. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy (nanotherapy), monoclonal antibody therapy, hormonal therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

In some embodiments, the additional therapy is administration of one or more small molecule enzyme inhibitors or one or more anti-metastatic agents. In some embodiments, the additional therapy is administration of side-effect limiting agents (e.g., agents intended to reduce the occurrence and/or severity of therapeutic side-effects, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma radiation. In some embodiments, the additional therapy is a therapy targeting the PBK/AKT/mTOR pathway, HSP90 inhibitors, tubulin inhibitors, apoptosis inhibitors, and/or one or more chemopreventive agents. The additional therapy may be one or more chemotherapeutic agents known in the art.

Immune cell therapies (in addition to the cell therapies of the present disclosure) may be administered before, during, after, or in various combinations relative to additional cancer therapies (such as immune checkpoint therapies). The interval between administrations may range from simultaneous to several minutes to days to weeks. In embodiments where immune cell therapy is provided to a patient separately from one or more compositions of the present disclosure, it is generally ensured that a significant period of time is not exceeded between each delivery, so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it is contemplated that the immunotherapy and disclosed compositions may be provided to the patient within about 12h to 24h or 72h of each other, more particularly within about 6-12h of each other. In some cases, it may be desirable to significantly extend the treatment time, with intervals between days (2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) to weeks (1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks) between each administration.

Administration of any compound or cell therapy of embodiments of the invention to a patient will follow the general regimen of administration of such compounds while taking into account the toxicity of the agent, if any. Thus, in some embodiments, there is a step of monitoring toxicity due to the combination therapy.

As used herein, the term "cancer" may be used to describe a solid tumor, a metastatic cancer, or a non-metastatic cancer. In certain embodiments, the cancer may originate from the bladder, blood, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testes, tongue, or uterus. In some embodiments, the cancer is a recurrent cancer. In some embodiments, the cancer is stage I cancer. In some embodiments, the cancer is stage II cancer. In some embodiments, the cancer is a stage III cancer. In some embodiments, the cancer is stage IV cancer.

The cancers may be specifically, but not limited to, the following histological types: neoplasms, malignancy; cancer; cancer, undifferentiated; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphatic epithelial cancer; basal cell carcinoma; hair matrix cancer; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinomas; gastrinomas, malignant; bile duct cancer; hepatocellular carcinoma; combining cholangiocarcinoma with hepatocellular carcinoma; small Liang Xianai; adenoid cystic carcinoma; adenocarcinomas among adenomatous polyps; adenocarcinomas, familial polyposis coli; solid cancer; carcinoid tumor, malignant; branchia acinar carcinoma; papillary adenocarcinoma; chromophobe cell cancer; eosinophilic cancer; eosinophilic adenocarcinoma; basophilic cancer; clear cell adenocarcinoma; granulocyte cancer; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerotic cancer; adrenal cortex cancer; endometrial-like cancer; skin accessory cancer; apocrine adenocarcinoma; sebaceous gland carcinoma; cerumen adenocarcinoma (ceruminous adenocarcinoma); epidermoid carcinoma of mucous; cystic adenocarcinoma; papillary cyst adenocarcinoma; papillary serous cystic adenocarcinoma; bursa adenocarcinoma; mucinous adenocarcinoma; printing ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory cancer; paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignancy; follicular membrane cytoma, malignant; granulocytoma, malignant; a male blastoma, malignancy; support cell carcinoma; stromal cell tumor, malignant; lipid cell neoplasms, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; hemangiosarcoma; malignant melanoma; non-pigmented melanoma; superficial diffuse melanoma; malignant melanoma in giant pigmented nevi; epithelioid cell melanoma; blue nevi, malignant; sarcoma; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryo-type rhabdomyosarcoma; acinar rhabdomyosarcoma; interstitial sarcoma; mixed tumor, malignant; miao Leguan mixed tumor; nephroblastoma; hepatoblastoma; carcinoma sarcoma; a mesenchymal neoplasm, malignancy; boehringer's tumor, malignant; she Zhuangliu, malignant; synovial sarcoma; mesothelioma, malignant; a vegetative cell tumor; embryonal carcinoma; teratoma, malignant; ovarian goiter, malignancy; choriocarcinoma; mesonephroma, malignancy; hemangiosarcoma; vascular endothelial tumor, malignant; kaposi's sarcoma; vascular epidermocytoma, malignant; lymphangiosarcoma; osteosarcoma; a cortical bone sarcoma; chondrosarcoma; chondroblastoma, malignant; interstitial chondrosarcoma; bone giant cell tumor; ewing's sarcoma; odontogenic tumors, malignancy; ameloblastic osteosarcoma; enameloblastoma, malignant; ameloblastic fibrosarcoma; pineal tumor, malignancy; chordoma; glioma, malignant; ventricular tube membranoma; astrocytoma; plasmatic astrocytomas; fibroastrocytoma; astrocytoma; glioblastoma; oligodendrogliomas; oligodendroglioma; primitive neuroectoderm; cerebellar sarcoma; ganglion neuroblastoma; neuroblastoma; retinoblastoma; an olfactory neurogenic tumor; meningioma, malignancy; neurofibrosarcoma; schwannoma, malignancy; granulocytoma, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; granuloma parades; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specific non-hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestine disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocyte leukemia; myeloid sarcoma; and hairy cell leukemia.

A. Chemotherapy treatment

According to this embodiment, a variety of chemotherapeutic agents may be used. The term "chemotherapy" refers to the treatment of cancer using drugs. "chemotherapeutic agent" is used to denote a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within the cell (e.g., whether and at which stage they affect the cell cycle). Alternatively, drugs may be characterized based on their ability to directly cross-link DNA, intercalate DNA, or induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, imperoshu and piposhu; aziridines such as phenobarbital, carbaquinone, methylurea dopa (metadropa) and ureidopa (uredropa); ethyleneimine and methamphetamine(s) including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, and trimethylol melamine; acetogenins (especially the stem bark contains bullatacin and bullatanone); camptothecins (including the synthetic analog topotecan); bryostatin; calysistatin; CC-1065 (including adori, carzelaixin, and bizepine synthetic analogs thereof); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (duocarmycin) (including synthetic analogs, KW-2189 and CB1-TM 1); soft corallool; a podocarpine (pancratistatin); stoloniferol; spongosine; nitrogen mustards such as chlorambucil, napthalene mustards, clodronamide, estramustine, ifosfamide, mechlorethamine hydrochloride, melphalan, mechlorethamine, chlorambucil cholesterol, prednisomustine, trefosamide and uracil mustards; nitrosoureas such as carmustine, chlorourectin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1 and calicheamicin ω1); daptomycin (dyneimicin), including daptomycin a; bisphosphonates, such as chlorophosphonate; epothilone; and neocarcinomycin chromophores and related chromophores such as enediyne-antibiotic chromophores, aclacinomycin, actinomycin, anthramycin (authrarnycin), diazoserine, bleomycin, actinomycin (cactomycin), carbicin, carminomycin, amphotericin, dactinomycin, daunorubicin, ditubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin, and deoxydoxorubicin), epirubicin, elsamycin, idarubicin, mitomycins such as mitomycin C, mycophenolic acid, norgabomycin (nogamycin), olivomycin, lomycin, pofexomycin, puromycin, quinimycin, rogubicin, streptozocin, spinosyn, benzoglicin, and zedoxycycline; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethyl folic acid, pterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamine, and thiopurine; pyrimidine analogs such as, for example, ambcitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as carbosterone, drotasone propionate, cyclothioandrol, emandrane, and testosterone; anti-epinephrine such as mitotane and trilostane; folic acid supplements such as folinic acid; acetoglucurolactone; aldehyde phosphoramide glycosides; aminolevulinic acid; enuracil; amsacrine; armustine (bestabucil); a specific group; edatraxate (edatrexate); refofamine; dimecoxin; deaquinone; elformimithin; ammonium elegance; epothilones; eggshell robust; gallium nitrate; hydroxyurea; lentinan; lonidamine (lonidamine); maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mo Pai dar alcohol; niterine; prastatin; phenamet; pirarubicin; losoxantrone; podophylloic acid; 2-ethyl hydrazide; procarbazine; PSK polysaccharide complex; carrying out a process of preparing the raw materials; rhizopus extract; cilaphland; spiral germanium; tenuazonic acid; triiminoquinone; 2,2',2 "-trichlorotriethylamine; trichothecenes (especially T-2 toxin, wart-sporine A (verracurin A), cyclosporin a, and serpentine) are described; uratam; vindesine; dacarbazine; mannitol nitrogen mustard; dibromomannitol; dibromodulcitol; pipobromine; a gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxanes, such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; no An Telong; teniposide; edatrase; daunomycin; aminopterin; hilded; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethyl ornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, novelties, farnesyl-protein transferase inhibitors, antiplatinum (transplatinum), pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

B. Radiation therapy

Other factors that lead to DNA damage and are widely used include what are commonly referred to as gamma rays, X-rays, and/or targeted delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam radiation (U.S. Pat. nos. 5,760,395 and 4,870,287), and ultraviolet radiation. Most likely, all of these factors produce extensive damage to DNA, precursors of DNA, replication and repair of DNA, and assembly and maintenance of chromosomes. The dose of the X-rays ranges from a dose of 50 to 200 rens to a single dose of 2000 to 6000 rens daily for a long period (3 to 4 weeks). The dosage range of the radioisotope varies widely and depends on the half-life of the isotope, the intensity and type of radiation, and the uptake by the tumor cells.

C. Immunotherapy

Those of skill in the art will appreciate that additional immunotherapy (beyond the disclosed cell therapies) may be combined or used in conjunction with the methods of the embodiments. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. RituximabThis is one example. The immune effector may be, for example, an antibody specific for certain markers on the surface of tumor cells. Antibodies alone may act as effectors of therapy, or they may recruit other cells to actually effect cell killing. Antibodies may also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin a chain, cholera toxin, pertussis toxin, etc.) and act as targeting agents. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells that are knockdown or knockdown outside of TGF- βr2 cells.

Antibody-drug conjugates have emerged as a breakthrough method for the development of cancer therapeutics. The antibody-drug conjugate (ADC) comprises a monoclonal antibody (MAb) covalently linked to a cell killing drug. This approach combines the high specificity of a MAb against its antigen target with a highly potent cytotoxic drug, resulting in an "armed" MAb that delivers a payload (drug) to tumor cells with enriched levels of antigen. Targeted delivery of the drug also minimizes its exposure to normal tissues, thereby reducing toxicity and increasing therapeutic index. Two ADC drugs approved by the FDA (approved in 2011)(dimension)Pertuzumab (brentuximab vedotin)) and +.about.approved in 2013>This method was validated by (enmeltrastuzumab (trastuzumab emtansine or T-DM 1)). Currently, there are more than 30 ADC drug candidates at various stages of the clinical trial for cancer treatment (Leal et al, 2014). As antibody engineering and linker-payload optimisation become more mature, the discovery and development of new ADCs is increasingly dependent on the identification and validation of new targets suitable for this approach and the generation of targeted mabs. Two criteria for ADC targets are up-regulated/high level expression and robust internalization in tumor cells.

In one aspect of immunotherapy, tumor cells must have some markers that are easily targeted, i.e., are not present on most other cells. There are many tumour markers, and any of these markers may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p 97), gp68, TAG-72, HMFG, sialyl Lewis antigen, mucA, mucB, PLAP, laminin receptor, erb B, and p155. Another aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligands.

Examples of immunotherapies currently under investigation or use are immunoadjuvants such as mycobacterium bovis (Mycobacterium bovis), plasmodium falciparum (Plasmodium falciparum), dinitrochlorobenzene, and aromatic compounds (U.S. Pat. nos. 5,801,005 and 5,739,169;Hui and Hashimoto,1998;Christodoulides et al, 1998); cytokine therapies, such as any type of interferon, IL-1, GM-CSF and TNF (Bukowski et al, 1998; davidson et al, 1998; hellstrand et al, 1998); gene therapy, such as TNF, IL-1, IL-2 and p53 (Qin et al, 1998; austin-Ward and Villaseca,1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD 20, anti-ganglioside GM2 and anti-p 185 (Hollander, 2012; hanibuchi et al, 1998; U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used with the antibody therapies described herein.

In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either up-regulate signals (e.g., costimulatory molecules) or down-regulate signals. Immune checkpoint blockade targetable inhibitory immune checkpoints include adenosine A2A receptor (A2 AR), B7-H3 (also known as CD 276), B and T lymphocyte attenuation factor (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD 152), indoleamine 2, 3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte-activating gene-3 (LAG 3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and T cell activated V domain Ig inhibitor (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.

D. Surgery

About 60% of cancer patients will undergo some type of surgery, including preventive, diagnostic or staging, curative and palliative surgery. Therapeutic surgery includes excision, where all or part of the cancerous tissue is physically removed, excised, and/or destroyed, and may be used in combination with other therapies, such as the treatment of this embodiment, chemotherapy, radiation therapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor ablation, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscope-controlled surgery (morse surgery).

After excision of some or all of the cancer cells, tissue or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusing the area with additional anti-cancer therapy, direct injection, or topical application. Such treatment may be repeated, for example, every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every 7 days, or every 1 week, every 2 weeks, every 3 weeks, every 4 weeks, and every 5 weeks, or every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months. These treatments may also have different dosages.

E. Other medicaments

It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to enhance the efficacy of the treatment. Such additional agents include agents that affect up-regulation of cell surface receptors and gap junctions, cytostatic and differentiating agents, cytostatic agents, agents that enhance the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions will increase the anti-hyperproliferative effect on neighboring hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of embodiments of the present invention to enhance the anti-hyperproliferative efficacy of the treatment. It is contemplated that cell adhesion inhibitors may enhance the efficacy of embodiments of the present invention. Examples of cell adhesion inhibitors are Focal Adhesion Kinase (FAK) inhibitors and lovastatin. It is also contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of embodiments of the present invention to enhance therapeutic effects.

VI. Carrier

The CD 70-targeted CAR can be delivered to the recipient cell, including NK cell, by any suitable vector, including by a viral vector or by a non-viral vector. Examples of viral vectors include at least retroviral vectors, lentiviral vectors, adenoviral vectors or adeno-associated viral vectors. Examples of non-viral vectors include at least plasmids, transposons, lipids, nanoparticles, liposomes, combinations thereof, and the like.

Where NK cells are transduced with a vector encoding a CD 70-targeted CAR and another gene or genes, such as a suicide gene and/or a cytokine and/or optionally a therapeutic gene product, are also required to be transduced into the cell, the CD 70-targeted CAR, optionally the suicide gene, optionally the cytokine or cytokines may or may not be contained on the same vector. In some cases, the CD 70-targeted CAR, suicide gene, and/or one or more cytokines are expressed by the same vector molecule, such as the same viral vector molecule. In such cases, the expression of the CD 70-targeted CAR, suicide gene, and/or one or more cytokines may or may not be regulated by the same regulatory element(s). When the CD70 targets the CAR, suicide gene and/or cytokine on the same vector, they may or may not be expressed as separate polypeptides. For example, where they are expressed as separate polypeptides, they may be separated on the vector by a 2A element or an IRES element (or both elements may be used one or more times on the same vector).

Those skilled in the art will be able to construct vectors for expressing the antigen receptors of the present disclosure by standard recombinant techniques (see, e.g., sambrook et al, 2001 and Ausubel et al, 1996, both incorporated herein by reference).

A. Regulatory element

Expression cassettes included in vectors useful in the present disclosure include, inter alia, eukaryotic transcription promoters operably linked (in the 5 '-to-3' direction) to protein coding sequences, splicing signals including intervening sequences, and transcription termination/polyadenylation sequences. Promoters and enhancers that control the transcription of a protein-encoding gene in eukaryotic cells may be composed of a plurality of genetic elements. Cellular mechanisms are capable of collecting and integrating the regulatory information transmitted by each element, allowing different genes to evolve different, often complex transcriptional regulatory patterns. For example, promoters used in the context of the present disclosure include constitutive, inducible, and tissue-specific promoters. Where the vector is used to produce a cancer therapy, the promoter may be effective under hypoxic conditions.

B. Promoters/enhancers

The expression constructs provided herein comprise a promoter that drives expression of antigen receptor and other cistron gene products. Promoters typically comprise sequences for locating the start site of RNA synthesis. The most well-known example of such a sequence is the TATA box, but in some promoters lacking a TATA box (e.g., promoters of mammalian terminal deoxynucleotidyl transferase genes and promoters of SV40 late genes), discrete elements overlaying the initiation site itself help to fix the initiation site. Additional promoter elements regulate the frequency of transcription initiation. Typically, these promoters are located in the upstream region of the initiation site, although many promoters are also shown to contain functional elements downstream of the initiation site. In order to place the coding sequence under the "control" of the promoter, the 5 'end of the transcription initiation site of the transcription reading frame is placed "downstream" (i.e., 3' thereof) of the selected promoter. An "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

The spacing between promoter elements is generally flexible such that promoter function is preserved when the elements are inverted or moved relative to each other. For example, in the tk promoter, the spacing between promoter elements may be increased to 50bp apart before activity begins to decrease. Depending on the promoter, it appears that individual elements may activate transcription synergistically or independently. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in transcriptional activation of a nucleic acid sequence.

The promoter may be one naturally associated with the nucleic acid sequence, which may be obtained by isolating 5' non-coding sequences located upstream of the coding segment and/or exon. Such promoters may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence, downstream or upstream of that sequence. Alternatively, certain advantages will be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers that are not normally associated with a nucleic acid sequence in their natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are not "naturally-occurring" (i.e., contain different elements of different transcriptional regulatory regions, and/or mutations that alter expression). For example, the most commonly used promoters in recombinant DNA construction include the beta-lactamase (penicillinase), lactose and tryptophan (trp-) promoter systems. In addition to the synthesis of In addition to the nucleic acid sequences of the promoters and enhancers, recombinant cloning and/or nucleic acid amplification techniques (including PCR can be used ^TM ) Sequences are generated in conjunction with the compositions disclosed herein. Furthermore, it is contemplated that control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like may also be used.

Naturally, it is important to use promoters and/or enhancers that are effective to direct the expression of a DNA segment in an organelle, cell type, tissue, organ, or organism selected for expression. The use of promoters, enhancers and cell type combinations for protein expression is generally known to those skilled in the art of molecular biology (see, e.g., sambrook et al, 1989, incorporated herein by reference). The promoters used may be constitutive, tissue-specific, inducible, and/or may be used to direct high levels of expression of the introduced DNA fragments under suitable conditions, e.g., to facilitate large-scale production of recombinant proteins and/or peptides. Promoters may be heterologous or endogenous.

In addition, any promoter/enhancer combination (e.g., by epd. Isb-sib. Ch/, in the world wide web, per eukaryotic promoter database EPDB) may also be used to drive expression. The use of T3, T7 or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if appropriate bacterial polymerases are provided (either as part of the delivery complex or as an additional gene expression construct).

Non-limiting examples of promoters include early or late viral promoters such as the SV40 early or late promoter, the Cytomegalovirus (CMV) immediate early promoter, the Rous Sarcoma Virus (RSV) early promoter; eukaryotic promoters such as the beta actin promoter, the GADPH promoter, and the metallothionein promoter; and tandem responsive element promoters near the minimal TATA box, such as cyclic AMP responsive element promoter (cre), serum responsive element promoter (sre), phorbol ester promoter (TPA), and responsive element promoter (tre). It is also possible to use human growth hormone promoter sequences (e.g., inThe human growth hormone minimal promoter described in (accession number X05244, nucleotides 283-341) or the mouse mammary tumor promoter (available from ATCC under accession number ATCC 45007). In certain embodiments, the promoter is a CMV IE, dectin-1, dectin-2, human CD11c, F4/80, SM22, RSV, SV40, ad MLP, beta-actin, MHC class I or MHC class II promoter, however, any other promoter for driving therapeutic gene expression is suitable for use in the practice of the invention.

In certain aspects, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that enhance promoter activity and have cis-acting potential even over relatively long distances (up to several kilobases from the target promoter) regardless of their orientation. However, the function of enhancers is not necessarily limited to such a long distance, as they may also function in the vicinity of a given promoter.

C. Initiation signal and associated expression

Specific initiation signals may also be used in the expression constructs provided by the present disclosure for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. It may be desirable to provide exogenous translational control signals, including the ATG initiation codon. One of ordinary skill in the art will be readily able to determine this and provide the necessary signals. It is well known that the initiation codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons can be natural or synthetic. By including appropriate transcription enhancer elements, expression efficiency can be improved.

In certain embodiments, internal Ribosome Entry Site (IRES) elements are used to generate polygenic or polycistronic information. IRES elements are able to bypass the ribosome scanning model of cap-dependent translation of 5' methylation and begin translation at internal sites. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described, as well as IRES from mammalian information. IRES elements may be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, resulting in polycistronic information. Due to the IRES element, ribosomes can proceed into each open reading frame for efficient translation. Multiple genes can be expressed efficiently using a single promoter/enhancer to transcribe a single message.

As detailed elsewhere herein, certain 2A sequence elements may be used to produce linked expression or co-expression of genes in the constructs provided in the present disclosure. For example, the cleavage sequences may be used to co-express genes by ligating open reading frames to form a single cistron. Exemplary cleavage sequences are equine rhinitis A virus (E2A) or F2A (2A foot-and-mouth disease virus) or "2A-like" sequences (e.g., thosea asigna virus 2A; T2A) or porcine teschovirus-1 (porcine teschovirus-1) (P2A). In particular embodiments, multiple 2A sequences are not identical in a single vector, although in alternative embodiments, the same vector utilizes two or more identical 2A sequences. Examples of 2A sequences are provided in US2011/0065779 (which is incorporated herein by reference in its entirety).

D. Origin of replication

For propagation of the vector in a host cell, it may contain one or more replication origin sites (commonly referred to as "ori"), e.g. a nucleic acid sequence corresponding to the oriP of EBV as described above, or a genetically engineered oriP with similar or improved function in programming, which is a specific nucleic acid sequence at which replication is initiated. Alternatively, the replication origin or Autonomous Replication Sequence (ARS) of other extrachromosomal replication viruses as described above may be used.

E. Selection and screenable markers

In some embodiments, cells, such as NK cells, comprising an anti-CD 70 CAR expression construct of the invention can be identified in vitro or in vivo by including a marker in the expression vector. Such markers will confer a recognizable change to the cells, allowing easy identification of cells containing the expression vector. In general, a selectable marker is a marker that confers a property that allows selection. A positive selection marker is a selection marker in which the presence of the marker allows its selection, while a negative selection marker is a selection marker in which its presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

The inclusion of a drug selection marker generally facilitates cloning and identification of transformants, e.g., genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, bleomycin (zeocin) and histidinol are useful selection markers. In addition to conferring markers that allow differentiation of the phenotype of the transformants depending on the implementation of the conditions, other types of markers are involved, including screenable markers such as GFP, the basis of which is colorimetric analysis. Alternatively, screenable enzymes, such as herpes simplex virus thymidine kinase (tk) or Chloramphenicol Acetyl Transferase (CAT), may be utilized as negative selection markers. The skilled person will also know how to use immunological markers, possibly in combination with FACS analysis. The marker used is not considered critical as long as it is capable of simultaneous expression with the nucleic acid encoding the gene product. Other examples of selectable and screenable markers are well known to those skilled in the art.

VII cells

The present disclosure encompasses any type of immune cell that contains a vector encoding a CD 70-targeted CAR, and may also encode at least one cytokine and at least one suicide gene. In some cases, the different vectors encode a CAR versus a suicide gene and/or cytokine. Although conventional T cells, NK cells, gamma-delta T cells, NKT and unchanged NK T cells, regulatory T cells, macrophages, B cells, tumor infiltrating lymphocytes, or mixtures thereof may be used, in particular cases, the cells are NK cells. NK cells may be derived from umbilical cord blood, peripheral blood, induced Pluripotent Stem Cells (iPSC), hematopoietic Stem Cells (HSC), or bone marrow. For example, NK cells may be derived from cell lines such as, but not limited to, NK-92 cells. The NK cells may be umbilical cord blood mononuclear cells such as CD56+ NK cells.

In some cases, the cells are not immortalized cell lines, but are cells obtained from an individual (e.g., primary cells). For example, in some cases, the cells are immune cells obtained from an individual. For example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cells are stem cells (e.g., peripheral blood stem cells) or progenitor cells obtained from an individual.

In particular embodiments, the cells of the present disclosure may be specifically formulated and/or they may be cultured in a particular medium. The cells may be formulated in a manner suitable for delivery to a recipient without deleterious effects.

In certain aspects, a medium for culturing animal cells, such as any one of AIM V, X-VIVO-15, neuroBasal, EGM2, teSR, BME, BGJb, CMRL 1066, glasgow MEM, modified MEM Zinc Option, IMDM, culture medium 199, ireger MEM, alpha MEM, DMEM, ham, RPMI-1640, and Fischer's Medium (any combination thereof), may be used as a basal medium for the culture of animal cells, but it is not particularly limited as long as the medium can be used for culturing animal cells. In particular, the medium may be xeno-free or chemically defined.

The medium may be a serum-containing or serum-free medium, or a medium free of heterologous components. From the standpoint of preventing contamination by components of heterologous animal origin, the serum may be derived from the same animal from which the stem cell or cells are derived. Serum-free medium refers to a medium that does not contain untreated or unpurified serum and thus may include a medium that contains purified blood-derived components or animal tissue-derived components (such as growth factors).

The medium may or may not contain any substitute for serum. Alternatives to serum may include materials suitably containing albumin (such as lipo-albumin, bovine albumin, albumin alternatives such as recombinant albumin or humanized albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transport proteins), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3' -mercaptoglycerol, or equivalents thereof. For example, a surrogate for serum may be prepared by the methods disclosed in, for example, international publication No. 98/30679, which is incorporated herein in its entirety. Alternatively, any commercially available material may be used for greater convenience. Commercially available materials include knock-out serum replacement (KSR), chemically defined concentrated lipids (Gibco) and Glutamax (Gibco).

In certain embodiments, the medium may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of: vitamins such as biotin; DL alpha tocopheryl acetate; DL alpha-tocopherol; vitamin a (acetate); proteins, such as BSA (bovine serum albumin) or human albumin, fraction V free of fatty acids; a catalase; human recombinant insulin; human transferrin; superoxide dismutase; other components such as corticosterone; d-galactose; ethanolamine HCl; glutathione (reduced); l-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and/or T3 (triiodothyronine). In particular embodiments, one or more of these may be explicitly excluded.

In some embodiments, the medium further comprises vitamins. In some embodiments, the medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 (and any range from which a producer can be derived): biotin, DL alpha tocopheryl acetate, DL alpha tocopherol, vitamin a, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium comprises a combination thereof or a salt thereof. In some embodiments, the culture medium comprises, or consists essentially of, biotin, DL alpha tocopheryl acetate, DL alpha tocopherol, vitamin a, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamin comprises, or consists essentially of, biotin, DL alpha-tocopheryl acetate, DL alpha-tocopherol, vitamin a, or a combination or salt thereof. In some embodiments, the medium further comprises a protein. In some embodiments, the protein comprises albumin or bovine serum albumin, a portion of BSA, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In some embodiments, the medium also Comprising one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodothyronine, or a combination thereof. In some embodiments, the culture medium comprises one or more of the following:supplement, heterologous-free +.>Supplements, GS21TM supplements, or combinations thereof. In some embodiments, the medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In some embodiments, the amino acid comprises arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof. In some embodiments, the inorganic ion comprises sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof. In some embodiments, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper or manganese or a combination thereof. In certain embodiments, the culture medium comprises, consists essentially of, or consists of one or more vitamins described herein and/or one or more proteins described herein and/or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite or triiodothyronine, - >Supplements, free of heterologous ingredientsSupplements, GS21TM supplements, amino acids (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine or valine), monosaccharides, inorganic ions (such as sodium, potassium, calcium, magnesiumNitrogen and/or phosphorus) or a salt thereof, and/or molybdenum, vanadium, iron, zinc, selenium, copper or manganese. In particular embodiments, one or more of these may be explicitly excluded.

The medium may also comprise one or more externally added fatty acids or lipids, amino acids (such as non-essential amino acids), one or more vitamins, growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffers and/or inorganic salts. In particular embodiments, one or more of these may be explicitly excluded.

One or more media components may be added at a concentration of at least, up to, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250ng/L, ng/ml, μg/ml, mg/ml, any range derivable therein.

In particular embodiments, the cells of the present disclosure are specifically formulated. They may or may not be formulated as cell suspensions. In certain cases, they are formulated into a single dosage form. They may be formulated for systemic or topical administration. In some cases, the cells are formulated for storage prior to use, and the cell preparation may contain one or more cryopreservative agents, such as DMSO (e.g., in 5% DMSO). The cell preparation may comprise albumin, including human albumin, wherein the specific preparation comprises 2.5% human albumin. Cells may be specifically formulated for intravenous administration; for example, they are formulated for intravenous administration in less than one hour. In certain embodiments, the cells are in a formulated cell suspension that is stable for 1, 2, 3, or 4 hours or more at room temperature from when thawed.

In some cases, the immune cells have been expanded prior to use and/or prior to manufacture. In some cases, the immune cells are NK cells that have been expanded in the presence of (including in any suitable ratio) an effective amount of antigen presenting cells, including Universal Antigen Presenting Cells (UAPCs). For example, the cells may be mixed with UAPC at a ratio of 10:1 to 1:10, 9:1 to 1:9;8:1 to 1:8, 7:1 to 1:7, 6:1 to 1:6, 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1:3, 2:1 to 1:2, or 1:1 ratio (including in a 1:2 ratio) together. In some cases, NK cells are expanded in the presence of IL-2, such as at a concentration of 10-500U/mL, 10-400U/mL, 10-300U/mL, 10-200U/mL, 10-100U/mL, 10-50U/mL, 100-500U/mL, 100-400U/mL, 100-300U/mL, 100-200U/mL, 200-500U/mL, 200-400U/mL, 200-300U/mL, 300-500U/mL, 300-400U/mL, or 400-500U/mL.

NK cells can be infused or stored immediately after genetic modification with the vector. In certain aspects, after genetic modification, cells can be propagated as a large population in vitro for days, weeks, or months about 1 day, 2 days, 3 days, 4 days, 5 days, or longer after gene transfer into the cells. In another aspect, the transfectants are cloned, the clones demonstrate the presence of a single integrated or episomally maintained expression cassette or plasmid, and CD 70-targeted CAR expression is amplified ex vivo. The clones selected for expansion demonstrated the ability to specifically recognize and lyse target cells expressing CD 70. Recombinant immune cells can be expanded by stimulation with IL-2 or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-12, IL-15, IL-21, etc.). Recombinant immune cells can be expanded by artificial antigen presenting cell stimulation. In another aspect, the genetically modified cells may be cryopreserved.

Embodiments of the present disclosure encompass cells expressing one or more CD 70-targeted CARs as encompassed herein. In particular embodiments, the NK cells comprise a recombinant nucleic acid encoding one or more CD 70-targeted CARs and one or more engineered non-secretable membrane-bound TNF-a mutant polypeptides. In particular embodiments, the cell comprises a nucleic acid encoding one or more therapeutic gene products in addition to expressing one or more CD 70-targeted CARs.

The cells may be obtained directly from the individual or may be obtained from a storage or other storage facility. For individuals to whom cells are provided as a therapy, the cells as a therapy may be autologous or allogeneic.

The cells may be from an individual in need of treatment for a medical disorder, and after manipulation of them to express a CD 70-targeted CAR, they may be provided back to the individual from which they were originally derived. In some cases, the cells are stored for later use by the individual or another individual. In such cases, the cells may or may not be further modified prior to use.

NK cells with CD 70-targeted CARs may be included in a population of cells, and a majority of the population may be transduced with one or more CD 70-targeted CARs. The cell population can comprise 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of cells that are targeted to the CAR with one or more CD 70.

NK cells with one or more CD 70-targeted CARs can be generated with the aim of being modular for a particular purpose. For example, cells expressing a CD 70-targeted CAR can be generated, including cells for commercial distribution, and can be modified by a user to express one or more other genes of interest (including therapeutic genes) according to one or more of their intended purposes. For example, a party interested in treating a CD 70-positive cancer can modify them to express a second CAR against the same or a different antigen. Other modifications to the cells may include modifications that adapt the cells to the needs of the individual receiving the cells, including the specific needs for the specific type of cancer the individual is suffering from.

Gene editing of cells

In certain embodiments, cells comprising an anti-CD 70 CAR are genetically edited to modify expression of one or more endogenous genes in the cells. In certain instances, the cell is modified to reduce the level of expression of one or more endogenous genes, including inhibiting the expression of one or more endogenous genes (which may be referred to as a knockout). Such cells may or may not be expanded.

In certain cases, one or more endogenous genes of the cell are modified, such as expression is disrupted, wherein expression is partially or fully reduced. In certain cases, one or more genes are knocked down or knocked out using the methods of the present disclosure. In certain cases, multiple genes are knocked down or knocked out, which may or may not occur in the same step in their production. The gene that is edited in the cell may be of any type, but in particular embodiments, the gene is one whose gene product inhibits the activity and/or proliferation of cells, including antigen-specific, e.g., CD 70-specific CAR NK cells, such as umbilical cord blood-derived cells, as an example. In certain cases, genes edited in antigen-specific (e.g., CD 70-specific) CAR cells allow antigen-specific (e.g., CD 70-specific) CAR cells to work more efficiently in the tumor microenvironment. In particular instances, the gene is one or more of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, and CD 7. In particular embodiments, the TGFBR2 gene is knocked out or knockdown in antigen specific (e.g., CD70 specific) CAR cells.

In some embodiments, gene editing is performed using one or more DNA binding nucleic acids, such as by RNA-guided endonuclease (RGEN) alteration. For example, alterations can be made using regularly spaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins; in some embodiments, cpF1 is used instead of Cas9. In general, "CRISPR system" refers to a collective term for transcripts and other elements involved in the expression of or directing the activity of a CRISPR-associated ("Cas") gene, including sequences encoding Cas genes, tracr (transactivation CRISPR) sequences (e.g., tracrRNA or active moiety tracrRNA), tracr-mate sequences (covering "forward repeats" and tracrRNA treated partial forward repeats in the context of an endogenous CRISPR system), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.

The CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include a non-coding RNA molecule (guide) RNA that sequence-specifically binds DNA, and a Cas protein (e.g., cas 9) with nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system may be derived from a type I, type II or type III CRISPR system, for example from a specific organism comprising an endogenous CRISPR system, such as streptococcus pyogenes (Streptococcus pyogenes).

In some aspects, cas nucleases and grnas (including fusions of crrnas specific for target sequences with immobilized tracrRNA) are introduced into cells. Typically, a target site at the 5' end of the gRNA targets the Cas nuclease to a target site, e.g., a gene, using complementary base pairing. The target site may be selected based on its position immediately 5' of the prosomain sequence adjacent motif (PAM) sequence, such as typically NGG or NAG. In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at target sequence sites. Typically, a "target sequence" generally refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence facilitates the formation of a CRISPR complex. Complete complementarity is not necessarily required if there is sufficient complementarity to cause hybridization and promote the formation of CRISPR complexes.

CRISPR systems can induce Double Strand Breaks (DSBs) at target sites followed by disruption or alteration as described herein. In other embodiments, cas9 variants that are considered "nickases" are used to create single-stranded nicks at a target site. Pairs of nicking enzymes may be used, for example, to increase specificity, each directed by a different pair of gRNA targeting sequences, such that when nicks are introduced simultaneously, 5' overhangs are introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm, such as within the organelle of a cell. In general, sequences or templates that can be used for recombination into a targeted locus comprising a target sequence are referred to as "editing templates" or "editing polynucleotides" or "editing sequences. In some aspects, the exogenous template polynucleotide may be referred to as an editing template. In certain aspects, the recombination is homologous recombination.

Generally, in the context of endogenous CRISPR systems, the formation of a CRISPR complex (comprising a guide sequence that hybridizes to a target sequence and that is complexed with one or more Cas proteins) results in cleavage of one or both strands in or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs). A tracr sequence that may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence) may also form part of a CRISPR complex, such as by hybridizing along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to a guide sequence. the tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned.

One or more vectors driving expression of one or more elements of a CRISPR system can be introduced into a cell such that expression of the CRISPR system elements directs the formation of a CRISPR complex at one or more target sites. The components may also be delivered to the cells in the form of proteins and/or RNAs. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence may each be operably linked to an independent regulatory element on an independent vector. Alternatively, two or more elements expressed by the same or different regulatory elements may be combined in a single vector, wherein one or more additional vectors provide any component of the CRISPR system not included in the first vector. The vector may comprise one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within a cell.

The vector may comprise a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme (such as a Cas protein). Non-limiting examples of Cas proteins include Cas1, cas1B, cas2, cas3, cas4, cas5, cas6, cas7, cas8, cas9 (also known as Csn1 and Csx 12), cas10, csy1, csy2, csy3, cse1, cse2, csc1, csc2, csa5, csn2, csm3, csm4, csm5, csm6, cmr1, cmr3, cmr4, cmr5, cmr6, csb1, csb2, csb3, csx17, csx14, csx10, csx16, csaX, csx3, csx1, csx15, csfl, csf2, csf3, csf4, cpf1 (Cas 12 a), homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of the streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW 2.

The CRISPR enzyme may be Cas9 (e.g. from streptococcus pyogenes or streptococcus pneumoniae (s)). In some cases, cpf1 (Cas 12 a) may be used as an endonuclease instead of Cas 9. CRISPR enzymes can direct cleavage of one or both strands at a location of a target sequence, such as within the target sequence and/or within a complementary sequence of the target sequence. The vector may encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide comprising a target sequence. For example, substitution of aspartic acid to alanine in the RuvC I catalytic domain of Cas9 from streptococcus pyogenes (D10A) converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves single strand). In some embodiments, cas9 nickase may be used in combination with one or more guide sequences (e.g., two guide sequences) that target the sense strand and the antisense strand of a DNA target, respectively. This combination allows both chains to be nicked and used to induce NHEJ or HDR.

In some embodiments, the enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in a particular cell, such as a eukaryotic cell. Eukaryotic cells may be cells of or derived from a particular organism, such as a mammal, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence with a more or most frequently used codon in the gene of the host cell, while maintaining the native amino acid sequence. Different species exhibit specific preferences for specific codons for specific amino acids. Codon bias (the difference in codon usage between organisms) is typically related to the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend on the nature of the codon being translated, availability of a particular transfer RNA (tRNA) molecule, etc. The advantage of the tRNA selected in the cell is typically the response of codons most commonly used in peptide synthesis. Thus, genes can be tailored based on codon optimization to achieve optimal gene expression in a given organism.

In general, a targeting sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more when optimally aligned using a suitable alignment algorithm.

Optimal alignment can be determined by using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burrow-Wheeler transform-based algorithm (e.g., burrows Wheeler Aligner), clustal W, clustal X, BLAT, novoalign (Novocraft Technologies), ELAND (Illumina, san Diego, calif.), SOAP (available at SOAP. Genemics. Org. Cn), and Maq (available at maq. Sourcefuge. Net).

The CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains. The CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza Hemagglutinin (HA) tags, myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol Acetyl Transferase (CAT) beta galactosidase, beta-glucuronidase, luciferase, green Fluorescent Protein (GFP), hcRed, dsRed, cyan Fluorescent Protein (CFP), yellow Fluorescent Protein (YFP), and autofluorescent proteins including Blue Fluorescent Protein (BFP). CRISPR enzymes can be fused to gene sequences encoding proteins or protein fragments that bind DNA molecules or bind other cellular molecules, including but not limited to Maltose Binding Protein (MBP), S-tag, lex a DNA Binding Domain (DBD) fusion, GAL4A DNA binding domain fusion, and Herpes Simplex Virus (HSV) BP16 protein fusion. Other domains that can form part of fusion proteins comprising CRISPR enzymes are described in US20110059502 (incorporated herein by reference).

IX. treatment method

In various embodiments, the CD 70-targeted CAR constructs, nucleic acid sequences, vectors, host cells, and the like contemplated herein and/or pharmaceutical compositions comprising the same are used to prevent, treat, or ameliorate cancerous diseases, such as neoplastic diseases. In particular embodiments, the pharmaceutical compositions of the present disclosure may be particularly useful for preventing, ameliorating and/or treating cancers, including, for example, cancers that express CD70 and cancers that may or may not be solid tumors. For example, an individual may utilize the disclosed methods of treatment as an initial treatment or after (or with) another treatment, such as after HSCT with the disclosed methods of treatment. Based on the type and/or stage of the cancer, the immunotherapy approach may be tailored to the needs of the individual suffering from the cancer, and in at least some cases, the immunotherapy may be modified during the course of the individual's treatment.

In some embodiments, the invention provides methods of immunotherapy comprising administering an effective amount of cells produced by the methods of the present disclosure. In one embodiment, the medical disease or disorder is treated by transferring a population of cells that are produced by the methods herein and elicit an immune response. In certain embodiments of the present disclosure, cancer is treated by transferring a population of cells that are produced by the methods of the present disclosure and elicit an immune response. Provided herein are methods for treating or slowing the progression of cancer in an individual comprising administering to the individual an effective amount of a CD 70-specific cell therapy. The method can be used for treating solid cancer or blood cancer.

Tumors for which the methods of treatment of the present invention are applicable include any malignant cell type, such as those found in solid tumors or hematological tumors. Exemplary solid tumors may include, but are not limited to, tumors of an organ selected from the group consisting of: acute myeloid leukemia, lymphoma, lung cancer, kidney cancer, bladder cancer, melanoma, glioblastoma, breast cancer, head and neck cancer, mesothelioma, multiple myeloma, and pancreatic cancer.

In certain embodiments of the present disclosure, immune cells expressing an anti-CD 70 CAR are delivered to an individual in need thereof, such as an individual with cancer. These cells then enhance the immune system of the individual to attack the cancer cells. In some cases, one or more doses of immune cells are provided to an individual. Where two or more doses of immune cells are provided to an individual, the duration between administrations should be sufficient to allow for propagation in the individual, and in particular embodiments, the duration between administrations is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more.

In particular embodiments, the NK cells utilizing the CD 70-targeted CAR can be NK cells, T cells, or unchanged NKT cells engineered for cell therapy in a mammal. In such cases where the cells are NK cells, the NK cell therapy may be of any type, and the NK cells may be of any type. In a specific embodiment, the fine The cells are NK cells that have been engineered to express one or more CD 70-targeted CARs and are expressed in a therapeutically effective amount (at 10 ³ To 10 ¹⁰ Within the scope of (d) is provided to the individual, the therapeutically effective amount improving at least one symptom associated with the CD70 expressing cells in the individual. In particular embodiments, the cell is an NK cell transduced with a CD 70-targeted CAR. The therapeutically effective amount may be 10 ³ To 10 ¹⁰ 10 pieces, 10 ³ To 10 ⁹ 10 pieces, 10 ³ To 10 ⁸ 10 pieces, 10 ³ To 10 ⁷ 10 pieces, 10 ³ To 10 ⁶ 10 pieces, 10 ³ To 10 ⁵ 10 pieces, 10 ³ To 10 ⁴ 10 pieces, 10 ⁴ To 10 ¹⁰ 10 pieces, 10 ⁴ To 10 ⁹ 10 pieces, 10 ⁴ To 10 ⁸ 10 pieces, 10 ⁴ To 10 ⁷ 10 pieces, 10 ⁴ To 10 ⁶ 10 pieces, 10 ⁴ To 10 ⁵ 10 pieces, 10 ⁵ To 10 ¹⁰ 10 pieces, 10 ⁵ To 10 ⁹ 10 pieces, 10 ⁵ To 10 ⁸ 10 pieces, 10 ⁵ To 10 ⁷ 10 pieces, 10 ⁵ To 10 ⁶ 10 pieces, 10 ⁶ To 10 ¹⁰ 10 pieces, 10 ⁶ To 10 ⁹ 10 pieces, 10 ⁶ To 10 ⁸ 10 pieces, 10 ⁶ To 10 ⁷ 10 pieces, 10 ⁷ To 10 ¹⁰ 10 pieces, 10 ⁷ To 10 ⁹ 10 pieces, 10 ⁷ To 10 ⁸ 10 pieces, 10 ⁸ To 10 ¹⁰ 10 pieces, 10 ⁸ To 10 ⁹ Or 10 ⁹ To 10 ¹⁰ Individual cells. Thus, in particular embodiments, one or more therapeutically effective amounts of cells (including NK cells) expressing one or more CD 70-targeted CARs are provided to an individual having a CD 70-positive cancer.

In particular embodiments, the present disclosure contemplates, in part, cells expressing a CD70 CAR, CD 70-targeted CAR constructs, CD 70-targeted CAR nucleic acid molecules, and CD 70-targeted CAR vectors, which may be administered alone or in any combination using standard vectors and/or gene delivery systems, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient. In certain embodiments, following administration, the nucleic acid molecule or vector may be stably integrated into the genome of the subject.

For example, in particular embodiments, viral vectors that are specific for certain cells or tissues and persist in NK cells may be used. Suitable pharmaceutical carriers and excipients are well known in the art. Compositions prepared according to the present disclosure are useful for preventing or treating or delaying the diseases identified above.

Furthermore, the present disclosure relates to a method for preventing, treating or ameliorating a neoplastic disease comprising the step of administering to a subject in need thereof an effective amount of cells expressing a CD 70-targeted CAR, nucleic acid sequence, vector as contemplated herein and/or produced by the methods contemplated herein.

For example, a possible indication for administration of one or more compositions of exemplary CD 70-targeted CAR cells is a cancerous disease, including a neoplastic disease, including, for example, a B cell malignancy, multiple myeloma, breast cancer, or lung cancer. An exemplary indication for administration of one or more compositions of CD 70-targeted CAR cells is a cancerous disease, including any malignancy that expresses CD 70. Administration of one or more compositions of the present disclosure may be useful for all stages and types of cancer, including, for example, minimal residual disease, early stage cancer, advanced cancer, and/or metastatic cancer, and/or refractory cancer.

The therapeutically effective amount of the produced cells can be administered by a variety of routes including parenteral administration, e.g., intravenous, intraperitoneal, intramuscular, intrasternal, intratumoral, intrathecal, intraventricular, by depot, intra-articular injection or infusion.

A therapeutically effective amount of the resulting cells for adoptive cell therapy is an amount that achieves the desired effect in the subject being treated. For example, this may be the amount of immune cells necessary to inhibit progression or cause regression of the cancer.

The resulting population of cells may be administered in a treatment regimen consistent with the disease, such as a single dose or several doses over a day to several days to improve the disease state, or periodic doses over an extended period of time to inhibit disease progression and prevent disease recurrence. The exact dosage used in the formulation will also depend on the route of administration and the diseaseThe severity of the disease or condition, and should be determined according to the judgment of the physician and the circumstances of each patient. The therapeutically effective amount of the cells will depend on the subject being treated, the severity and type of the condition, and the mode of administration. In some embodiments, the dosage range useful for treating a human subject is at least 1x10 ³ Individual T cells/m ² At least 1x10 ⁴ Individual T cells/m ² At least 3.8X10 ⁴ Individual T cells/m ² At least 3.8X10 ⁵ Individual T cells/m ² At least 3.8X10 ⁶ Individual T cells/m ² At least 3.8X10 ⁷ Individual T cells/m ² At least 3.8X10 ⁸ Individual T cells/m ² At least 3.8X10 ⁹ Individual T cells/m ² Or at least 3.8X10 ¹⁰ Individual T cells/m ² . In a certain embodiment, the dosage range for treating a human subject is about 3.8X10 ⁹ Individual T cells/m ² To about 3.8X10 ¹⁰ Individual T cells/m ² . In further embodiments, a therapeutically effective amount of T cells may be from about 5X 10 ⁶ Individual cells/kg body weight change to about 7.5x10 ⁸ Individual cells/kg body weight, such as about 2X 10 ⁷ Individual cells to about 5X 10 ⁸ Individual cells/kg body weight, or about 5X 10 ⁷ From about 2X 10 cells ⁸ Individual cells/kg body weight. The exact amount of T cells can be readily determined by one skilled in the art based on the age, weight, sex, and physiological condition of the subject. The effective dose can be deduced from dose response curves obtained from in vitro or animal model test systems.

The present disclosure also encompasses co-administration regimens with other compounds (e.g., bispecific antibody constructs, targeted toxins, or other compounds that act through immune cells). Clinical protocols for co-administration of one or more compounds of the invention may involve co-administration simultaneously, before or after administration of the other components. Specific combination therapies include chemotherapy, radiation therapy, surgery, hormonal therapy, or other types of immunotherapy.

Embodiments relate to a kit comprising a CD 70-targeted CAR construct as defined herein, a nucleic acid sequence as defined herein, a vector as defined herein, and/or a host as defined herein. It is also contemplated that the kits of the present disclosure comprise a pharmaceutical composition as described above, alone or in combination with other drugs to be administered to an individual in need of drug treatment or intervention.

X. the medicine box of the present disclosure

Any of the compositions described herein may be contained in a kit. In one non-limiting example, the kit comprises an anti-CD 70 CAR molecule, a cell comprising the molecule, and/or an agent that produces the molecule, and any of these can be contained in a suitable container means of the kit of the present disclosure. The kit may comprise immune cells, including NK cells, vectors, expression construct polynucleotides for insertion into a vector (whether viral or not), any type of CD27 sequence (as contemplated herein), and the like. Primers for amplifying any polynucleotide may be included. In some cases, the kit comprises cryopreserved cells, including NK cells. Any agent for transfection or transduction of cells may be included.

The composition of the kit may be packaged in an aqueous medium or in lyophilized form. The container means of the kit typically comprises at least one vial, test tube, flask, bottle, syringe or other container means into which one or more components may be placed, and preferably the components are suitably aliquoted. When more than one component is present in the kit, the kit may also typically include a second, third or other additional container into which additional components may be separately placed. However, various combinations of components may be contained in one vial. The kits of the present disclosure will also typically include means for including therein an anti-CD 70 CAR molecule, cells comprising an anti-CD 70 CAR molecule, and/or reagents for producing an anti-CD 70 CAR molecule for commercial sale. Such containers may include injection containers or blow molded plastic containers with the desired vials retained therein.

When the components of the kit are provided in the form of one and/or more liquid solutions, the liquid solutions are aqueous solutions, particularly contemplated are sterile aqueous solutions. The composition may also be formulated as an injectable composition. In this case, the container means itself may be a syringe, pipette and/or other similar device whereby the formulation may be applied to the infected area of the body, injected into the animal, and/or even applied to and/or mixed with the further components of the kit.

However, the components of the kit may be provided in one or more dry powder forms. When the reagents and/or components are provided in dry powder form, the powder may be reconstituted by the addition of a suitable solvent. It is envisaged that the solvent may also be provided in another container means.

Regardless of the number and/or type of containers, the kits of the present invention may also include and/or be packaged with devices for aiding in the injection/administration and/or placement of the final composition within an animal. Such a device may be a syringe, pipette, forceps, and/or any such medically approved delivery vehicle. In some embodiments, the kit includes reagents or devices or containers for ex vivo use.

Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

CD 27-containing CAR targeting CD70 positive tumors

The inventors first demonstrated the expression of CD70 in AML patient samples. Tsne plots of mass cytometry data showed high expression of CD70 on primary AML samples (n=54) but not on healthy CB cd34+ cells (n=10) (fig. 1A). As shown in fig. 1B, the Tsne plot also shows various bone marrow markers on lineage neg cells of CB (upper layer CB population of fig. 1A) and AML samples (lower layer AML population of fig. 1A). CD70 expression in AML samples was similar to other well-characterized bone marrow markers.

Figure 3 shows that TrCD27 constructs show higher transfection efficiency than flCD27 constructs. As shown, 293T cells were transfected with various CD27 constructs using genejet as transfection reagent. Transfection efficiency was determined by examining the surface expression of CD27 constructs in 293T cells using flow cytometry after virus collection. Non-transduced (NT) cells were used as controls. We also include constructs containing only IL15 (i.e., IL 15) and CD70scFv CAR constructs containing single heavy and light chains derived from commercially available CD70 antibody sequences, as well as IL15 (i.e., CD70-IL 15). Surface expression of IgG was used to determine the transfection efficiency of the latter two constructs.

Figure 4 demonstrates that TrCD27 constructs exhibit higher transduction efficiencies than flCD27 constructs. Retrovirus supernatant (fig. 3) collected from transfection experiments was used to transduce umbilical cord blood-derived NK (CBNK) cells, and RetroNectin was used to enhance transduction efficiency. Transduction efficiency was measured 48 hours after transduction by examining the surface expression of CD27 on CBNK cells using flow cytometry. Supernatants from non-transduced (NT) cells were used as controls. For IL15 and CD70 CAR-IL15 CAR constructs, surface expression of IgG on CBNK cells was used to determine transduction efficiency. Emphasis was placed on trCD27 constructs because flCD27 constructs showed poor transfection and transduction efficiency under these conditions.

In FIGS. 5A-5C, CBNK cells transduced with trCD27 constructs induced cancer cell apoptosis. Performing annexin V-LIVE/DEAD ^TM The fixble Aqua staining test was performed to investigate whether CBNK cells transduced with various trCD27 constructs could induce CD70 expressing cancer cell death. Raji and Karpas cells were used as targets because both had relatively high CD70 surface expression (fig. 5A, 5B, respectively). NK cells and cancer cells transduced with the TrCD27 construct were co-cultured at a ratio of 1:1 for 4hr and subjected to annexin V-LIVE/DEAD ^TM And (3) dyeing the Fixable Aqua. Various trCD70 transduced CBNK cells (such as #3, #13, #15, #17, #19, #20, etc.) produced better killing of both Raji cells (black bars) and Karpas (gray bars) cells compared to NT, IL15 or CD70-IL15 CAR transduced CBNK cells (FIG. 5C).

In FIG. 6, CB-NK cells transduced with trCD27 constructs showed enhanced cytotoxic activity against cancer cells. Degranulation marker CD107a is a widely accepted marker of NK cell cytotoxic activity. trCD27 CAR transduced CBNK cells and cancer cells (Raji and Karpas) were co-cultured in the presence of CD107a antibody at a ratio of 1:1 for 6hr. After incubation, CD107a expression was assessed by flow cytometry. Most trCD 70-transduced CB-NK cells show enhanced cytotoxic activity against Raji cells (middle black bars) and Karpas cells (right gray bars) when compared to NT, IL15 or CD70-IL15 CAR-transduced CB-NK cells. CB-NK cells not co-cultured with cancer cells showed the lowest basal expression level of CD107a (left green column).

FIGS. 7A-7B demonstrate that CB-NK cells transduced with trCD27 constructs exhibit enhanced cytotoxicity against cancer cells. To analyze the real-time cytotoxic activity of trCD27 CAR transduced CBNK cells on cancer cells, an IncuCyte cytotoxicity assay was performed. trCD27 CAR transduced CB-NK cells were co-cultured with Raji cells (FIG. 7A) or Karpas cells (FIG. 7B) at a ratio of 1:1, and real-time cytotoxicity of NK cells against Raji cells and Karpas cells was measured every hour over 12 hours. All trCD70 CAR-transduced CB-NK cells used in the assay showed enhanced cytotoxic activity against Raji cells (left panel) and Karpas cells (right panel) when compared to NT, IL15 or CD70-IL15 CAR-transduced CB-NK cells.

Figures 8A-8B demonstrate that CB-NK cells transduced with trCD27CAR have potent anti-tumor activity against CD70 positive Raji cells in vivo. Raji cells (Raji-FFluc) labeled with firefly luciferase with high CD70 expression were transplanted into NSG mice. Mice were injected with 20,000 Raji cells/mouse and 5M CBNK cells/mouse. Bioluminescence imaging of tumors showed that CB-NK cells transduced with trCD27CAR were able to reduce tumor burden due to CD70 high Raji cells (fig. 8A). In addition, survival curves showed significant survival benefit for Raji-bearing NSG mice when infused with a single dose of CB-NK cells transduced with various trCD27 CARs, compared to non-transduced (NT) cells and cells transduced with IL15 or CD70-IL15 CARs (fig. 8B). the trcd27#23CAR structure showed the most significant survival benefit. p=0.002 (raji+nt versus raji+cd27#23); p=0.0018 (raji+il15 vs raji+cd27#23); p=0.001 (raji+cd70-IL 15 vs raji+cd27#23). The Mantel-Cox test was used to measure p-values.

Figures 9A-9B demonstrate that CB-NK cells transduced with trCD27CAR have potent anti-tumor activity against CD70 positive AML cells in vivo. THP-1 cells labeled with firefly luciferase (THP 1-FFluc), which have high CD70 expression, were transplanted into NSG mice. Mice were injected with 100,000 THP1 cells/mouse and 5M CBNK cells/mouse. Bioluminescence imaging of tumors showed that CB-NK cells transduced with trCD27CAR were able to reduce tumor burden due to CD70 high THP-1 cells (fig. 9A). In addition, survival curves showed significant survival benefit for NSG mice with THP-1 when infused with a single dose of CB-NK cells transduced with various trCD27 CARs, compared to non-transduced (NT) cells and cells transduced with IL15 or CD70-IL15 CARs (fig. 9B). trcd27#15 and trcd27#23CAR constructs showed the most significant survival benefits. p=0.0027 (THP-1+nt vs THP-1+cd27#15 or#23); p=0.001 (THP-1+il15 vs THP-1+cd27#15 or#23); p=0.001 (THP-1+cd70-IL 15 vs THP-1+cd27#15 or#23). The Mantel-Cox test was used to measure p-values.

Although the present disclosure and its advantageous aspects have been described in detail, it should be understood that various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the design as defined by the appended claims. Furthermore, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Sequence listing

<110> Board of Regents, The University of Texas System

<120> CD27 ectodomain CAR targeting CD70 positive tumors

<130> MDAC.P1261WO/1001195363

<140>

<141> 2022-01-24

<150> 63/141,016

<151> 2021-01-25

<150> 63/270,414

<151> 2021-10-21

<160> 74

<170> PatentIn version 3.5

<210> 1

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<213> artificial sequence

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Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val

1 5 10 15

Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr

20 25 30

Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe

35 40 45

Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro

50 55 60

Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His

65 70 75 80

Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys

85 90 95

Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys

100 105 110

Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu

115 120 125

Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His

130 135 140

Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met

145 150 155 160

Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr

165 170 175

His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Phe

180 185 190

Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu

195 200 205

Val Thr Val Ala Phe Ile Ile Phe Trp Val

210 215

<210> 2

<211> 191

<212> PRT

<213> artificial sequence

<220>

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Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val

1 5 10 15

Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr

20 25 30

Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe

35 40 45

Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro

50 55 60

Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His

65 70 75 80

Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys

85 90 95

Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys

100 105 110

Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu

115 120 125

Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His

130 135 140

Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met

145 150 155 160

Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr

165 170 175

His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

180 185 190

<210> 3

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<213> artificial sequence

<220>

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<400> 3

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 4

<211> 260

<212> PRT

<213> artificial sequence

<220>

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<400> 4

Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val

1 5 10 15

Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr

20 25 30

Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe

35 40 45

Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro

50 55 60

Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His

65 70 75 80

Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys

85 90 95

Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys

100 105 110

Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu

115 120 125

Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His

130 135 140

Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met

145 150 155 160

Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr

165 170 175

His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile

180 185 190

Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala

195 200 205

Leu Phe Leu His Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser

210 215 220

Pro Val Glu Pro Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu

225 230 235 240

Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro

245 250 255

Ala Cys Ser Pro

260

<210> 5

<211> 172

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 5

Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala Gln

1 5 10 15

Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val Lys

20 25 30

Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile Pro

35 40 45

Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu Ser

50 55 60

Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile Thr

65 70 75 80

Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp Lys

85 90 95

Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala Arg

100 105 110

Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro Tyr

115 120 125

Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr Leu

130 135 140

Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp Pro

145 150 155 160

Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

165 170

<210> 6

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 6

Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val

1 5 10 15

Gly Leu Ser

<210> 7

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 7

Ile Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly

1 5 10 15

Ala Leu Phe Leu His

20

<210> 8

<211> 48

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 8

Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro

1 5 10 15

Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr

20 25 30

Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro

35 40 45

<210> 9

<211> 783

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 9

atggcacggc cacatccctg gtggctgtgc gttctgggga ccctggtggg gctctcagct 60

actccagccc ccaagagctg cccagagagg cactactggg ctcagggaaa gctgtgctgc 120

cagatgtgtg agccaggaac attcctcgtg aaggactgtg accagcatag aaaggctgct 180

cagtgtgatc cttgcatacc gggggtctcc ttctctcctg accaccacac ccggccccac 240

tgtgagagct gtcggcactg taactctggt cttctcgttc gcaactgcac catcactgcc 300

aatgctgagt gtgcctgtcg caatggctgg cagtgcaggg acaaggagtg caccgagtgt 360

gatcctcttc caaacccttc gctgaccgct cggtcgtctc aggccctgag cccacaccct 420

cagcccaccc acttacctta tgtcagtgag atgctggagg ccaggacagc tgggcacatg 480

cagactctgg ctgacttcag gcagctgcct gcccggactc tctctaccca ctggccaccc 540

caaagatccc tgtgcagctc cgattttatt cgcatccttg tgatcttctc tggaatgttc 600

cttgttttca ccctggccgg ggccctgttc ctccatcaac gaaggaaata tagatcaaac 660

aaaggagaaa gtcctgtgga gcctgcagag ccttgtcgtt acagctgccc cagggaggag 720

gagggcagca ccatccccat ccaggaggat taccgaaaac cggagcctgc ctgctccccc 780

tga 783

<210> 10

<211> 113

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 10

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

Gly

<210> 11

<211> 41

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 11

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 12

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 12

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 13

<211> 780

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 13

atggcgagac cacacccttg gtggctgtgc gtactcggca cacttgtagg tctgtccgct 60

acaccggctc cgaagtcctg cccggagcgg cattattggg cacagggcaa gttgtgttgt 120

caaatgtgtg agccgggaac ctttctcgtg aaggattgcg atcagcatcg gaaggccgcg 180

cagtgcgacc catgtatacc aggggtctca ttttccccag atcaccatac gaggccgcac 240

tgtgagtctt gcaggcattg taattccggc ttgttggtcc gcaactgtac tattactgcg 300

aatgcagagt gtgcttgtag aaacggatgg cagtgcaggg acaaagaatg tacggagtgt 360

gatccactgc ctaaccccag tcttacagca agatcttcac aggccctcag cccgcatcct 420

caaccaacac atcttcctta cgtgtcagaa atgttggagg cgcgaaccgc aggccatatg 480

cagaccctgg cggactttcg gcagctgcca gcacgcacac ttagtacaca ctggccacca 540

caacgcagct tgtgctcttc cgatttcatc cgcatactgg tcatcttttc tggaatgttc 600

cttgtgttca ccctggcagg agccctgttc cttcaccaga gacgcaagta caggtcaaac 660

aagggtgaga gccccgttga acccgcagag ccgtgtagat actcatgtcc tagagaagaa 720

gagggctcta ctatccctat tcaggaagat tatagaaaac ccgaacccgc gtgcagcccc 780

<210> 14

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 14

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 15

<211> 654

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 15

atggcacggc cacatccctg gtggctgtgc gttctgggga ccctggtggg gctctcagct 60

actccagccc ccaagagctg cccagagagg cactactggg ctcagggaaa gctgtgctgc 120

cagatgtgtg agccaggaac attcctcgtg aaggactgtg accagcatag aaaggctgct 180

cagtgtgatc cttgcatacc gggggtctcc ttctctcctg accaccacac ccggccccac 240

tgtgagagct gtcggcactg taactctggt cttctcgttc gcaactgcac catcactgcc 300

aatgctgagt gtgcctgtcg caatggctgg cagtgcaggg acaaggagtg caccgagtgt 360

gatcctcttc caaacccttc gctgaccgct cggtcgtctc aggccctgag cccacaccct 420

cagcccaccc acttacctta tgtcagtgag atgctggagg ccaggacagc tgggcacatg 480

cagactctgg ctgacttcag gcagctgcct gcccggactc tctctaccca ctggccaccc 540

caaagatccc tgtgcagctc cgattttatt cgcttttggg tgctggtggt ggttggtgga 600

gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtg 654

<210> 16

<211> 2004

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 16

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atggcacggc cacatccctg gtggctgtgc 360

gttctgggga ccctggtggg gctctcagct actccagccc ccaagagctg cccagagagg 420

cactactggg ctcagggaaa gctgtgctgc cagatgtgtg agccaggaac attcctcgtg 480

aaggactgtg accagcatag aaaggctgct cagtgtgatc cttgcatacc gggggtctcc 540

ttctctcctg accaccacac ccggccccac tgtgagagct gtcggcactg taactctggt 600

cttctcgttc gcaactgcac catcactgcc aatgctgagt gtgcctgtcg caatggctgg 660

cagtgcaggg acaaggagtg caccgagtgt gatcctcttc caaacccttc gctgaccgct 720

cggtcgtctc aggccctgag cccacaccct cagcccaccc acttacctta tgtcagtgag 780

atgctggagg ccaggacagc tgggcacatg cagactctgg ctgacttcag gcagctgcct 840

gcccggactc tctctaccca ctggccaccc caaagatccc tgtgcagctc cgattttatt 900

cgcttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 960

gtggccttta ttattttctg ggtgaaacgg ggcagaaaga aactcctgta tatattcaaa 1020

caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 1080

ccagaagaag aagaaggagg atgtgaactg aaacgcgtga agttcagcag gagcgcagac 1140

gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1200

gaggagtacg atgttttgga caaaagacgt ggccgggacc ctgagatggg gggaaagccg 1260

agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1320

gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1380

taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1440

ccccctcgcg gaccgcagtg tactaattat gctctcttga aattggctgg agatgttgag 1500

agcaatcccg ggcccatgcg cattagcaag ccccacctgc ggagcatcag catccagtgc 1560

tacctgtgcc tgctgctgaa cagccacttc ctgaccgagg ccggcatcca cgtgttcatc 1620

ctgggctgct tcagcgccgg actgcccaag accgaggcca actgggtgaa cgtgatcagc 1680

gacctgaaga agatcgagga cctgatccag agcatgcaca tcgacgccac cctgtacacc 1740

gagagcgacg tgcaccccag ctgcaaggtg accgccatga agtgctttct gctggaactg 1800

caggtgatca gcctggaaag cggcgacgcc agcatccacg acaccgtgga gaacctgatc 1860

atcctggcca acaacagcct gagcagcaac ggcaacgtga ccgagagcgg ctgcaaagag 1920

tgcgaggaac tggaagagaa gaacatcaaa gagtttctgc agagcttcgt gcacatcgtg 1980

cagatgttca tcaacaccag ctga 2004

<210> 17

<211> 2013

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 17

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atgcttctcc tggtgacaag ccttctgctc 360

tgtgagttac cacacccagc attcctcctg atcccagcta caccggctcc gaagtcctgc 420

ccggagcggc attattgggc acagggcaag ttgtgttgtc aaatgtgtga gccgggaacc 480

tttctcgtga aggattgcga tcagcatcgg aaggccgcgc agtgcgaccc atgtatacca 540

ggggtctcat tttccccaga tcaccatacg aggccgcact gtgagtcttg caggcattgt 600

aattccggct tgttggtccg caactgtact attactgcga atgcagagtg tgcttgtaga 660

aacggatggc agtgcaggga caaagaatgt acggagtgtg atccactgcc taaccccagt 720

cttacagcaa gatcttcaca ggccctcagc ccgcatcctc aaccaacaca tcttccttac 780

gtgtcagaaa tgttggaggc gcgaaccgca ggccatatgc agaccctggc ggactttcgg 840

cagctgccag cacgcacact tagtacacac tggccaccac aacgcagctt gtgctcttcc 900

gatttcatcc gcttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 960

ctagtaacag tggcctttat tattttctgg gtgaaacggg gcagaaagaa actcctgtat 1020

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1080

tgccgatttc cagaagaaga agaaggagga tgtgaactga aacgcgtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aaaagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg accgcagtgt actaattatg ctctcttgaa attggctgga 1500

gatgttgaga gcaatcccgg gcccatgcgc attagcaagc cccacctgcg gagcatcagc 1560

atccagtgct acctgtgcct gctgctgaac agccacttcc tgaccgaggc cggcatccac 1620

gtgttcatcc tgggctgctt cagcgccgga ctgcccaaga ccgaggccaa ctgggtgaac 1680

gtgatcagcg acctgaagaa gatcgaggac ctgatccaga gcatgcacat cgacgccacc 1740

ctgtacaccg agagcgacgt gcaccccagc tgcaaggtga ccgccatgaa gtgctttctg 1800

ctggaactgc aggtgatcag cctggaaagc ggcgacgcca gcatccacga caccgtggag 1860

aacctgatca tcctggccaa caacagcctg agcagcaacg gcaacgtgac cgagagcggc 1920

tgcaaagagt gcgaggaact ggaagagaag aacatcaaag agtttctgca gagcttcgtg 1980

cacatcgtgc agatgttcat caacaccagc tga 2013

<210> 18

<211> 1950

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 18

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atggcacggc cacatccctg gtggctgtgc 360

gttctgggga ccctggtggg gctctcagct actccagccc ccaagagctg cccagagagg 420

cactactggg ctcagggaaa gctgtgctgc cagatgtgtg agccaggaac attcctcgtg 480

aaggactgtg accagcatag aaaggctgct cagtgtgatc cttgcatacc gggggtctcc 540

ttctctcctg accaccacac ccggccccac tgtgagagct gtcggcactg taactctggt 600

cttctcgttc gcaactgcac catcactgcc aatgctgagt gtgcctgtcg caatggctgg 660

cagtgcaggg acaaggagtg caccgagtgt gatcctcttc caaacccttc gctgaccgct 720

cggtcgtctc aggccctgag cccacaccct cagcccaccc acttacctta tgtcagtgag 780

atgctggagg ccaggacagc tgggcacatg cagactctgg ctgacttcag gcagctgcct 840

gcccggactc tctctaccca ctggccaccc caaagatccc tgtgcagctc cgattttatt 900

cgcttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 960

gtggccttta ttattttctg ggtgctttgc gcacgcccac gccgcagccc cgcccaagaa 1020

gatggcaaag tctacatcaa catgccaggc aggggcaaac gcgtgaagtt cagcaggagc 1080

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1140

cgaagagagg agtacgatgt tttggacaaa agacgtggcc gggaccctga gatgggggga 1200

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1260

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1320

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1380

gccctgcccc ctcgcggacc gcagtgtact aattatgctc tcttgaaatt ggctggagat 1440

gttgagagca atcccgggcc catgcgcatt agcaagcccc acctgcggag catcagcatc 1500

cagtgctacc tgtgcctgct gctgaacagc cacttcctga ccgaggccgg catccacgtg 1560

ttcatcctgg gctgcttcag cgccggactg cccaagaccg aggccaactg ggtgaacgtg 1620

atcagcgacc tgaagaagat cgaggacctg atccagagca tgcacatcga cgccaccctg 1680

tacaccgaga gcgacgtgca ccccagctgc aaggtgaccg ccatgaagtg ctttctgctg 1740

gaactgcagg tgatcagcct ggaaagcggc gacgccagca tccacgacac cgtggagaac 1800

ctgatcatcc tggccaacaa cagcctgagc agcaacggca acgtgaccga gagcggctgc 1860

aaagagtgcg aggaactgga agagaagaac atcaaagagt ttctgcagag cttcgtgcac 1920

atcgtgcaga tgttcatcaa caccagctga 1950

<210> 19

<211> 1959

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 19

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atgcttctcc tggtgacaag ccttctgctc 360

tgtgagttac cacacccagc attcctcctg atcccagcta caccggctcc gaagtcctgc 420

ccggagcggc attattgggc acagggcaag ttgtgttgtc aaatgtgtga gccgggaacc 480

tttctcgtga aggattgcga tcagcatcgg aaggccgcgc agtgcgaccc atgtatacca 540

ggggtctcat tttccccaga tcaccatacg aggccgcact gtgagtcttg caggcattgt 600

aattccggct tgttggtccg caactgtact attactgcga atgcagagtg tgcttgtaga 660

aacggatggc agtgcaggga caaagaatgt acggagtgtg atccactgcc taaccccagt 720

cttacagcaa gatcttcaca ggccctcagc ccgcatcctc aaccaacaca tcttccttac 780

gtgtcagaaa tgttggaggc gcgaaccgca ggccatatgc agaccctggc ggactttcgg 840

cagctgccag cacgcacact tagtacacac tggccaccac aacgcagctt gtgctcttcc 900

gatttcatcc gcttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 960

ctagtaacag tggcctttat tattttctgg gtgctttgcg cacgcccacg ccgcagcccc 1020

gcccaagaag atggcaaagt ctacatcaac atgccaggca ggggcaaacg cgtgaagttc 1080

agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1140

aatctaggac gaagagagga gtacgatgtt ttggacaaaa gacgtggccg ggaccctgag 1200

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1260

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1320

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1380

cacatgcagg ccctgccccc tcgcggaccg cagtgtacta attatgctct cttgaaattg 1440

gctggagatg ttgagagcaa tcccgggccc atgcgcatta gcaagcccca cctgcggagc 1500

atcagcatcc agtgctacct gtgcctgctg ctgaacagcc acttcctgac cgaggccggc 1560

atccacgtgt tcatcctggg ctgcttcagc gccggactgc ccaagaccga ggccaactgg 1620

gtgaacgtga tcagcgacct gaagaagatc gaggacctga tccagagcat gcacatcgac 1680

gccaccctgt acaccgagag cgacgtgcac cccagctgca aggtgaccgc catgaagtgc 1740

tttctgctgg aactgcaggt gatcagcctg gaaagcggcg acgccagcat ccacgacacc 1800

gtggagaacc tgatcatcct ggccaacaac agcctgagca gcaacggcaa cgtgaccgag 1860

agcggctgca aagagtgcga ggaactggaa gagaagaaca tcaaagagtt tctgcagagc 1920

ttcgtgcaca tcgtgcagat gttcatcaac accagctga 1959

<210> 20

<211> 2034

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 20

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atggcacggc cacatccctg gtggctgtgc 360

gttctgggga ccctggtggg gctctcagct actccagccc ccaagagctg cccagagagg 420

cactactggg ctcagggaaa gctgtgctgc cagatgtgtg agccaggaac attcctcgtg 480

aaggactgtg accagcatag aaaggctgct cagtgtgatc cttgcatacc gggggtctcc 540

ttctctcctg accaccacac ccggccccac tgtgagagct gtcggcactg taactctggt 600

cttctcgttc gcaactgcac catcactgcc aatgctgagt gtgcctgtcg caatggctgg 660

cagtgcaggg acaaggagtg caccgagtgt gatcctcttc caaacccttc gctgaccgct 720

cggtcgtctc aggccctgag cccacaccct cagcccaccc acttacctta tgtcagtgag 780

atgctggagg ccaggacagc tgggcacatg cagactctgg ctgacttcag gcagctgcct 840

gcccggactc tctctaccca ctggccaccc caaagatccc tgtgcagctc cgattttatt 900

cgcttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 960

gtggccttta ttattttctg ggtgtacttc ctgggccggc tggtccctcg ggggcgaggg 1020

gctgcggagg cagcgacccg gaaacagcgt atcactgaga ccgagtcgcc ttatcaggag 1080

ctccagggtc agaggtcgga tgtctacagc gacctcaaca cacagaggcc gtattacaaa 1140

aaacgcgtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 1200

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caaaagacgt 1260

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1320

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1380

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1440

acctacgacg cccttcacat gcaggccctg ccccctcgcg gaccgcagtg tactaattat 1500

gctctcttga aattggctgg agatgttgag agcaatcccg ggcccatgcg cattagcaag 1560

ccccacctgc ggagcatcag catccagtgc tacctgtgcc tgctgctgaa cagccacttc 1620

ctgaccgagg ccggcatcca cgtgttcatc ctgggctgct tcagcgccgg actgcccaag 1680

accgaggcca actgggtgaa cgtgatcagc gacctgaaga agatcgagga cctgatccag 1740

agcatgcaca tcgacgccac cctgtacacc gagagcgacg tgcaccccag ctgcaaggtg 1800

accgccatga agtgctttct gctggaactg caggtgatca gcctggaaag cggcgacgcc 1860

agcatccacg acaccgtgga gaacctgatc atcctggcca acaacagcct gagcagcaac 1920

ggcaacgtga ccgagagcgg ctgcaaagag tgcgaggaac tggaagagaa gaacatcaaa 1980

gagtttctgc agagcttcgt gcacatcgtg cagatgttca tcaacaccag ctga 2034

<210> 21

<211> 2043

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 21

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atgcttctcc tggtgacaag ccttctgctc 360

tgtgagttac cacacccagc attcctcctg atcccagcta caccggctcc gaagtcctgc 420

ccggagcggc attattgggc acagggcaag ttgtgttgtc aaatgtgtga gccgggaacc 480

tttctcgtga aggattgcga tcagcatcgg aaggccgcgc agtgcgaccc atgtatacca 540

ggggtctcat tttccccaga tcaccatacg aggccgcact gtgagtcttg caggcattgt 600

aattccggct tgttggtccg caactgtact attactgcga atgcagagtg tgcttgtaga 660

aacggatggc agtgcaggga caaagaatgt acggagtgtg atccactgcc taaccccagt 720

cttacagcaa gatcttcaca ggccctcagc ccgcatcctc aaccaacaca tcttccttac 780

gtgtcagaaa tgttggaggc gcgaaccgca ggccatatgc agaccctggc ggactttcgg 840

cagctgccag cacgcacact tagtacacac tggccaccac aacgcagctt gtgctcttcc 900

gatttcatcc gcttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 960

ctagtaacag tggcctttat tattttctgg gtgtacttcc tgggccggct ggtccctcgg 1020

gggcgagggg ctgcggaggc agcgacccgg aaacagcgta tcactgagac cgagtcgcct 1080

tatcaggagc tccagggtca gaggtcggat gtctacagcg acctcaacac acagaggccg 1140

tattacaaaa aacgcgtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc 1200

cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1260

aaaagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1320

ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1380

aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1440

accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcgg accgcagtgt 1500

actaattatg ctctcttgaa attggctgga gatgttgaga gcaatcccgg gcccatgcgc 1560

attagcaagc cccacctgcg gagcatcagc atccagtgct acctgtgcct gctgctgaac 1620

agccacttcc tgaccgaggc cggcatccac gtgttcatcc tgggctgctt cagcgccgga 1680

ctgcccaaga ccgaggccaa ctgggtgaac gtgatcagcg acctgaagaa gatcgaggac 1740

ctgatccaga gcatgcacat cgacgccacc ctgtacaccg agagcgacgt gcaccccagc 1800

tgcaaggtga ccgccatgaa gtgctttctg ctggaactgc aggtgatcag cctggaaagc 1860

ggcgacgcca gcatccacga caccgtggag aacctgatca tcctggccaa caacagcctg 1920

agcagcaacg gcaacgtgac cgagagcggc tgcaaagagt gcgaggaact ggaagagaag 1980

aacatcaaag agtttctgca gagcttcgtg cacatcgtgc agatgttcat caacaccagc 2040

tga 2043

<210> 22

<211> 2031

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 22

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atggcacggc cacatccctg gtggctgtgc 360

gttctgggga ccctggtggg gctctcagct actccagccc ccaagagctg cccagagagg 420

cactactggg ctcagggaaa gctgtgctgc cagatgtgtg agccaggaac attcctcgtg 480

aaggactgtg accagcatag aaaggctgct cagtgtgatc cttgcatacc gggggtctcc 540

ttctctcctg accaccacac ccggccccac tgtgagagct gtcggcactg taactctggt 600

cttctcgttc gcaactgcac catcactgcc aatgctgagt gtgcctgtcg caatggctgg 660

cagtgcaggg acaaggagtg caccgagtgt gatcctcttc caaacccttc gctgaccgct 720

cggtcgtctc aggccctgag cccacaccct cagcccaccc acttacctta tgtcagtgag 780

atgctggagg ccaggacagc tgggcacatg cagactctgg ctgacttcag gcagctgcct 840

gcccggactc tctctaccca ctggccaccc caaagatccc tgtgcagctc cgattttatt 900

cgcttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 960

gtggccttta ttattttctg ggtgagcgcg aacgaacgct gcaaaagcaa agtggtgccg 1020

tgccgccaga aacagtggcg caccagcttt gatagcaaaa aactggatct gaactataac 1080

cattttgaaa gcatggaatg gagccatcgc agccgccgcg gccgcatttg gggcatgaaa 1140

cgcgtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 1200

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa aagacgtggc 1260

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1320

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1380

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1440

tacgacgccc ttcacatgca ggccctgccc cctcgcggac cgcagtgtac taattatgct 1500

ctcttgaaat tggctggaga tgttgagagc aatcccgggc ccatgcgcat tagcaagccc 1560

cacctgcgga gcatcagcat ccagtgctac ctgtgcctgc tgctgaacag ccacttcctg 1620

accgaggccg gcatccacgt gttcatcctg ggctgcttca gcgccggact gcccaagacc 1680

gaggccaact gggtgaacgt gatcagcgac ctgaagaaga tcgaggacct gatccagagc 1740

atgcacatcg acgccaccct gtacaccgag agcgacgtgc accccagctg caaggtgacc 1800

gccatgaagt gctttctgct ggaactgcag gtgatcagcc tggaaagcgg cgacgccagc 1860

atccacgaca ccgtggagaa cctgatcatc ctggccaaca acagcctgag cagcaacggc 1920

aacgtgaccg agagcggctg caaagagtgc gaggaactgg aagagaagaa catcaaagag 1980

tttctgcaga gcttcgtgca catcgtgcag atgttcatca acaccagctg a 2031

<210> 23

<211> 2040

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 23

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atgcttctcc tggtgacaag ccttctgctc 360

tgtgagttac cacacccagc attcctcctg atcccagcta caccggctcc gaagtcctgc 420

ccggagcggc attattgggc acagggcaag ttgtgttgtc aaatgtgtga gccgggaacc 480

tttctcgtga aggattgcga tcagcatcgg aaggccgcgc agtgcgaccc atgtatacca 540

ggggtctcat tttccccaga tcaccatacg aggccgcact gtgagtcttg caggcattgt 600

aattccggct tgttggtccg caactgtact attactgcga atgcagagtg tgcttgtaga 660

aacggatggc agtgcaggga caaagaatgt acggagtgtg atccactgcc taaccccagt 720

cttacagcaa gatcttcaca ggccctcagc ccgcatcctc aaccaacaca tcttccttac 780

gtgtcagaaa tgttggaggc gcgaaccgca ggccatatgc agaccctggc ggactttcgg 840

cagctgccag cacgcacact tagtacacac tggccaccac aacgcagctt gtgctcttcc 900

gatttcatcc gcttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 960

ctagtaacag tggcctttat tattttctgg gtgagcgcga acgaacgctg caaaagcaaa 1020

gtggtgccgt gccgccagaa acagtggcgc accagctttg atagcaaaaa actggatctg 1080

aactataacc attttgaaag catggaatgg agccatcgca gccgccgcgg ccgcatttgg 1140

ggcatgaaac gcgtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 1200

aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaaa 1260

agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1320

ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1380

ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1440

aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgcggacc gcagtgtact 1500

aattatgctc tcttgaaatt ggctggagat gttgagagca atcccgggcc catgcgcatt 1560

agcaagcccc acctgcggag catcagcatc cagtgctacc tgtgcctgct gctgaacagc 1620

cacttcctga ccgaggccgg catccacgtg ttcatcctgg gctgcttcag cgccggactg 1680

cccaagaccg aggccaactg ggtgaacgtg atcagcgacc tgaagaagat cgaggacctg 1740

atccagagca tgcacatcga cgccaccctg tacaccgaga gcgacgtgca ccccagctgc 1800

aaggtgaccg ccatgaagtg ctttctgctg gaactgcagg tgatcagcct ggaaagcggc 1860

gacgccagca tccacgacac cgtggagaac ctgatcatcc tggccaacaa cagcctgagc 1920

agcaacggca acgtgaccga gagcggctgc aaagagtgcg aggaactgga agagaagaac 1980

atcaaagagt ttctgcagag cttcgtgcac atcgtgcaga tgttcatcaa caccagctga 2040

<210> 24

<211> 1986

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 24

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atggcacggc cacatccctg gtggctgtgc 360

gttctgggga ccctggtggg gctctcagct actccagccc ccaagagctg cccagagagg 420

cactactggg ctcagggaaa gctgtgctgc cagatgtgtg agccaggaac attcctcgtg 480

aaggactgtg accagcatag aaaggctgct cagtgtgatc cttgcatacc gggggtctcc 540

ttctctcctg accaccacac ccggccccac tgtgagagct gtcggcactg taactctggt 600

cttctcgttc gcaactgcac catcactgcc aatgctgagt gtgcctgtcg caatggctgg 660

cagtgcaggg acaaggagtg caccgagtgt gatcctcttc caaacccttc gctgaccgct 720

cggtcgtctc aggccctgag cccacaccct cagcccaccc acttacctta tgtcagtgag 780

atgctggagg ccaggacagc tgggcacatg cagactctgg ctgacttcag gcagctgcct 840

gcccggactc tctctaccca ctggccaccc caaagatccc tgtgcagctc cgattttatt 900

cgcatccttg tgatcttctc tggaatgttc cttgttttca ccctggccgg ggccctgttc 960

ctccataaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 1020

gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 1080

ggatgtgaac tgaaacgcgt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 1140

ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1200

gacaaaagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1260

gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1320

atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1380

gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg cggaccgcag 1440

tgtactaatt atgctctctt gaaattggct ggagatgttg agagcaatcc cgggcccatg 1500

cgcattagca agccccacct gcggagcatc agcatccagt gctacctgtg cctgctgctg 1560

aacagccact tcctgaccga ggccggcatc cacgtgttca tcctgggctg cttcagcgcc 1620

ggactgccca agaccgaggc caactgggtg aacgtgatca gcgacctgaa gaagatcgag 1680

gacctgatcc agagcatgca catcgacgcc accctgtaca ccgagagcga cgtgcacccc 1740

agctgcaagg tgaccgccat gaagtgcttt ctgctggaac tgcaggtgat cagcctggaa 1800

agcggcgacg ccagcatcca cgacaccgtg gagaacctga tcatcctggc caacaacagc 1860

ctgagcagca acggcaacgt gaccgagagc ggctgcaaag agtgcgagga actggaagag 1920

aagaacatca aagagtttct gcagagcttc gtgcacatcg tgcagatgtt catcaacacc 1980

agctga 1986

<210> 25

<211> 1995

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 25

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atgcttctcc tggtgacaag ccttctgctc 360

tgtgagttac cacacccagc attcctcctg atcccagcta caccggctcc gaagtcctgc 420

ccggagcggc attattgggc acagggcaag ttgtgttgtc aaatgtgtga gccgggaacc 480

tttctcgtga aggattgcga tcagcatcgg aaggccgcgc agtgcgaccc atgtatacca 540

ggggtctcat tttccccaga tcaccatacg aggccgcact gtgagtcttg caggcattgt 600

aattccggct tgttggtccg caactgtact attactgcga atgcagagtg tgcttgtaga 660

aacggatggc agtgcaggga caaagaatgt acggagtgtg atccactgcc taaccccagt 720

cttacagcaa gatcttcaca ggccctcagc ccgcatcctc aaccaacaca tcttccttac 780

gtgtcagaaa tgttggaggc gcgaaccgca ggccatatgc agaccctggc ggactttcgg 840

cagctgccag cacgcacact tagtacacac tggccaccac aacgcagctt gtgctcttcc 900

gatttcatcc gcatactggt catcttttct ggaatgttcc ttgtgttcac cctggcagga 960

gccctgttcc ttcacaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 1020

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1080

gaagaaggag gatgtgaact gaaacgcgtg aagttcagca ggagcgcaga cgcccccgcg 1140

taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 1200

gatgttttgg acaaaagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 1260

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1320

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1380

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1440

ggaccgcagt gtactaatta tgctctcttg aaattggctg gagatgttga gagcaatccc 1500

gggcccatgc gcattagcaa gccccacctg cggagcatca gcatccagtg ctacctgtgc 1560

ctgctgctga acagccactt cctgaccgag gccggcatcc acgtgttcat cctgggctgc 1620

ttcagcgccg gactgcccaa gaccgaggcc aactgggtga acgtgatcag cgacctgaag 1680

aagatcgagg acctgatcca gagcatgcac atcgacgcca ccctgtacac cgagagcgac 1740

gtgcacccca gctgcaaggt gaccgccatg aagtgctttc tgctggaact gcaggtgatc 1800

agcctggaaa gcggcgacgc cagcatccac gacaccgtgg agaacctgat catcctggcc 1860

aacaacagcc tgagcagcaa cggcaacgtg accgagagcg gctgcaaaga gtgcgaggaa 1920

ctggaagaga agaacatcaa agagtttctg cagagcttcg tgcacatcgt gcagatgttc 1980

atcaacacca gctga 1995

<210> 26

<211> 1860

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 26

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atggcacggc cacatccctg gtggctgtgc 360

gttctgggga ccctggtggg gctctcagct actccagccc ccaagagctg cccagagagg 420

cactactggg ctcagggaaa gctgtgctgc cagatgtgtg agccaggaac attcctcgtg 480

aaggactgtg accagcatag aaaggctgct cagtgtgatc cttgcatacc gggggtctcc 540

ttctctcctg accaccacac ccggccccac tgtgagagct gtcggcactg taactctggt 600

cttctcgttc gcaactgcac catcactgcc aatgctgagt gtgcctgtcg caatggctgg 660

cagtgcaggg acaaggagtg caccgagtgt gatcctcttc caaacccttc gctgaccgct 720

cggtcgtctc aggccctgag cccacaccct cagcccaccc acttacctta tgtcagtgag 780

atgctggagg ccaggacagc tgggcacatg cagactctgg ctgacttcag gcagctgcct 840

gcccggactc tctctaccca ctggccaccc caaagatccc tgtgcagctc cgattttatt 900

cgcatccttg tgatcttctc tggaatgttc cttgttttca ccctggccgg ggccctgttc 960

ctccataaac gcgtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 1020

aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaaa 1080

agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1140

ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1200

ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1260

aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgcggacc gcagtgtact 1320

aattatgctc tcttgaaatt ggctggagat gttgagagca atcccgggcc catgcgcatt 1380

agcaagcccc acctgcggag catcagcatc cagtgctacc tgtgcctgct gctgaacagc 1440

cacttcctga ccgaggccgg catccacgtg ttcatcctgg gctgcttcag cgccggactg 1500

cccaagaccg aggccaactg ggtgaacgtg atcagcgacc tgaagaagat cgaggacctg 1560

atccagagca tgcacatcga cgccaccctg tacaccgaga gcgacgtgca ccccagctgc 1620

aaggtgaccg ccatgaagtg ctttctgctg gaactgcagg tgatcagcct ggaaagcggc 1680

gacgccagca tccacgacac cgtggagaac ctgatcatcc tggccaacaa cagcctgagc 1740

agcaacggca acgtgaccga gagcggctgc aaagagtgcg aggaactgga agagaagaac 1800

atcaaagagt ttctgcagag cttcgtgcac atcgtgcaga tgttcatcaa caccagctga 1860

<210> 27

<211> 1866

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 27

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atgcttctcc tggtgacaag ccttctgctc 360

tgtgagttac cacacccagc attcctcctg atcccagcta caccggctcc gaagtcctgc 420

ccggagcggc attattgggc acagggcaag ttgtgttgtc aaatgtgtga gccgggaacc 480

tttctcgtga aggattgcga tcagcatcgg aaggccgcgc agtgcgaccc atgtatacca 540

ggggtctcat tttccccaga tcaccatacg aggccgcact gtgagtcttg caggcattgt 600

aattccggct tgttggtccg caactgtact attactgcga atgcagagtg tgcttgtaga 660

aacggatggc agtgcaggga caaagaatgt acggagtgtg atccactgcc taaccccagt 720

cttacagcaa gatcttcaca ggccctcagc ccgcatcctc aaccaacaca tcttccttac 780

gtgtcagaaa tgttggaggc gcgaaccgca ggccatatgc agaccctggc ggactttcgg 840

cagctgccag cacgcacact tagtacacac tggccaccac aacgcagctt gtgctcttcc 900

gatttcatcc gcatactggt catcttttct ggaatgttcc ttgtgttcac cctggcagga 960

gccctgttcc ttcaccgcgt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 1020

ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1080

gacaaaagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1140

gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1200

atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1260

gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg cggaccgcag 1320

tgtactaatt atgctctctt gaaattggct ggagatgttg agagcaatcc cgggcccatg 1380

cgcattagca agccccacct gcggagcatc agcatccagt gctacctgtg cctgctgctg 1440

aacagccact tcctgaccga ggccggcatc cacgtgttca tcctgggctg cttcagcgcc 1500

ggactgccca agaccgaggc caactgggtg aacgtgatca gcgacctgaa gaagatcgag 1560

gacctgatcc agagcatgca catcgacgcc accctgtaca ccgagagcga cgtgcacccc 1620

agctgcaagg tgaccgccat gaagtgcttt ctgctggaac tgcaggtgat cagcctggaa 1680

agcggcgacg ccagcatcca cgacaccgtg gagaacctga tcatcctggc caacaacagc 1740

ctgagcagca acggcaacgt gaccgagagc ggctgcaaag agtgcgagga actggaagag 1800

aagaacatca aagagtttct gcagagcttc gtgcacatcg tgcagatgtt catcaacacc 1860

agctga 1866

<210> 28

<211> 1980

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 28

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atggcacggc cacatccctg gtggctgtgc 360

gttctgggga ccctggtggg gctctcagct actccagccc ccaagagctg cccagagagg 420

cactactggg ctcagggaaa gctgtgctgc cagatgtgtg agccaggaac attcctcgtg 480

aaggactgtg accagcatag aaaggctgct cagtgtgatc cttgcatacc gggggtctcc 540

ttctctcctg accaccacac ccggccccac tgtgagagct gtcggcactg taactctggt 600

cttctcgttc gcaactgcac catcactgcc aatgctgagt gtgcctgtcg caatggctgg 660

cagtgcaggg acaaggagtg caccgagtgt gatcctcttc caaacccttc gctgaccgct 720

cggtcgtctc aggccctgag cccacaccct cagcccaccc acttacctta tgtcagtgag 780

atgctggagg ccaggacagc tgggcacatg cagactctgg ctgacttcag gcagctgcct 840

gcccggactc tctctaccca ctggccaccc caaagatccc tgtgcagctc cgattttatt 900

cgcatccttg tgatcttctc tggaatgttc cttgttttca ccctggccgg ggccctgttc 960

ctccatagga gtaagaggag caggctcctg cacagtgact acatgaacat gactccccgc 1020

cgccccgggc ccacccgcaa gcattaccag ccctatgccc caccacgcga cttcgcagcc 1080

tatcgctcac gcgtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 1140

aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaaa 1200

agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1260

ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1320

ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1380

aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgcggacc gcagtgtact 1440

aattatgctc tcttgaaatt ggctggagat gttgagagca atcccgggcc catgcgcatt 1500

agcaagcccc acctgcggag catcagcatc cagtgctacc tgtgcctgct gctgaacagc 1560

cacttcctga ccgaggccgg catccacgtg ttcatcctgg gctgcttcag cgccggactg 1620

cccaagaccg aggccaactg ggtgaacgtg atcagcgacc tgaagaagat cgaggacctg 1680

atccagagca tgcacatcga cgccaccctg tacaccgaga gcgacgtgca ccccagctgc 1740

aaggtgaccg ccatgaagtg ctttctgctg gaactgcagg tgatcagcct ggaaagcggc 1800

gacgccagca tccacgacac cgtggagaac ctgatcatcc tggccaacaa cagcctgagc 1860

agcaacggca acgtgaccga gagcggctgc aaagagtgcg aggaactgga agagaagaac 1920

atcaaagagt ttctgcagag cttcgtgcac atcgtgcaga tgttcatcaa caccagctga 1980

<210> 29

<211> 1989

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 29

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atgcttctcc tggtgacaag ccttctgctc 360

tgtgagttac cacacccagc attcctcctg atcccagcta caccggctcc gaagtcctgc 420

ccggagcggc attattgggc acagggcaag ttgtgttgtc aaatgtgtga gccgggaacc 480

tttctcgtga aggattgcga tcagcatcgg aaggccgcgc agtgcgaccc atgtatacca 540

ggggtctcat tttccccaga tcaccatacg aggccgcact gtgagtcttg caggcattgt 600

aattccggct tgttggtccg caactgtact attactgcga atgcagagtg tgcttgtaga 660

aacggatggc agtgcaggga caaagaatgt acggagtgtg atccactgcc taaccccagt 720

cttacagcaa gatcttcaca ggccctcagc ccgcatcctc aaccaacaca tcttccttac 780

gtgtcagaaa tgttggaggc gcgaaccgca ggccatatgc agaccctggc ggactttcgg 840

cagctgccag cacgcacact tagtacacac tggccaccac aacgcagctt gtgctcttcc 900

gatttcatcc gcatactggt catcttttct ggaatgttcc ttgtgttcac cctggcagga 960

gccctgttcc ttcacaggag taagaggagc aggctcctgc acagtgacta catgaacatg 1020

actccccgcc gccccgggcc cacccgcaag cattaccagc cctatgcccc accacgcgac 1080

ttcgcagcct atcgctcacg cgtgaagttc agcaggagcg cagacgcccc cgcgtaccag 1140

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1200

ttggacaaaa gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1260

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1320

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1380

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgcggaccg 1440

cagtgtacta attatgctct cttgaaattg gctggagatg ttgagagcaa tcccgggccc 1500

atgcgcatta gcaagcccca cctgcggagc atcagcatcc agtgctacct gtgcctgctg 1560

ctgaacagcc acttcctgac cgaggccggc atccacgtgt tcatcctggg ctgcttcagc 1620

gccggactgc ccaagaccga ggccaactgg gtgaacgtga tcagcgacct gaagaagatc 1680

gaggacctga tccagagcat gcacatcgac gccaccctgt acaccgagag cgacgtgcac 1740

cccagctgca aggtgaccgc catgaagtgc tttctgctgg aactgcaggt gatcagcctg 1800

gaaagcggcg acgccagcat ccacgacacc gtggagaacc tgatcatcct ggccaacaac 1860

agcctgagca gcaacggcaa cgtgaccgag agcggctgca aagagtgcga ggaactggaa 1920

gagaagaaca tcaaagagtt tctgcagagc ttcgtgcaca tcgtgcagat gttcatcaac 1980

accagctga 1989

<210> 30

<211> 1998

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 30

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atggcacggc cacatccctg gtggctgtgc 360

gttctgggga ccctggtggg gctctcagct actccagccc ccaagagctg cccagagagg 420

cactactggg ctcagggaaa gctgtgctgc cagatgtgtg agccaggaac attcctcgtg 480

aaggactgtg accagcatag aaaggctgct cagtgtgatc cttgcatacc gggggtctcc 540

ttctctcctg accaccacac ccggccccac tgtgagagct gtcggcactg taactctggt 600

cttctcgttc gcaactgcac catcactgcc aatgctgagt gtgcctgtcg caatggctgg 660

cagtgcaggg acaaggagtg caccgagtgt gatcctcttc caaacccttc gctgaccgct 720

cggtcgtctc aggccctgag cccacaccct cagcccaccc acttacctta tgtcagtgag 780

atgctggagg ccaggacagc tgggcacatg cagactctgg ctgacttcag gcagctgcct 840

gcccggactc tctctaccca ctggccaccc caaagatccc tgtgcagctc cgattttatt 900

cgcttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 960

gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 1020

atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 1080

ccacgcgact tcgcagccta tcgctcacgc gtgaagttca gcaggagcgc agacgccccc 1140

gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1200

tacgatgttt tggacaaaag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1260

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1320

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1380

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1440

cgcggaccgc agtgtactaa ttatgctctc ttgaaattgg ctggagatgt tgagagcaat 1500

cccgggccca tgcgcattag caagccccac ctgcggagca tcagcatcca gtgctacctg 1560

tgcctgctgc tgaacagcca cttcctgacc gaggccggca tccacgtgtt catcctgggc 1620

tgcttcagcg ccggactgcc caagaccgag gccaactggg tgaacgtgat cagcgacctg 1680

aagaagatcg aggacctgat ccagagcatg cacatcgacg ccaccctgta caccgagagc 1740

gacgtgcacc ccagctgcaa ggtgaccgcc atgaagtgct ttctgctgga actgcaggtg 1800

atcagcctgg aaagcggcga cgccagcatc cacgacaccg tggagaacct gatcatcctg 1860

gccaacaaca gcctgagcag caacggcaac gtgaccgaga gcggctgcaa agagtgcgag 1920

gaactggaag agaagaacat caaagagttt ctgcagagct tcgtgcacat cgtgcagatg 1980

ttcatcaaca ccagctga 1998

<210> 31

<211> 2007

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 31

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atgcttctcc tggtgacaag ccttctgctc 360

tgtgagttac cacacccagc attcctcctg atcccagcta caccggctcc gaagtcctgc 420

ccggagcggc attattgggc acagggcaag ttgtgttgtc aaatgtgtga gccgggaacc 480

tttctcgtga aggattgcga tcagcatcgg aaggccgcgc agtgcgaccc atgtatacca 540

ggggtctcat tttccccaga tcaccatacg aggccgcact gtgagtcttg caggcattgt 600

aattccggct tgttggtccg caactgtact attactgcga atgcagagtg tgcttgtaga 660

aacggatggc agtgcaggga caaagaatgt acggagtgtg atccactgcc taaccccagt 720

cttacagcaa gatcttcaca ggccctcagc ccgcatcctc aaccaacaca tcttccttac 780

gtgtcagaaa tgttggaggc gcgaaccgca ggccatatgc agaccctggc ggactttcgg 840

cagctgccag cacgcacact tagtacacac tggccaccac aacgcagctt gtgctcttcc 900

gatttcatcc gcttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 960

ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 1020

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 1080

tatgccccac cacgcgactt cgcagcctat cgctcacgcg tgaagttcag caggagcgca 1140

gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1200

agagaggagt acgatgtttt ggacaaaaga cgtggccggg accctgagat ggggggaaag 1260

ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 1320

gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 1380

ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 1440

ctgccccctc gcggaccgca gtgtactaat tatgctctct tgaaattggc tggagatgtt 1500

gagagcaatc ccgggcccat gcgcattagc aagccccacc tgcggagcat cagcatccag 1560

tgctacctgt gcctgctgct gaacagccac ttcctgaccg aggccggcat ccacgtgttc 1620

atcctgggct gcttcagcgc cggactgccc aagaccgagg ccaactgggt gaacgtgatc 1680

agcgacctga agaagatcga ggacctgatc cagagcatgc acatcgacgc caccctgtac 1740

accgagagcg acgtgcaccc cagctgcaag gtgaccgcca tgaagtgctt tctgctggaa 1800

ctgcaggtga tcagcctgga aagcggcgac gccagcatcc acgacaccgt ggagaacctg 1860

atcatcctgg ccaacaacag cctgagcagc aacggcaacg tgaccgagag cggctgcaaa 1920

gagtgcgagg aactggaaga gaagaacatc aaagagtttc tgcagagctt cgtgcacatc 1980

gtgcagatgt tcatcaacac cagctga 2007

<210> 32

<211> 1932

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 32

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atggcacggc cacatccctg gtggctgtgc 360

gttctgggga ccctggtggg gctctcagct actccagccc ccaagagctg cccagagagg 420

cactactggg ctcagggaaa gctgtgctgc cagatgtgtg agccaggaac attcctcgtg 480

aaggactgtg accagcatag aaaggctgct cagtgtgatc cttgcatacc gggggtctcc 540

ttctctcctg accaccacac ccggccccac tgtgagagct gtcggcactg taactctggt 600

cttctcgttc gcaactgcac catcactgcc aatgctgagt gtgcctgtcg caatggctgg 660

cagtgcaggg acaaggagtg caccgagtgt gatcctcttc caaacccttc gctgaccgct 720

cggtcgtctc aggccctgag cccacaccct cagcccaccc acttacctta tgtcagtgag 780

atgctggagg ccaggacagc tgggcacatg cagactctgg ctgacttcag gcagctgcct 840

gcccggactc tctctaccca ctggccaccc caaagatccc tgtgcagctc cgattttatt 900

cgcatactgg tcatcttttc tggaatgttc cttgtgttca ccctggcagg agccctgttc 960

cttcaccttt gcgcacgccc acgccgcagc cccgcccaag aagatggcaa agtctacatc 1020

aacatgccag gcaggggcaa acgcgtgaag ttcagcagga gcgcagacgc ccccgcgtac 1080

cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1140

gttttggaca aaagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 1200

cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1260

attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1320

agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgcgga 1380

ccgcagtgta ctaattatgc tctcttgaaa ttggctggag atgttgagag caatcccggg 1440

cccatgcgca ttagcaagcc ccacctgcgg agcatcagca tccagtgcta cctgtgcctg 1500

ctgctgaaca gccacttcct gaccgaggcc ggcatccacg tgttcatcct gggctgcttc 1560

agcgccggac tgcccaagac cgaggccaac tgggtgaacg tgatcagcga cctgaagaag 1620

atcgaggacc tgatccagag catgcacatc gacgccaccc tgtacaccga gagcgacgtg 1680

caccccagct gcaaggtgac cgccatgaag tgctttctgc tggaactgca ggtgatcagc 1740

ctggaaagcg gcgacgccag catccacgac accgtggaga acctgatcat cctggccaac 1800

aacagcctga gcagcaacgg caacgtgacc gagagcggct gcaaagagtg cgaggaactg 1860

gaagagaaga acatcaaaga gtttctgcag agcttcgtgc acatcgtgca gatgttcatc 1920

aacaccagct ga 1932

<210> 33

<211> 1941

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 33

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atgcttctcc tggtgacaag ccttctgctc 360

tgtgagttac cacacccagc attcctcctg atcccagcta caccggctcc gaagtcctgc 420

ccggagcggc attattgggc acagggcaag ttgtgttgtc aaatgtgtga gccgggaacc 480

tttctcgtga aggattgcga tcagcatcgg aaggccgcgc agtgcgaccc atgtatacca 540

ggggtctcat tttccccaga tcaccatacg aggccgcact gtgagtcttg caggcattgt 600

aattccggct tgttggtccg caactgtact attactgcga atgcagagtg tgcttgtaga 660

aacggatggc agtgcaggga caaagaatgt acggagtgtg atccactgcc taaccccagt 720

cttacagcaa gatcttcaca ggccctcagc ccgcatcctc aaccaacaca tcttccttac 780

gtgtcagaaa tgttggaggc gcgaaccgca ggccatatgc agaccctggc ggactttcgg 840

cagctgccag cacgcacact tagtacacac tggccaccac aacgcagctt gtgctcttcc 900

gatttcatcc gcatactggt catcttttct ggaatgttcc ttgtgttcac cctggcagga 960

gccctgttcc ttcacctttg cgcacgccca cgccgcagcc ccgcccaaga agatggcaaa 1020

gtctacatca acatgccagg caggggcaaa cgcgtgaagt tcagcaggag cgcagacgcc 1080

cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1140

gagtacgatg ttttggacaa aagacgtggc cgggaccctg agatgggggg aaagccgaga 1200

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1260

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1320

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1380

cctcgcggac cgcagtgtac taattatgct ctcttgaaat tggctggaga tgttgagagc 1440

aatcccgggc ccatgcgcat tagcaagccc cacctgcgga gcatcagcat ccagtgctac 1500

ctgtgcctgc tgctgaacag ccacttcctg accgaggccg gcatccacgt gttcatcctg 1560

ggctgcttca gcgccggact gcccaagacc gaggccaact gggtgaacgt gatcagcgac 1620

ctgaagaaga tcgaggacct gatccagagc atgcacatcg acgccaccct gtacaccgag 1680

agcgacgtgc accccagctg caaggtgacc gccatgaagt gctttctgct ggaactgcag 1740

gtgatcagcc tggaaagcgg cgacgccagc atccacgaca ccgtggagaa cctgatcatc 1800

ctggccaaca acagcctgag cagcaacggc aacgtgaccg agagcggctg caaagagtgc 1860

gaggaactgg aagagaagaa catcaaagag tttctgcaga gcttcgtgca catcgtgcag 1920

atgttcatca acaccagctg a 1941

<210> 34

<211> 2016

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 34

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atggcacggc cacatccctg gtggctgtgc 360

gttctgggga ccctggtggg gctctcagct actccagccc ccaagagctg cccagagagg 420

cactactggg ctcagggaaa gctgtgctgc cagatgtgtg agccaggaac attcctcgtg 480

aaggactgtg accagcatag aaaggctgct cagtgtgatc cttgcatacc gggggtctcc 540

ttctctcctg accaccacac ccggccccac tgtgagagct gtcggcactg taactctggt 600

cttctcgttc gcaactgcac catcactgcc aatgctgagt gtgcctgtcg caatggctgg 660

cagtgcaggg acaaggagtg caccgagtgt gatcctcttc caaacccttc gctgaccgct 720

cggtcgtctc aggccctgag cccacaccct cagcccaccc acttacctta tgtcagtgag 780

atgctggagg ccaggacagc tgggcacatg cagactctgg ctgacttcag gcagctgcct 840

gcccggactc tctctaccca ctggccaccc caaagatccc tgtgcagctc cgattttatt 900

cgcatccttg tgatcttctc tggaatgttc cttgttttca ccctggccgg ggccctgttc 960

ctccattact tcctgggccg gctggtccct cgggggcgag gggctgcgga ggcagcgacc 1020

cggaaacagc gtatcactga gaccgagtcg ccttatcagg agctccaggg tcagaggtcg 1080

gatgtctaca gcgacctcaa cacacagagg ccgtattaca aaaaacgcgt gaagttcagc 1140

aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1200

ctaggacgaa gagaggagta cgatgttttg gacaaaagac gtggccggga ccctgagatg 1260

gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1320

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440

atgcaggccc tgccccctcg cggaccgcag tgtactaatt atgctctctt gaaattggct 1500

ggagatgttg agagcaatcc cgggcccatg cgcattagca agccccacct gcggagcatc 1560

agcatccagt gctacctgtg cctgctgctg aacagccact tcctgaccga ggccggcatc 1620

cacgtgttca tcctgggctg cttcagcgcc ggactgccca agaccgaggc caactgggtg 1680

aacgtgatca gcgacctgaa gaagatcgag gacctgatcc agagcatgca catcgacgcc 1740

accctgtaca ccgagagcga cgtgcacccc agctgcaagg tgaccgccat gaagtgcttt 1800

ctgctggaac tgcaggtgat cagcctggaa agcggcgacg ccagcatcca cgacaccgtg 1860

gagaacctga tcatcctggc caacaacagc ctgagcagca acggcaacgt gaccgagagc 1920

ggctgcaaag agtgcgagga actggaagag aagaacatca aagagtttct gcagagcttc 1980

gtgcacatcg tgcagatgtt catcaacacc agctga 2016

<210> 35

<211> 2025

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 35

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atgcttctcc tggtgacaag ccttctgctc 360

tgtgagttac cacacccagc attcctcctg atcccagcta caccggctcc gaagtcctgc 420

ccggagcggc attattgggc acagggcaag ttgtgttgtc aaatgtgtga gccgggaacc 480

tttctcgtga aggattgcga tcagcatcgg aaggccgcgc agtgcgaccc atgtatacca 540

ggggtctcat tttccccaga tcaccatacg aggccgcact gtgagtcttg caggcattgt 600

aattccggct tgttggtccg caactgtact attactgcga atgcagagtg tgcttgtaga 660

aacggatggc agtgcaggga caaagaatgt acggagtgtg atccactgcc taaccccagt 720

cttacagcaa gatcttcaca ggccctcagc ccgcatcctc aaccaacaca tcttccttac 780

gtgtcagaaa tgttggaggc gcgaaccgca ggccatatgc agaccctggc ggactttcgg 840

cagctgccag cacgcacact tagtacacac tggccaccac aacgcagctt gtgctcttcc 900

gatttcatcc gcatactggt catcttttct ggaatgttcc ttgtgttcac cctggcagga 960

gccctgttcc ttcactactt cctgggccgg ctggtccctc gggggcgagg ggctgcggag 1020

gcagcgaccc ggaaacagcg tatcactgag accgagtcgc cttatcagga gctccagggt 1080

cagaggtcgg atgtctacag cgacctcaac acacagaggc cgtattacaa aaaacgcgtg 1140

aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1200

gagctcaatc taggacgaag agaggagtac gatgttttgg acaaaagacg 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 ggaccgcagt gtactaatta tgctctcttg 1500

aaattggctg gagatgttga gagcaatccc gggcccatgc gcattagcaa gccccacctg 1560

cggagcatca gcatccagtg ctacctgtgc ctgctgctga acagccactt cctgaccgag 1620

gccggcatcc acgtgttcat cctgggctgc ttcagcgccg gactgcccaa gaccgaggcc 1680

aactgggtga acgtgatcag cgacctgaag aagatcgagg acctgatcca gagcatgcac 1740

atcgacgcca ccctgtacac cgagagcgac gtgcacccca gctgcaaggt gaccgccatg 1800

aagtgctttc tgctggaact gcaggtgatc agcctggaaa gcggcgacgc cagcatccac 1860

gacaccgtgg agaacctgat catcctggcc aacaacagcc tgagcagcaa cggcaacgtg 1920

accgagagcg gctgcaaaga gtgcgaggaa ctggaagaga agaacatcaa agagtttctg 1980

cagagcttcg tgcacatcgt gcagatgttc atcaacacca gctga 2025

<210> 36

<211> 2013

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 36

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atggcacggc cacatccctg gtggctgtgc 360

gttctgggga ccctggtggg gctctcagct actccagccc ccaagagctg cccagagagg 420

cactactggg ctcagggaaa gctgtgctgc cagatgtgtg agccaggaac attcctcgtg 480

aaggactgtg accagcatag aaaggctgct cagtgtgatc cttgcatacc gggggtctcc 540

ttctctcctg accaccacac ccggccccac tgtgagagct gtcggcactg taactctggt 600

cttctcgttc gcaactgcac catcactgcc aatgctgagt gtgcctgtcg caatggctgg 660

cagtgcaggg acaaggagtg caccgagtgt gatcctcttc caaacccttc gctgaccgct 720

cggtcgtctc aggccctgag cccacaccct cagcccaccc acttacctta tgtcagtgag 780

atgctggagg ccaggacagc tgggcacatg cagactctgg ctgacttcag gcagctgcct 840

gcccggactc tctctaccca ctggccaccc caaagatccc tgtgcagctc cgattttatt 900

cgcatccttg tgatcttctc tggaatgttc cttgttttca ccctggccgg ggccctgttc 960

ctccatagcg cgaacgaacg ctgcaaaagc aaagtggtgc cgtgccgcca gaaacagtgg 1020

cgcaccagct ttgatagcaa aaaactggat ctgaactata accattttga aagcatggaa 1080

tggagccatc gcagccgccg cggccgcatt tggggcatga aacgcgtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aaaagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg accgcagtgt actaattatg ctctcttgaa attggctgga 1500

gatgttgaga gcaatcccgg gcccatgcgc attagcaagc cccacctgcg gagcatcagc 1560

atccagtgct acctgtgcct gctgctgaac agccacttcc tgaccgaggc cggcatccac 1620

gtgttcatcc tgggctgctt cagcgccgga ctgcccaaga ccgaggccaa ctgggtgaac 1680

gtgatcagcg acctgaagaa gatcgaggac ctgatccaga gcatgcacat cgacgccacc 1740

ctgtacaccg agagcgacgt gcaccccagc tgcaaggtga ccgccatgaa gtgctttctg 1800

ctggaactgc aggtgatcag cctggaaagc ggcgacgcca gcatccacga caccgtggag 1860

aacctgatca tcctggccaa caacagcctg agcagcaacg gcaacgtgac cgagagcggc 1920

tgcaaagagt gcgaggaact ggaagagaag aacatcaaag agtttctgca gagcttcgtg 1980

cacatcgtgc agatgttcat caacaccagc tga 2013

<210> 37

<211> 2022

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 37

atgacaagag ttactaacag cccctctctc caagctcact tacaggctct ctacttagtc 60

cagcacgaag tctggagacc tctggcggca gcctaccaag aacaactgga ccgaccggtg 120

gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc gccgacacca gactaagaac 180

ctagaacctc gctggaaagg accttacaca gtcctgctga ccacccccac cgccctcaaa 240

gtagacggca tcgcagcttg gatacacgcc gcccacgtga aggctgccga ccccgggggt 300

ggaccatcct ctagactgcc atgctcgagg atgcttctcc tggtgacaag ccttctgctc 360

tgtgagttac cacacccagc attcctcctg atcccagcta caccggctcc gaagtcctgc 420

ccggagcggc attattgggc acagggcaag ttgtgttgtc aaatgtgtga gccgggaacc 480

tttctcgtga aggattgcga tcagcatcgg aaggccgcgc agtgcgaccc atgtatacca 540

ggggtctcat tttccccaga tcaccatacg aggccgcact gtgagtcttg caggcattgt 600

aattccggct tgttggtccg caactgtact attactgcga atgcagagtg tgcttgtaga 660

aacggatggc agtgcaggga caaagaatgt acggagtgtg atccactgcc taaccccagt 720

cttacagcaa gatcttcaca ggccctcagc ccgcatcctc aaccaacaca tcttccttac 780

gtgtcagaaa tgttggaggc gcgaaccgca ggccatatgc agaccctggc ggactttcgg 840

cagctgccag cacgcacact tagtacacac tggccaccac aacgcagctt gtgctcttcc 900

gatttcatcc gcatactggt catcttttct ggaatgttcc ttgtgttcac cctggcagga 960

gccctgttcc ttcacagcgc gaacgaacgc tgcaaaagca aagtggtgcc gtgccgccag 1020

aaacagtggc gcaccagctt tgatagcaaa aaactggatc tgaactataa ccattttgaa 1080

agcatggaat ggagccatcg cagccgccgc ggccgcattt ggggcatgaa acgcgtgaag 1140

ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1200

ctcaatctag gacgaagaga ggagtacgat gttttggaca aaagacgtgg ccgggaccct 1260

gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440

cttcacatgc aggccctgcc ccctcgcgga ccgcagtgta ctaattatgc tctcttgaaa 1500

ttggctggag atgttgagag caatcccggg cccatgcgca ttagcaagcc ccacctgcgg 1560

agcatcagca tccagtgcta cctgtgcctg ctgctgaaca gccacttcct gaccgaggcc 1620

ggcatccacg tgttcatcct gggctgcttc agcgccggac tgcccaagac cgaggccaac 1680

tgggtgaacg tgatcagcga cctgaagaag atcgaggacc tgatccagag catgcacatc 1740

gacgccaccc tgtacaccga gagcgacgtg caccccagct gcaaggtgac cgccatgaag 1800

tgctttctgc tggaactgca ggtgatcagc ctggaaagcg gcgacgccag catccacgac 1860

accgtggaga acctgatcat cctggccaac aacagcctga gcagcaacgg caacgtgacc 1920

gagagcggct gcaaagagtg cgaggaactg gaagagaaga acatcaaaga gtttctgcag 1980

agcttcgtgc acatcgtgca gatgttcatc aacaccagct ga 2022

<210> 38

<211> 218

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 38

Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val

1 5 10 15

Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr

20 25 30

Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe

35 40 45

Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro

50 55 60

Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His

65 70 75 80

Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys

85 90 95

Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys

100 105 110

Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu

115 120 125

Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His

130 135 140

Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met

145 150 155 160

Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr

165 170 175

His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Phe

180 185 190

Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu

195 200 205

Val Thr Val Ala Phe Ile Ile Phe Trp Val

210 215

<210> 39

<211> 667

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 39

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Ala

100 105 110

Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val Gly Leu

115 120 125

Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala

130 135 140

Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val

145 150 155 160

Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile

165 170 175

Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu

180 185 190

Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile

195 200 205

Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp

210 215 220

Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala

225 230 235 240

Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro

245 250 255

Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr

260 265 270

Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp

275 280 285

Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Phe Trp Val

290 295 300

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

305 310 315 320

Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu

325 330 335

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

340 345 350

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

355 360 365

Glu Leu Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

370 375 380

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

385 390 395 400

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

405 410 415

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

420 425 430

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

435 440 445

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

450 455 460

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

465 470 475 480

Pro Pro Arg Gly Pro Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala

485 490 495

Gly Asp Val Glu Ser Asn Pro Gly Pro Met Arg Ile Ser Lys Pro His

500 505 510

Leu Arg Ser Ile Ser Ile Gln Cys Tyr Leu Cys Leu Leu Leu Asn Ser

515 520 525

His Phe Leu Thr Glu Ala Gly Ile His Val Phe Ile Leu Gly Cys Phe

530 535 540

Ser Ala Gly Leu Pro Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser

545 550 555 560

Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala

565 570 575

Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala

580 585 590

Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly

595 600 605

Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn

610 615 620

Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu

625 630 635 640

Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe

645 650 655

Val His Ile Val Gln Met Phe Ile Asn Thr Ser

660 665

<210> 40

<211> 670

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 40

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Leu

100 105 110

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

115 120 125

Leu Leu Ile Pro Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His

130 135 140

Tyr Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr

145 150 155 160

Phe Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp

165 170 175

Pro Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro

180 185 190

His Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn

195 200 205

Cys Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln

210 215 220

Cys Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser

225 230 235 240

Leu Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr

245 250 255

His Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His

260 265 270

Met Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser

275 280 285

Thr His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

290 295 300

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

305 310 315 320

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys

325 330 335

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

340 345 350

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

355 360 365

Gly Gly Cys Glu Leu Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg Gly Pro Gln Cys Thr Asn Tyr Ala Leu Leu

485 490 495

Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Arg Ile Ser

500 505 510

Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr Leu Cys Leu Leu

515 520 525

Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His Val Phe Ile Leu

530 535 540

Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala Asn Trp Val Asn

545 550 555 560

Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His

565 570 575

Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys

580 585 590

Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu

595 600 605

Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile

610 615 620

Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly

625 630 635 640

Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu

645 650 655

Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

660 665 670

<210> 41

<211> 649

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 41

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Ala

100 105 110

Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val Gly Leu

115 120 125

Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala

130 135 140

Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val

145 150 155 160

Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile

165 170 175

Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu

180 185 190

Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile

195 200 205

Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp

210 215 220

Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala

225 230 235 240

Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro

245 250 255

Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr

260 265 270

Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp

275 280 285

Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Phe Trp Val

290 295 300

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

305 310 315 320

Val Ala Phe Ile Ile Phe Trp Val Leu Cys Ala Arg Pro Arg Arg Ser

325 330 335

Pro Ala Gln Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly

340 345 350

Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

355 360 365

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

370 375 380

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

385 390 395 400

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

405 410 415

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

420 425 430

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

435 440 445

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

450 455 460

Arg Gly Pro Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

465 470 475 480

Val Glu Ser Asn Pro Gly Pro Met Arg Ile Ser Lys Pro His Leu Arg

485 490 495

Ser Ile Ser Ile Gln Cys Tyr Leu Cys Leu Leu Leu Asn Ser His Phe

500 505 510

Leu Thr Glu Ala Gly Ile His Val Phe Ile Leu Gly Cys Phe Ser Ala

515 520 525

Gly Leu Pro Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu

530 535 540

Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu

545 550 555 560

Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys

565 570 575

Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala

580 585 590

Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser

595 600 605

Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu

610 615 620

Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His

625 630 635 640

Ile Val Gln Met Phe Ile Asn Thr Ser

645

<210> 42

<211> 652

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 42

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Leu

100 105 110

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

115 120 125

Leu Leu Ile Pro Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His

130 135 140

Tyr Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr

145 150 155 160

Phe Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp

165 170 175

Pro Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro

180 185 190

His Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn

195 200 205

Cys Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln

210 215 220

Cys Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser

225 230 235 240

Leu Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr

245 250 255

His Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His

260 265 270

Met Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser

275 280 285

Thr His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

290 295 300

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

305 310 315 320

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Leu Cys Ala Arg Pro

325 330 335

Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val Tyr Ile Asn Met Pro

340 345 350

Gly Arg Gly Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

355 360 365

Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

370 375 380

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

385 390 395 400

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

405 410 415

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

420 425 430

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

435 440 445

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

450 455 460

Leu Pro Pro Arg Gly Pro Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu

465 470 475 480

Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Arg Ile Ser Lys Pro

485 490 495

His Leu Arg Ser Ile Ser Ile Gln Cys Tyr Leu Cys Leu Leu Leu Asn

500 505 510

Ser His Phe Leu Thr Glu Ala Gly Ile His Val Phe Ile Leu Gly Cys

515 520 525

Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala Asn Trp Val Asn Val Ile

530 535 540

Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp

545 550 555 560

Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr

565 570 575

Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser

580 585 590

Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala

595 600 605

Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys

610 615 620

Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser

625 630 635 640

Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

645 650

<210> 43

<211> 677

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 43

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Ala

100 105 110

Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val Gly Leu

115 120 125

Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala

130 135 140

Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val

145 150 155 160

Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile

165 170 175

Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu

180 185 190

Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile

195 200 205

Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp

210 215 220

Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala

225 230 235 240

Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro

245 250 255

Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr

260 265 270

Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp

275 280 285

Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Phe Trp Val

290 295 300

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

305 310 315 320

Val Ala Phe Ile Ile Phe Trp Val Tyr Phe Leu Gly Arg Leu Val Pro

325 330 335

Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr

340 345 350

Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val

355 360 365

Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys Lys Arg Val Lys

370 375 380

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

385 390 395 400

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

405 410 415

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

420 425 430

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

435 440 445

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

450 455 460

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

465 470 475 480

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Pro Gln

485 490 495

Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn

500 505 510

Pro Gly Pro Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile

515 520 525

Gln Cys Tyr Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala

530 535 540

Gly Ile His Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys

545 550 555 560

Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu

565 570 575

Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser

580 585 590

Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu

595 600 605

Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp

610 615 620

Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn

625 630 635 640

Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu

645 650 655

Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met

660 665 670

Phe Ile Asn Thr Ser

675

<210> 44

<211> 680

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 44

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Leu

100 105 110

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

115 120 125

Leu Leu Ile Pro Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His

130 135 140

Tyr Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr

145 150 155 160

Phe Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp

165 170 175

Pro Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro

180 185 190

His Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn

195 200 205

Cys Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln

210 215 220

Cys Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser

225 230 235 240

Leu Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr

245 250 255

His Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His

260 265 270

Met Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser

275 280 285

Thr His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

290 295 300

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

305 310 315 320

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Tyr Phe Leu Gly Arg

325 330 335

Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln

340 345 350

Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg

355 360 365

Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys Lys

370 375 380

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

385 390 395 400

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

405 410 415

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

420 425 430

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

435 440 445

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

450 455 460

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

465 470 475 480

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

Gly Pro Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val

500 505 510

Glu Ser Asn Pro Gly Pro Met Arg Ile Ser Lys Pro His Leu Arg Ser

515 520 525

Ile Ser Ile Gln Cys Tyr Leu Cys Leu Leu Leu Asn Ser His Phe Leu

530 535 540

Thr Glu Ala Gly Ile His Val Phe Ile Leu Gly Cys Phe Ser Ala Gly

545 550 555 560

Leu Pro Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys

565 570 575

Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr

580 585 590

Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys

595 600 605

Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser

610 615 620

Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu

625 630 635 640

Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu

645 650 655

Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile

660 665 670

Val Gln Met Phe Ile Asn Thr Ser

675 680

<210> 45

<211> 676

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 45

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Ala

100 105 110

Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val Gly Leu

115 120 125

Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala

130 135 140

Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val

145 150 155 160

Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile

165 170 175

Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu

180 185 190

Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile

195 200 205

Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp

210 215 220

Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala

225 230 235 240

Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro

245 250 255

Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr

260 265 270

Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp

275 280 285

Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Phe Trp Val

290 295 300

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

305 310 315 320

Val Ala Phe Ile Ile Phe Trp Val Ser Ala Asn Glu Arg Cys Lys Ser

325 330 335

Lys Val Val Pro Cys Arg Gln Lys Gln Trp Arg Thr Ser Phe Asp Ser

340 345 350

Lys Lys Leu Asp Leu Asn Tyr Asn His Phe Glu Ser Met Glu Trp Ser

355 360 365

His Arg Ser Arg Arg Gly Arg Ile Trp Gly Met Lys Arg Val Lys Phe

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

385 390 395 400

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

405 410 415

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

420 425 430

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

435 440 445

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

450 455 460

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

465 470 475 480

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Pro Gln Cys

485 490 495

Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro

500 505 510

Gly Pro Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln

515 520 525

Cys Tyr Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly

530 535 540

Ile His Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr

545 550 555 560

Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp

565 570 575

Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp

580 585 590

Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu

595 600 605

Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr

610 615 620

Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly

625 630 635 640

Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys

645 650 655

Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe

660 665 670

Ile Asn Thr Ser

675

<210> 46

<211> 679

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 46

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Leu

100 105 110

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

115 120 125

Leu Leu Ile Pro Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His

130 135 140

Tyr Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr

145 150 155 160

Phe Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp

165 170 175

Pro Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro

180 185 190

His Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn

195 200 205

Cys Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln

210 215 220

Cys Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser

225 230 235 240

Leu Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr

245 250 255

His Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His

260 265 270

Met Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser

275 280 285

Thr His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

290 295 300

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

305 310 315 320

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Ser Ala Asn Glu Arg

325 330 335

Cys Lys Ser Lys Val Val Pro Cys Arg Gln Lys Gln Trp Arg Thr Ser

340 345 350

Phe Asp Ser Lys Lys Leu Asp Leu Asn Tyr Asn His Phe Glu Ser Met

355 360 365

Glu Trp Ser His Arg Ser Arg Arg Gly Arg Ile Trp Gly Met Lys Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

435 440 445

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

450 455 460

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

465 470 475 480

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly

485 490 495

Pro Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu

500 505 510

Ser Asn Pro Gly Pro Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile

515 520 525

Ser Ile Gln Cys Tyr Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr

530 535 540

Glu Ala Gly Ile His Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu

545 550 555 560

Pro Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys

565 570 575

Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr

580 585 590

Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe

595 600 605

Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile

610 615 620

His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser

625 630 635 640

Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu

645 650 655

Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val

660 665 670

Gln Met Phe Ile Asn Thr Ser

675

<210> 47

<211> 661

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 47

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Ala

100 105 110

Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val Gly Leu

115 120 125

Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala

130 135 140

Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val

145 150 155 160

Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile

165 170 175

Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu

180 185 190

Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile

195 200 205

Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp

210 215 220

Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala

225 230 235 240

Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro

245 250 255

Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr

260 265 270

Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp

275 280 285

Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile Leu Val

290 295 300

Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala Leu Phe

305 310 315 320

Leu His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

325 330 335

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

340 345 350

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Lys Arg Val Lys

355 360 365

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

370 375 380

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

385 390 395 400

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

405 410 415

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

420 425 430

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

435 440 445

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

450 455 460

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Pro Gln

465 470 475 480

Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn

485 490 495

Pro Gly Pro Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile

500 505 510

Gln Cys Tyr Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala

515 520 525

Gly Ile His Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys

530 535 540

Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu

545 550 555 560

Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser

565 570 575

Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu

580 585 590

Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp

595 600 605

Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn

610 615 620

Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu

625 630 635 640

Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met

645 650 655

Phe Ile Asn Thr Ser

660

<210> 48

<211> 664

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 48

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Leu

100 105 110

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

115 120 125

Leu Leu Ile Pro Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His

130 135 140

Tyr Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr

145 150 155 160

Phe Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp

165 170 175

Pro Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro

180 185 190

His Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn

195 200 205

Cys Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln

210 215 220

Cys Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser

225 230 235 240

Leu Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr

245 250 255

His Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His

260 265 270

Met Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser

275 280 285

Thr His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

290 295 300

Ile Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly

305 310 315 320

Ala Leu Phe Leu His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

325 330 335

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

340 345 350

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Lys

355 360 365

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

370 375 380

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

385 390 395 400

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

405 410 415

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

420 425 430

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

435 440 445

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

450 455 460

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

465 470 475 480

Gly Pro Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val

485 490 495

Glu Ser Asn Pro Gly Pro Met Arg Ile Ser Lys Pro His Leu Arg Ser

500 505 510

Ile Ser Ile Gln Cys Tyr Leu Cys Leu Leu Leu Asn Ser His Phe Leu

515 520 525

Thr Glu Ala Gly Ile His Val Phe Ile Leu Gly Cys Phe Ser Ala Gly

530 535 540

Leu Pro Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys

545 550 555 560

Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr

565 570 575

Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys

580 585 590

Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser

595 600 605

Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu

610 615 620

Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu

625 630 635 640

Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile

645 650 655

Val Gln Met Phe Ile Asn Thr Ser

660

<210> 49

<211> 619

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 49

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Ala

100 105 110

Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val Gly Leu

115 120 125

Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala

130 135 140

Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val

145 150 155 160

Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile

165 170 175

Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu

180 185 190

Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile

195 200 205

Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp

210 215 220

Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala

225 230 235 240

Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro

245 250 255

Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr

260 265 270

Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp

275 280 285

Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile Leu Val

290 295 300

Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala Leu Phe

305 310 315 320

Leu His Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

325 330 335

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

340 345 350

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

355 360 365

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

370 375 380

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

385 390 395 400

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

405 410 415

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

420 425 430

Pro Pro Arg Gly Pro Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala

435 440 445

Gly Asp Val Glu Ser Asn Pro Gly Pro Met Arg Ile Ser Lys Pro His

450 455 460

Leu Arg Ser Ile Ser Ile Gln Cys Tyr Leu Cys Leu Leu Leu Asn Ser

465 470 475 480

His Phe Leu Thr Glu Ala Gly Ile His Val Phe Ile Leu Gly Cys Phe

485 490 495

Ser Ala Gly Leu Pro Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser

500 505 510

Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala

515 520 525

Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala

530 535 540

Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly

545 550 555 560

Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn

565 570 575

Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu

580 585 590

Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe

595 600 605

Val His Ile Val Gln Met Phe Ile Asn Thr Ser

610 615

<210> 50

<211> 622

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 50

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Leu

100 105 110

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

115 120 125

Leu Leu Ile Pro Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His

130 135 140

Tyr Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr

145 150 155 160

Phe Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp

165 170 175

Pro Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro

180 185 190

His Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn

195 200 205

Cys Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln

210 215 220

Cys Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser

225 230 235 240

Leu Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr

245 250 255

His Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His

260 265 270

Met Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser

275 280 285

Thr His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

290 295 300

Ile Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly

305 310 315 320

Ala Leu Phe Leu His Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

325 330 335

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

340 345 350

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

355 360 365

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

370 375 380

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

385 390 395 400

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

405 410 415

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

420 425 430

Gln Ala Leu Pro Pro Arg Gly Pro Gln Cys Thr Asn Tyr Ala Leu Leu

435 440 445

Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Arg Ile Ser

450 455 460

Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr Leu Cys Leu Leu

465 470 475 480

Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His Val Phe Ile Leu

485 490 495

Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala Asn Trp Val Asn

500 505 510

Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His

515 520 525

Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys

530 535 540

Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu

545 550 555 560

Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile

565 570 575

Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly

580 585 590

Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu

595 600 605

Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

610 615 620

<210> 51

<211> 659

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 51

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Ala

100 105 110

Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val Gly Leu

115 120 125

Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala

130 135 140

Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val

145 150 155 160

Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile

165 170 175

Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu

180 185 190

Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile

195 200 205

Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp

210 215 220

Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala

225 230 235 240

Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro

245 250 255

Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr

260 265 270

Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp

275 280 285

Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile Leu Val

290 295 300

Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala Leu Phe

305 310 315 320

Leu His Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn

325 330 335

Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr

340 345 350

Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser

355 360 365

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

370 375 380

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

385 390 395 400

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

405 410 415

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

420 425 430

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

435 440 445

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

450 455 460

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Pro Gln Cys Thr

465 470 475 480

Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly

485 490 495

Pro Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys

500 505 510

Tyr Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile

515 520 525

His Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu

530 535 540

Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

545 550 555 560

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

565 570 575

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

580 585 590

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

595 600 605

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

610 615 620

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

625 630 635 640

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

645 650 655

Asn Thr Ser

<210> 52

<211> 662

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 52

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Leu

100 105 110

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

115 120 125

Leu Leu Ile Pro Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His

130 135 140

Tyr Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr

145 150 155 160

Phe Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp

165 170 175

Pro Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro

180 185 190

His Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn

195 200 205

Cys Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln

210 215 220

Cys Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser

225 230 235 240

Leu Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr

245 250 255

His Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His

260 265 270

Met Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser

275 280 285

Thr His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

290 295 300

Ile Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly

305 310 315 320

Ala Leu Phe Leu His Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

325 330 335

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

340 345 350

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val

355 360 365

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

370 375 380

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

385 390 395 400

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

405 410 415

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

420 425 430

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

435 440 445

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

450 455 460

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Pro

465 470 475 480

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

485 490 495

Asn Pro Gly Pro Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser

500 505 510

Ile Gln Cys Tyr Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu

515 520 525

Ala Gly Ile His Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro

530 535 540

Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile

545 550 555 560

Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu

565 570 575

Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu

580 585 590

Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His

595 600 605

Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser

610 615 620

Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu

625 630 635 640

Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln

645 650 655

Met Phe Ile Asn Thr Ser

660

<210> 53

<211> 665

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 53

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Ala

100 105 110

Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val Gly Leu

115 120 125

Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala

130 135 140

Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val

145 150 155 160

Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile

165 170 175

Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu

180 185 190

Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile

195 200 205

Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp

210 215 220

Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala

225 230 235 240

Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro

245 250 255

Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr

260 265 270

Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp

275 280 285

Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Phe Trp Val

290 295 300

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

305 310 315 320

Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu

325 330 335

His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg

340 345 350

Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg

355 360 365

Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

370 375 380

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

385 390 395 400

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

405 410 415

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

420 425 430

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

435 440 445

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

450 455 460

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

465 470 475 480

Arg Gly Pro Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

485 490 495

Val Glu Ser Asn Pro Gly Pro Met Arg Ile Ser Lys Pro His Leu Arg

500 505 510

Ser Ile Ser Ile Gln Cys Tyr Leu Cys Leu Leu Leu Asn Ser His Phe

515 520 525

Leu Thr Glu Ala Gly Ile His Val Phe Ile Leu Gly Cys Phe Ser Ala

530 535 540

Gly Leu Pro Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu

545 550 555 560

Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu

565 570 575

Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys

580 585 590

Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala

595 600 605

Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser

610 615 620

Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu

625 630 635 640

Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His

645 650 655

Ile Val Gln Met Phe Ile Asn Thr Ser

660 665

<210> 54

<211> 668

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 54

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Leu

100 105 110

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

115 120 125

Leu Leu Ile Pro Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His

130 135 140

Tyr Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr

145 150 155 160

Phe Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp

165 170 175

Pro Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro

180 185 190

His Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn

195 200 205

Cys Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln

210 215 220

Cys Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser

225 230 235 240

Leu Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr

245 250 255

His Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His

260 265 270

Met Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser

275 280 285

Thr His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

290 295 300

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

305 310 315 320

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser

325 330 335

Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly

340 345 350

Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala

355 360 365

Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

370 375 380

Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

385 390 395 400

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

405 410 415

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

420 425 430

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

435 440 445

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

450 455 460

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

465 470 475 480

Leu Pro Pro Arg Gly Pro Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu

485 490 495

Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Arg Ile Ser Lys Pro

500 505 510

His Leu Arg Ser Ile Ser Ile Gln Cys Tyr Leu Cys Leu Leu Leu Asn

515 520 525

Ser His Phe Leu Thr Glu Ala Gly Ile His Val Phe Ile Leu Gly Cys

530 535 540

Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala Asn Trp Val Asn Val Ile

545 550 555 560

Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp

565 570 575

Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr

580 585 590

Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser

595 600 605

Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala

610 615 620

Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys

625 630 635 640

Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser

645 650 655

Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

660 665

<210> 55

<211> 643

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 55

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Ala

100 105 110

Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val Gly Leu

115 120 125

Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala

130 135 140

Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val

145 150 155 160

Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile

165 170 175

Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu

180 185 190

Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile

195 200 205

Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp

210 215 220

Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala

225 230 235 240

Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro

245 250 255

Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr

260 265 270

Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp

275 280 285

Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile Leu Val

290 295 300

Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala Leu Phe

305 310 315 320

Leu His Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly

325 330 335

Lys Val Tyr Ile Asn Met Pro Gly Arg Gly Lys Arg Val Lys Phe Ser

340 345 350

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

355 360 365

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

370 375 380

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

385 390 395 400

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

405 410 415

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

420 425 430

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

435 440 445

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Pro Gln Cys Thr

450 455 460

Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly

465 470 475 480

Pro Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys

485 490 495

Tyr Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile

500 505 510

His Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu

515 520 525

Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

530 535 540

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

545 550 555 560

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

565 570 575

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

580 585 590

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

595 600 605

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

610 615 620

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

625 630 635 640

Asn Thr Ser

<210> 56

<211> 646

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 56

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Leu

100 105 110

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

115 120 125

Leu Leu Ile Pro Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His

130 135 140

Tyr Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr

145 150 155 160

Phe Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp

165 170 175

Pro Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro

180 185 190

His Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn

195 200 205

Cys Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln

210 215 220

Cys Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser

225 230 235 240

Leu Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr

245 250 255

His Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His

260 265 270

Met Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser

275 280 285

Thr His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

290 295 300

Ile Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly

305 310 315 320

Ala Leu Phe Leu His Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln

325 330 335

Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly Lys Arg Val

340 345 350

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

355 360 365

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

370 375 380

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

385 390 395 400

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

405 410 415

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

420 425 430

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

435 440 445

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Pro

450 455 460

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

465 470 475 480

Asn Pro Gly Pro Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser

485 490 495

Ile Gln Cys Tyr Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu

500 505 510

Ala Gly Ile His Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro

515 520 525

Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile

530 535 540

Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu

545 550 555 560

Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu

565 570 575

Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His

580 585 590

Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser

595 600 605

Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu

610 615 620

Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln

625 630 635 640

Met Phe Ile Asn Thr Ser

645

<210> 57

<211> 671

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 57

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Ala

100 105 110

Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val Gly Leu

115 120 125

Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala

130 135 140

Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val

145 150 155 160

Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile

165 170 175

Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu

180 185 190

Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile

195 200 205

Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp

210 215 220

Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala

225 230 235 240

Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro

245 250 255

Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr

260 265 270

Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp

275 280 285

Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile Leu Val

290 295 300

Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala Leu Phe

305 310 315 320

Leu His Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala

325 330 335

Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr

340 345 350

Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr

355 360 365

Gln Arg Pro Tyr Tyr Lys Lys Arg Val Lys Phe Ser Arg Ser Ala Asp

370 375 380

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

385 390 395 400

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

405 410 415

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

420 425 430

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

435 440 445

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

450 455 460

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

465 470 475 480

Met Gln Ala Leu Pro Pro Arg Gly Pro Gln Cys Thr Asn Tyr Ala Leu

485 490 495

Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Arg Ile

500 505 510

Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr Leu Cys Leu

515 520 525

Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His Val Phe Ile

530 535 540

Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala Asn Trp Val

545 550 555 560

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

565 570 575

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

580 585 590

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

595 600 605

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile

610 615 620

Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

625 630 635 640

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

645 650 655

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

660 665 670

<210> 58

<211> 674

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 58

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Leu

100 105 110

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

115 120 125

Leu Leu Ile Pro Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His

130 135 140

Tyr Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr

145 150 155 160

Phe Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp

165 170 175

Pro Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro

180 185 190

His Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn

195 200 205

Cys Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln

210 215 220

Cys Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser

225 230 235 240

Leu Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr

245 250 255

His Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His

260 265 270

Met Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser

275 280 285

Thr His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

290 295 300

Ile Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly

305 310 315 320

Ala Leu Phe Leu His Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg

325 330 335

Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu

340 345 350

Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp

355 360 365

Leu Asn Thr Gln Arg Pro Tyr Tyr Lys Lys Arg Val Lys Phe Ser Arg

370 375 380

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

385 390 395 400

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

405 410 415

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

420 425 430

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

435 440 445

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

450 455 460

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Pro Gln Cys Thr Asn

485 490 495

Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro

500 505 510

Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr

515 520 525

Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His

530 535 540

Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala

545 550 555 560

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

565 570 575

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

580 585 590

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

595 600 605

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

610 615 620

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

625 630 635 640

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

645 650 655

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

660 665 670

Thr Ser

<210> 59

<211> 670

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 59

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Ala

100 105 110

Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val Gly Leu

115 120 125

Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala

130 135 140

Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val

145 150 155 160

Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile

165 170 175

Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu

180 185 190

Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile

195 200 205

Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp

210 215 220

Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala

225 230 235 240

Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro

245 250 255

Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr

260 265 270

Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp

275 280 285

Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile Leu Val

290 295 300

Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala Leu Phe

305 310 315 320

Leu His Ser Ala Asn Glu Arg Cys Lys Ser Lys Val Val Pro Cys Arg

325 330 335

Gln Lys Gln Trp Arg Thr Ser Phe Asp Ser Lys Lys Leu Asp Leu Asn

340 345 350

Tyr Asn His Phe Glu Ser Met Glu Trp Ser His Arg Ser Arg Arg Gly

355 360 365

Arg Ile Trp Gly Met Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg Gly Pro Gln Cys Thr Asn Tyr Ala Leu Leu

485 490 495

Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Arg Ile Ser

500 505 510

Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr Leu Cys Leu Leu

515 520 525

Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His Val Phe Ile Leu

530 535 540

Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala Asn Trp Val Asn

545 550 555 560

Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His

565 570 575

Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys

580 585 590

Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu

595 600 605

Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile

610 615 620

Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly

625 630 635 640

Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu

645 650 655

Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

660 665 670

<210> 60

<211> 673

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 60

Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala His Leu Gln Ala

1 5 10 15

Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro Leu Ala Ala Ala Tyr

20 25 30

Gln Glu Gln Leu Asp Arg Pro Val Val Pro His Pro Tyr Arg Val Gly

35 40 45

Asp Thr Val Trp Val Arg Arg His Gln Thr Lys Asn Leu Glu Pro Arg

50 55 60

Trp Lys Gly Pro Tyr Thr Val Leu Leu Thr Thr Pro Thr Ala Leu Lys

65 70 75 80

Val Asp Gly Ile Ala Ala Trp Ile His Ala Ala His Val Lys Ala Ala

85 90 95

Asp Pro Gly Gly Gly Pro Ser Ser Arg Leu Pro Cys Ser Arg Met Leu

100 105 110

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

115 120 125

Leu Leu Ile Pro Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His

130 135 140

Tyr Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr

145 150 155 160

Phe Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp

165 170 175

Pro Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro

180 185 190

His Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn

195 200 205

Cys Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln

210 215 220

Cys Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser

225 230 235 240

Leu Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr

245 250 255

His Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His

260 265 270

Met Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser

275 280 285

Thr His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

290 295 300

Ile Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly

305 310 315 320

Ala Leu Phe Leu His Ser Ala Asn Glu Arg Cys Lys Ser Lys Val Val

325 330 335

Pro Cys Arg Gln Lys Gln Trp Arg Thr Ser Phe Asp Ser Lys Lys Leu

340 345 350

Asp Leu Asn Tyr Asn His Phe Glu Ser Met Glu Trp Ser His Arg Ser

355 360 365

Arg Arg Gly Arg Ile Trp Gly Met Lys Arg Val Lys Phe Ser Arg Ser

370 375 380

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

420 425 430

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

435 440 445

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

450 455 460

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg Gly Pro Gln Cys Thr Asn Tyr

485 490 495

Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met

500 505 510

Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr Leu

515 520 525

Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His Val

530 535 540

Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala Asn

545 550 555 560

Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln

565 570 575

Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro

580 585 590

Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val

595 600 605

Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn

610 615 620

Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr

625 630 635 640

Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys

645 650 655

Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr

660 665 670

Ser

<210> 61

<211> 57

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 61

atggcacggc cacatccctg gtggctgtgc gttctgggga ccctggtggg gctctca 57

<210> 62

<211> 66

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 62

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atccca 66

<210> 63

<211> 516

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 63

gctactccag cccccaagag ctgcccagag aggcactact gggctcaggg aaagctgtgc 60

tgccagatgt gtgagccagg aacattcctc gtgaaggact gtgaccagca tagaaaggct 120

gctcagtgtg atccttgcat accgggggtc tccttctctc ctgaccacca cacccggccc 180

cactgtgaga gctgtcggca ctgtaactct ggtcttctcg ttcgcaactg caccatcact 240

gccaatgctg agtgtgcctg tcgcaatggc tggcagtgca gggacaagga gtgcaccgag 300

tgtgatcctc ttccaaaccc ttcgctgacc gctcggtcgt ctcaggccct gagcccacac 360

cctcagccca cccacttacc ttatgtcagt gagatgctgg aggccaggac agctgggcac 420

atgcagactc tggctgactt caggcagctg cctgcccgga ctctctctac ccactggcca 480

ccccaaagat ccctgtgcag ctccgatttt attcgc 516

<210> 64

<211> 516

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 64

gctacaccgg ctccgaagtc ctgcccggag cggcattatt gggcacaggg caagttgtgt 60

tgtcaaatgt gtgagccggg aacctttctc gtgaaggatt gcgatcagca tcggaaggcc 120

gcgcagtgcg acccatgtat accaggggtc tcattttccc cagatcacca tacgaggccg 180

cactgtgagt cttgcaggca ttgtaattcc ggcttgttgg tccgcaactg tactattact 240

gcgaatgcag agtgtgcttg tagaaacgga tggcagtgca gggacaaaga atgtacggag 300

tgtgatccac tgcctaaccc cagtcttaca gcaagatctt cacaggccct cagcccgcat 360

cctcaaccaa cacatcttcc ttacgtgtca gaaatgttgg aggcgcgaac cgcaggccat 420

atgcagaccc tggcggactt tcggcagctg ccagcacgca cacttagtac acactggcca 480

ccacaacgca gcttgtgctc ttccgatttc atccgc 516

<210> 65

<211> 81

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 65

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81

<210> 66

<211> 63

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 66

atccttgtga tcttctctgg aatgttcctt gttttcaccc tggccggggc cctgttcctc 60

cat 63

<210> 67

<211> 340

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 67

cgcgtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa aagacgtggc 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 cctcgcggac 340

<210> 68

<211> 126

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 68

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 69

<211> 72

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 69

ctttgcgcac gcccacgccg cagccccgcc caagaagatg gcaaagtcta catcaacatg 60

ccaggcaggg gc 72

<210> 70

<211> 153

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 70

agcgcgaacg aacgctgcaa aagcaaagtg gtgccgtgcc gccagaaaca gtggcgcacc 60

agctttgata gcaaaaaact ggatctgaac tataaccatt ttgaaagcat ggaatggagc 120

catcgcagcc gccgcggccg catttggggc atg 153

<210> 71

<211> 156

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<400> 71

tacttcctgg gccggctggt ccctcggggg cgaggggctg cggaggcagc gacccggaaa 60

cagcgtatca ctgagaccga gtcgccttat caggagctcc agggtcagag gtcggatgtc 120

tacagcgacc tcaacacaca gaggccgtat tacaaa 156

<210> 72

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 72

Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val

1 5 10 15

Tyr Ile Asn Met Pro Gly Arg Gly

20

<210> 73

<211> 51

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 73

Ser Ala Asn Glu Arg Cys Lys Ser Lys Val Val Pro Cys Arg Gln Lys

1 5 10 15

Gln Trp Arg Thr Ser Phe Asp Ser Lys Lys Leu Asp Leu Asn Tyr Asn

20 25 30

His Phe Glu Ser Met Glu Trp Ser His Arg Ser Arg Arg Gly Arg Ile

35 40 45

Trp Gly Met

50

<210> 74

<211> 52

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<400> 74

Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala

1 5 10 15

Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu

20 25 30

Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg

35 40 45

Pro Tyr Tyr Lys

50

Claims

1. A polynucleotide encoding an anti-CD 70 Chimeric Antigen Receptor (CAR), the CAR comprising a signal peptide, an anti-CD 70 antigen binding domain, a transmembrane domain, and at least one intracellular domain, wherein the anti-CD 70 antigen binding domain does not comprise an antibody, and optionally wherein the transmembrane domain of the CAR is not a CD27 transmembrane domain.

2. The polynucleotide of claim 1, wherein the anti-CD 70 antigen binding domain is comprised in the extracellular domain of CD 27.

3. The polynucleotide of claim 1 or 2, wherein the CAR comprises the antigen binding domain of codon optimized CD 27.

4. The polynucleotide of claim 1, 2 or 3, wherein the antigen binding domain comprises, consists of or consists essentially of SEQ ID No. 2.

5. The polynucleotide of any one of claims 1, 2 or 3, wherein the antigen binding domain and transmembrane domain comprise, consist of or consist essentially of SEQ ID No. 1.

6. The polynucleotide of any one of claims 1-5, wherein the sequence encoding the transmembrane domain is codon optimized.

7. The method of any one of claims 1-4, wherein the transmembrane domain is from CD28, the alpha chain of a T cell receptor, the beta chain of a T cell receptor, the zeta chain of a T cell receptor, CD3 ζ, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS/CD278, GITR/CD357, NKG2D, DAP, or DAP12.

8. The method of claim 7, wherein the CD28 transmembrane domain comprises, consists of, or consists essentially of SEQ ID NO 3.

9. The polynucleotide of any one of claims 1-8, wherein the CAR comprises two or more intracellular domains.

10. The polynucleotide of any one of claims 1-8, wherein the CAR comprises three or more intracellular domains.

11. The polynucleotide of any one of claims 1-10, wherein the intracellular domain comprises cd3ζ.

12. The polynucleotide of any one of claims 1-8, wherein the intracellular domain is an intracellular co-stimulatory domain.

13. The polynucleotide of claim 9, wherein the intracellular domain comprises an ITAM-containing signaling domain.

14. The polynucleotide of claim 9, wherein the intracellular co-stimulatory domain is selected from the group consisting of: CD27, CD28, 4-1BB, DAP12, NKG2D, OX-40 (CD 134), DAP10, CD40L, 2B4, DNAM, CS1, CD48, NKp30, NKp44, NKp46, NKp80, and combinations thereof.

15. The polynucleotide of any one of claims 1-14, wherein the intracellular domain is a co-stimulatory domain.

16. The polynucleotide of any one of claims 1-15, wherein the signal peptide is from CD27 or granulocyte-macrophage colony-stimulating factor receptor (GMSCF-R).

17. The polynucleotide of claim 16, wherein the sequence of the CD27 signal peptide comprises, consists of, or consists essentially of SEQ ID No. 6.

18. The polynucleotide of claim 1, wherein the signal peptide is from CD27, the anti-CD 70 antigen binding domain is from CD27, the transmembrane domain is from CD27, and the intracellular domain is from CD27.

19. The polynucleotide of any one of claims 1-18, wherein the signal peptide comprises, consists of, or consists essentially of SEQ ID No. 6.

20. The polynucleotide of any one of claims 1-19, wherein the CAR comprises, consists of, or consists essentially of SEQ ID No. 4.

21. The polynucleotide of any one of claims 1-20, wherein the transmembrane domain comprises, consists of, or consists essentially of SEQ ID No. 3 or SEQ ID No. 7.

22. The polynucleotide of any one of claims 1-21, wherein the intracellular domain comprises one or more of SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 11, or SEQ ID No. 12.

23. The polynucleotide of any one of claims 1-22, wherein said CAR comprises, consists of, or consists essentially of SEQ ID No. 5.

24. The polynucleotide of any one of claims 1-23, wherein said CAR comprises, consists of, or consists essentially of SEQ ID No. 1.

25. The polynucleotide of any one of claims 2-24, wherein the CAR comprises one or more of the following:

(b) GMSCF-R SP, codon optimized CD27 EC, codon optimized CD27 TMD, DAP12 ICD;

(c) CD27 SP, CD27 EC, CD28 TMD, DAP12 ICD and cd3ζ;

(d) GMSCF-R SP, codon optimized CD27 EC, CD28 TMD, DAP12 ICD and cd3ζ;

(h) GMSCF-R SP, codon optimized CD27 EC, CD28 TMD, NKG2D ICD and cd3ζ;

(i) CD27 SP, CD27 EC, CD27 TMD, 4-1BB ICD and CD3 ζ;

(k) CD27 SP, CD27 EC, CD28 TMD, 4-1BB ICD and CD3 ζ;

(l) GMSCF-R SP, codon optimized CD27 EC, CD28 TMD, 4-1BB ICD and cd3ζ;

(m) CD27 SP, CD27 EC, CD27 TMD, DAP10 ICD, and cd3ζ;

(o) CD27 SP, CD27 EC, CD28 TMD, DAP10 ICD, and cd3ζ;

(p) GMSCF-R SP, codon optimized CD27 EC, CD28 TMD, DAP10 ICD and cd3ζ;

(q) CD27 SP, CD27 Full Length (FL), CD27 TMD, CD27 ICD, and CD3 ζ;

(s) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, CD28 ICD and CD3 ζ;

(u) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, 4-1BB ICD and CD3 ζ;

(w) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, DAP10 ICD, and cd3ζ;

(x) GMSCF-R SP, CD27 FL, CD27 TMD, CD27 ICD, DAP10 ICD and cd3ζ;

(y) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, DAP12 ICD, and cd3ζ;

(aa) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, NKG2D ICD and cd3ζ;

(cc) CD27 SP, CD27 EC, CD27 TMD and CD3 ζ;

(dd) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD and cd3ζ;

(ee) CD27 SP, CD27 EC, CD28 TMD and CD3 ζ;

(ff) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD28 TMD and cd3ζ;

(gg) CD27 SP, CD27 EC, CD27 TMD, CD28 ICD and cd3ζ;

(ii) CD27 SP, CD27 EC, CD28 TMD, CD28 ICD and cd3ζ; or (b)

(jj) GMSCF-R SP, codon optimized CD27 EC, CD28 TMD and CD3 ζ.

26. A cell comprising the polynucleotide of any one of claims 1-25.

27. The cell of claim 26, wherein the cell is an immune cell.

28. The immune cell of claim 27, wherein the immune cell is a Natural Killer (NK) cell, a T cell, a γδ T cell, a Invariant NKT (iNKT) cell, a B cell, a macrophage, an MSC, or a dendritic cell.

29. The immune cell of claim 27 or 28, wherein the cell is an NK cell.

30. The cell of claim 28 or 29, wherein the NK cell is derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, derived from a cell line, or a mixture thereof.

31. The immune cell of claim 30, wherein the NK cell line is an NK-92 cell line or another NK cell line derived from a tumor or from a healthy NK cell, or a progenitor cell.

32. The immune cell of any one of claims 29-31, wherein the NK cell is derived from an umbilical cord blood mononuclear cell.

33. The immune cell of any one of claims 28-32, wherein the NK cell is a cd56+ NK cell.

34. The immune cell of any one of claims 28-33, wherein the NK cell expresses one or more exogenously supplied cytokines.

35. The immune cell of claim 34, wherein the cytokine is IL-15, IL-2, IL-12, IL-18, IL-21, IL-7, or a combination thereof.

36. The population of immune cells of any one of claims 27-35, said cells being present in a suitable medium.

37. The population of claim 36, wherein the immune cells are NK cells.

38. A method of killing CD70 positive cells in an individual comprising the step of administering to the individual a therapeutically effective amount of cells comprising the polynucleotide of any one of claims 1-25.

39. The method of claim 38, wherein the cell is an NK cell, a T cell, a γδ T cell, a Invariant NKT (iNKT) cell, a B cell, a macrophage, a γδ T cell, or a dendritic cell.

40. The method of claim 39, wherein the NK cells are derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, or from cell lines.

41. The method of any one of claims 39-40, wherein the NK cells are derived from umbilical cord blood mononuclear cells.

42. The method of any one of claims 38-41, wherein the CD 70-positive cells are not cancer cells.

43. The method of claim 42, wherein the CD70 positive cells are regulatory T cells.

44. The method of any one of claims 28-41, wherein the individual has a CD70 expressing cancer.

45. The method of any one of claims 28-41, wherein the subject has acute myeloid leukemia, lymphoma, lung cancer, kidney cancer, bladder cancer, melanoma, glioblastoma, breast cancer, head and neck cancer, mesothelioma, multiple myeloma, pancreatic cancer, or a combination thereof.

46. The method of any one of claims 28-45, wherein the cells are allogeneic with respect to the individual.

47. The method of any one of claims 28-45, wherein the cells are autologous to the subject.

48. The method of any one of claims 28-47, wherein the individual is a human.

49. The method of any one of claims 28-48, wherein the cells are administered to the individual one or more times.

50. The method of claim 49, wherein the duration between administrations of the cells to the individual is 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, or one or more years.

51. The method of any one of claims 28-50, further comprising the step of providing an effective amount of additional therapy to the individual.

52. The method of claim 51, wherein the additional therapy comprises surgery, radiation, gene therapy, immunotherapy, or hormonal therapy.

53. The method of claim 51 or 52, wherein the additional therapy comprises one or more antibodies.

54. The method of any one of claims 28-53, wherein the cells are administered to the individual intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, transdermally, subcutaneously, topically, by perfusion in a tumor microenvironment, or a combination thereof.

55. The method of any one of claims 28-54, further comprising the step of identifying CD70 positive cells in the individual.