RU2798348C2 - Compositions and methods of immunotherapy - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an immunoreactive cell including an antigen-recognizing receptor that binds an antigen, and binding of the antigen-recognizing receptor to an antigen activates the immunoreactive cell, and a soluble single-chain variable fragment (scFv), as well as to a composition containing it. Also the following is disclosed: a method for producing an immunoreactive cell that includes introducing into the immunoreactive cell a nucleic acid sequence that encodes a single-chain variable fragment (scFv), where the immunoreactive cell contains an antigen-recognizing receptor that binds the antigen.

EFFECT: invention is effective for reducing tumor mass and increasing the survival of an individual with neoplasia.

37 cl, 29 dwg, 6 ex

Description

PRIORITY CLAIMS

This application claims priority to United States Provisional Application No. 61/769543, filed Feb. 26, 2013, the contents of which are incorporated herein by reference in their entirety herein.

INFORMATION ABOUT GRANT SUPPORT

This invention was made with government support under grants Nos. CA95152, CA138738, CA059350 and CA08748 from the National Institutes of Health. The government has certain rights to the invention.

Introduction

The present invention provides methods and compositions for enhancing the immune response against cancers and pathogens. This refers to immunoreactive cells bearing antigen receptors, which may be chimeric antigen receptors (CARs) that express introduced ligands for immunomodulatory molecules. In specific embodiments, the engineered immunoreactive cells are antigen-targeted and resistant to immunosuppression and/or have improved immune-activating properties.

BACKGROUND OF THE INVENTION

Most mature B-cell malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, and non-Hodgkin's lymphoma, are incurable despite currently available treatments. Adoptive therapies using engineered autologous T cells have shown evidence of therapeutic efficacy in melanoma and indolent B cell malignancies. T cells can be modified to target tumor-associated antigens by introducing genes encoding T cell receptors, called chimeric antigen receptors (CARs), specific for such antigens. Immunotherapy is an open radionuclide therapy that has the potential to provide cancer treatment.

In doing so, malignant cells adapt to create an immunosuppressive microenvironment to protect themselves from immune recognition and elimination. This "hostile" tumor microenvironment poses a challenge to immune stimulation therapies, such as targeted T-cell therapies. Accordingly, new therapeutic strategies for the treatment of neoplasia are urgently required.

SUMMARY OF THE INVENTION

The present invention generally provides immunoreactive cells (e.g., T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and regulatory T cells) expressing an antigen-binding receptor (e.g., CAR or TCR) having activity immune cell activation, and a single chain variable fragment (scFv) that binds an antigen having immunosuppressive activity (eg, CD47, PD-1, CTLA-4 and their ligands), thereby reducing or eliminating the immunosuppressive activity of the antigen.

The invention further provides immunoreactive cells (e.g., T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and regulatory T cells) expressing an antigen-binding receptor (e.g., CAR or TCR) having immune activation activity. cells, and a single chain variable fragment (scFv) that binds an antigen having immunostimulatory or pro-inflammatory activity (eg, CD28, OX-40, 4-1BB, CD40 and their ligands), thereby increasing the immunostimulatory activity of the antigen.

The invention further provides immunoreactive cells (e.g., T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL) and regulatory T cells) expressing an antigen-binding receptor (e.g., CAR or TCR) having immune activation activity. cells, and CD40L, e.g., exogenous CD40L (CD40L that has been introduced directly or indirectly into a cell (e.g., via an unprotected nucleic acid vector comprising a nucleic acid sequence encoding CD40L), compared to endogenous CD40L originating in the cell itself), thereby increasing the immunostimulatory activity of the antigen.

Accordingly, the invention provides methods for using such immunoreactive cells to treat neoplasia, infectious disease, and other pathologies.

In one aspect, the invention provides an isolated immunoreactive cell having an antigen recognition receptor that binds an antigen, the binding activating the immunoreactive cell, and a soluble single chain variable fragment (scFv) that binds to a polypeptide having immunosuppressive activity or immunostimulatory activity.

In another aspect, the invention provides a method for treating or preventing neoplasia in an individual, comprising administering to the individual an effective amount of an immunoreactive cell having an antigen recognition receptor that binds to a first antigen, the binding activating the immunoreactive cell, and a soluble single chain variable fragment (scFv) that binds to a polypeptide that has immunosuppressive activity or immunostimulatory activity, thereby providing treatment or prevention of neoplasia in a single individual. In non-limiting embodiments, the antigen recognition receptor is CAR.

In another aspect, the invention provides a method for reducing tumor burden in an individual comprising administering to the individual an effective amount of an immunoreactive cell having an antigen recognition receptor that binds to a first antigen, the binding activating the immunoreactive cell, and a soluble single chain variable fragment (scFv) that binds with a polypeptide that has immunosuppressive activity or immunostimulatory activity, thereby causing the death of tumor cells in the subject. In yet another aspect, the invention provides a method of increasing the survival of an individual having neoplasia, comprising administering to the individual an effective amount of an immunoreactive cell with an antigen recognition receptor that binds to a first antigen, the binding activating the immunoreactive cell, and a soluble single chain variable fragment (scFv) that binds to a polypeptide that has immunosuppressive activity or immunostimulatory activity, thereby increasing the survival of the individual. In non-limiting embodiments, the antigen recognition receptor is CAR.

In various non-limiting embodiments, the invention provides a method for increasing the production of immune-activating cytokines in response to a cancer cell in an individual, comprising administering to the individual an immunoreactive cell having an antigen-recognizing receptor that binds the cancer cell antigen and further expressing exogenous CD40L. In a specific non-limiting embodiment, the immune-activating cytokine is IL-12. In non-limiting embodiments, the antigen recognition receptor is CAR.

In various non-limiting embodiments, the invention provides a method for increasing the production of immune-activating cytokines in response to a pathogen in an individual, comprising administering to the individual an immunoreactive cell having an antigen-recognizing receptor that binds the pathogen antigen and further expressing exogenous CD40L. In a specific non-limiting embodiment, the immune-activating cytokine is IL-12. In non-limiting embodiments, the antigen recognition receptor is CAR.

In various non-limiting embodiments, the invention provides a method of enhancing the CD8 ⁺ cytotoxic T cell response to a cancer cell in an individual, comprising administering to the individual an immunoreactive cell having an antigen recognition receptor that binds the cancer cell antigen and further expressing exogenous CD40L. In non-limiting embodiments, the antigen recognition receptor is CAR.

In various non-limiting embodiments, the invention provides a method for enhancing a CD8 ⁺ cytotoxic T cell response to a pathogen in an individual, comprising administering to the individual an immunoreactive cell having an antigen recognition receptor that binds the pathogen antigen and further expressing exogenous CD40L. In non-limiting embodiments, the antigen recognition receptor is CAR.

In various non-limiting embodiments, the invention provides a method of promoting maturation of dendritic cells in an individual having cancer, comprising administering to the individual an immunoreactive cell having an antigen recognition receptor that binds a cancer cell antigen and further expressing exogenous CD40L. In non-limiting embodiments, the antigen recognition receptor is CAR.

In various non-limiting embodiments, the invention provides a method of promoting dendritic cell maturation in an individual having a disease caused by a pathogen, comprising administering to the individual an immunoreactive cell having an antigen recognition receptor that binds the pathogen antigen and further expressing exogenous CD40L. In non-limiting embodiments, the antigen recognition receptor is CAR.

In yet another aspect, the invention provides a method for treating or preventing neoplasia in an individual, comprising administering to the individual an effective amount of an immunoreactive cell having an antigen recognition receptor that binds to a first antigen, the binding activating the immunoreactive cell, and expressing exogenous CD40L, thereby providing treatment or preventing neoplasia in an individual. In non-limiting embodiments, the antigen recognition receptor is CAR.

In another aspect, the invention provides a method for reducing tumor burden in an individual, comprising administering to the individual an effective amount of an immunoreactive cell having an antigen-recognizing receptor that binds to a first antigen, the binding activating the immunoreactive cell, and expressing exogenous CD40L, thereby causing tumor cell death. at the individual. In yet another aspect, the invention provides a method for increasing survival in an individual having neoplasia, comprising administering to the individual an effective amount of an immunoreactive cell having an antigen recognition receptor that binds to a first antigen, the binding activating the immunoreactive cell, and expressing exogenous CD40L, thereby increasing survival of the individual.

In yet another aspect, the invention provides a method of treating blood cancer in an individual in need thereof, a paw of an individual in need thereof, comprising administering to the individual a therapeutically effective amount of T cells having an antigen recognition receptor that binds CD19, wherein the binding activates an immunoreactive cell , and a soluble single chain variable fragment (scFv) that binds one or more of CD47, PD-1, CTLA-4 and their ligands, thereby providing treatment for blood cancer in an individual. In non-limiting embodiments, the antigen recognition receptor is CAR.

In yet another aspect, the invention provides a method of treating blood cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a T cell having an antigen recognition receptor that binds CD19, the binding activating an immunoreactive cell, and expressing exogenous CD40L, providing thereby treating blood cancer in an individual. In non-limiting embodiments, the antigen recognition receptor is CAR.

In one aspect, the invention provides a method for producing an antigen-specific immunoreactive cell, comprising introducing into the immunoreactive cell a nucleotide sequence encoding a single-chain variable fragment (scFv) that binds to a polypeptide having immunosuppressive activity or immunostimulatory activity, wherein the immunoreactive cell has an antigen-recognizing receptor that binds the antigen. In non-limiting embodiments, the antigen recognition receptor is CAR.

In one aspect, the invention provides a method for producing an antigen-specific immunoreactive cell, comprising introducing into the immunoreactive cell a nucleic acid sequence that encodes CD40L, the immunoreactive cell having an antigen-recognizing receptor that binds the antigen. In non-limiting embodiments, a nucleic acid sequence that encodes for CD40L is operably linked to a promoter element constitutively or inducibly expressed in an immunoreactive cell, further contained in the vector. In non-limiting embodiments, the antigen recognition receptor is CAR. In non-limiting embodiments, the invention provides a nucleic acid comprising a sequence encoding a CAR and encoding a CD40L, each of which is optionally operably linked to a promoter element constitutively or inducibly expressed in an immunoreactive cell, and said nucleic acid may be further contained in a vector. In one aspect, the invention provides a vector having a nucleic acid sequence encoding an antigen recognition receptor that binds an antigen and a nucleic acid sequence encoding a soluble single chain variable fragment (scFv) that binds to a polypeptide having immunosuppressive activity or immunostimulatory activity. In non-limiting embodiments, the antigen recognition receptor is CAR.

In one aspect, the invention provides a vector containing a nucleic acid sequence encoding an antigen recognition receptor that binds an antigen and a nucleic acid sequence encoding CD40L. In non-limiting embodiments, the antigen recognition receptor is CAR. In one specific non-limiting embodiment, the invention provides a retroviral vector containing an anti-CD19 CAR (1928z)- and CD40L-encoding nucleic acid.

In a related aspect, the invention provides a pharmaceutical composition comprising an effective amount of an immunoreactive cell according to any aspect of the invention referred to herein, in a pharmaceutically acceptable excipient. In another related aspect, the invention provides a pharmaceutical composition for the treatment of neoplasia, comprising an effective amount of a tumor antigen-specific T cell of any aspect of the invention referred to herein, in a pharmaceutically acceptable excipient.

In a further aspect, the invention provides a kit for the treatment of neoplasia, pathogenic infection, autoimmune disorder, or allogeneic transplantation, comprising an immunoreactive cell having an antigen recognition receptor that binds an antigen and activates the immunoreactive cell, and a soluble single chain variable fragment (scFv) that binds to a polypeptide that has immunosuppressive activity or immunostimulatory activity. In non-limiting embodiments, the antigen recognition receptor is CAR. In specific embodiments, the kit further comprises written instructions for using the cell to treat an individual having neoplasia, a pathogenic infection, an autoimmune disorder, or an allogeneic transplant.

In a further aspect, the invention provides a kit for the treatment of neoplasia, pathogenic infection, autoimmune disorder, or allogeneic transplantation, comprising an immunoreactive cell having an antigen-recognizing receptor that binds to an antigen and activates the immunoreactive cell and expresses exogenous CD40L. In non-limiting embodiments, the antigen recognition receptor is CAR. In specific embodiments, the kit further comprises written instructions for using the cells to treat an individual having neoplasia, a pathogenic infection, an autoimmune disorder, or an allogeneic transplant.

In a further aspect, the invention provides a kit for the treatment of neoplasia, a pathogenic infection, an autoimmune disorder, or an allogeneic transplant, comprising a nucleic acid encoding a CAR that recognizes an antigen of a neoplasia, an infection pathogen, an autoimmune disorder, or a transplant being treated, and a nucleic acid encoding a soluble single-stranded a variable fragment (scFv) that binds to a polypeptide having immunosuppressive activity or immunostimulatory activity. If desired, one or both nucleic acids may be included in the vector, which may be the same vector (bicistronic) or separate vectors. The CAR-encoding nucleic acid and/or the scFv-encoding nucleic acid may be operably linked to a promoter, which may be the same promoter or different promoters. In specific embodiments, the kit further comprises written instructions for using the cells to treat an individual having neoplasia, a pathogenic infection, an autoimmune disorder, or an allogeneic transplant.

In a further aspect, the invention provides a cancer treatment kit comprising a nucleic acid encoding a CAR that recognizes a cancer antigen and a nucleic acid encoding a soluble single chain variable fragment (scFv) that binds to a polypeptide having immunosuppressive activity or immunostimulatory activity. If desired, one or both nucleic acids may be included in the vector, which may be the same vector (bicistronic) or separate vectors. The CAR-encoding nucleic acid and/or the scFv-encoding nucleic acid may be operably linked to a promoter, which may be the same promoter or different promoters. In specific embodiments, the kit further comprises written instructions for using the cells to treat an individual having cancer.

In a further aspect, the invention provides a kit for the treatment of cancer or a pathogen-mediated disorder, comprising a nucleic acid encoding CAR 67 that recognizes a cancer or pathogen antigen and a nucleic acid encoding a soluble single chain variable fragment (scFv) that binds to a polypeptide having immunosuppressive activity or immunostimulatory activity. If desired, one or both nucleic acids may be included in the vector, which may be the same vector (bicistronic) or separate vectors. The CAR-encoding nucleic acid and/or the scFv-encoding nucleic acid may be operably linked to a promoter, which may be the same promoter or different promoters. In specific embodiments, the kit further comprises written instructions for using the cells to treat an individual having a cancer or disorder. In a further aspect, the invention provides a kit for the treatment of cancer or a pathogen-mediated disorder, comprising a nucleic acid encoding a CAR that recognizes a cancer or pathogen antigen and a nucleic acid encoding CD40L. If desired, one or both nucleic acids may be included in the vector, which may be the same vector (bicistronic) or separate vectors. A nucleic acid encoding a CAR and/or a nucleic acid encoding a CD40L may be operably linked to a promoter, which may be the same promoter or different promoters. In specific embodiments, the kit further comprises written instructions for using the cells to treat an individual having a cancer or disorder.

In various embodiments of any of the aspects defined herein, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a human embryonic stem cell, and a pluripotent stem cell from which lymphoid cells can be differentiated. In various embodiments of any of the aspects defined herein, the immunoreactive cell is autologous.

In various embodiments of any of the aspects defined herein, the antigen recognition receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In various embodiments of any of the aspects defined herein, the antigen recognition receptor is exogenous or endogenous. In various embodiments of any of the aspects defined herein, the antigen recognition receptor is recombinantly expressed. In various embodiments of any of the aspects defined herein, the antigen recognition receptor is expressed from a vector. In various embodiments of any of the aspects defined herein, the intracellular signaling domain of an antigen recognition receptor is the signaling domain of the CDζ chain, CD3ζ chain, CD97, CD11a-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-IBB, CD28, parts thereof or combinations thereof. In non-limiting embodiments, the antigen recognition receptor is a CAR including at least a portion of CD28, 4-IBB and/or CD3ζ chains (see e.g. Zhong et al., 2010, Molecular Ther. 18(2) :413-420), together with the antigen-binding portion. In non-limiting embodiments, the antigen recognition receptor is CAR as described in Kohn et al., 2011, Molecular Ther. 19(3):432-438) wherein the antigen-binding portion is optionally replaced with an amino acid sequence that binds to another tumor or pathogenic antigen. In various embodiments, the cell expresses a recombinant or endogenous antigen receptor that is 1928z or 4H1128z.

In various embodiments of any of the aspects defined herein, the antigen is a tumor or pathogenic antigen. In various embodiments of any of the aspects defined herein, the tumor antigen is one or more of CD19, MUC16, MUC1, CA1X, CEA, CDS, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41 , CD44, CD49f, CD56, CD74, CD133, CD138, antigen of cells infected with cytomegalovirus (CMV), EGP-2, EGP-40, EpCAM, eRB-B2,3,4, FBP, embryonic acetylcholine receptor, folate receptor-α , GD2, GD3, HER-2, hTERT, IL-13R-a2, κ-light chain, KDR, LeY, cell adhesion molecules L1, MAGE-A1, mesothelin, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4 ), PSCA, PSMA, ROR1, TAG-72, VEGF-R2 or WT-1. In specific embodiments, the antigen is CD19 or MUC16. Amino acid sequences that specifically bind to said antigens are known in the art or can be prepared using methods known in the art; examples include immunoglobulins, immunoglobulin variable regions (eg, variable fragment ("Fv") or bivalent variable fragment ("Fab")), single chain antibodies, and others.

In various embodiments of any of the aspects defined herein, the soluble scFv is secreted. In various embodiments of any of the aspects defined herein, the scFv is expressed from a vector. In various embodiments of any of the aspects defined herein, the immunosuppressive polypeptide is one or more of CD47, PD-1, CTLA-4, and their ligands. In various embodiments of any of the aspects defined herein, the immunostimulatory polypeptide is one or more of CD28, OX-40, 4-1BB, and their ligands. In various embodiments of any of the aspects defined herein, the soluble scFv enhances the immune response of the immunoreactive cell.

In various embodiments of any of the aspects defined herein, the immunoreactive cell secretes a cytokine. In various embodiments of any of the aspects defined herein, the cytokine is expressed from a vector. In various embodiments of any of the aspects defined herein, a pharmaceutical composition containing an immunoreactive cell of the invention contains a cytokine. In various embodiments of any of the aspects defined herein, a cytokine is introduced into an immunoreactive cell of the invention. In various embodiments of any of the aspects defined herein, the cytokine is one or more of IL-2, IL-3, IL-6, IL-11, IL7, IL12, IL15, IL21, granulocyte-macrophage colony-stimulating factor, alpha, beta or gamma interferon and erythropoietin.

In various embodiments of any of the aspects defined herein, the method reduces the number of tumor cells, reduces the size of the tumor, destroys the tumor in the individual, reduces the volume of the tumor lesion in the individual, and/or eradicates the tumor mass in the individual.

Illustrative neoplasms for which the invention may be applied include, without limitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, myelomonocytic acute leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia , chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma , sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, Nile duct carcinoma, choriocarcinoma, seminoma, fetal carcinoma, Wilm's tumor, cervical cancer, cancer uterus Yaichk cancer, lung cancer, lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medical, craniophastic nibble, ependymom, pinealoma, hemangito -cheese, neurinoma of the auditory nerve, oligodendroglioma, sluttiom Stoma and retinoblastoma).

In various non-limiting embodiments of any of the aspects defined herein, the neoplasia is one or more of blood cancer, B-cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma, and ovarian cancer. In some embodiments, the blood cancer is one or more of B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. In specific embodiments, the neoplasia is B-cell leukemia, the antigen is CD19, and the polypeptide that has immunosuppressive activity is one or more of CD47, PD-1, CTLA-4, and their ligands. In specific embodiments, the neoplasia is multiple myeloma, the antigen is CD19, and the polypeptide that has immunosuppressive activity is one or more of CD47, PD-1, CTLA-4, and their ligands. In specific embodiments, the neoplasia is acute lymphoblastic leukemia (ALL), the antigen is CD19, and the polypeptide that has immunosuppressive activity is one or more of CD47, PD-1, CTLA-4, and their ligands. In specific embodiments, the neoplasia is chronic lymphocytic leukemia, the antigen is CD19, and the polypeptide that has immunosuppressive activity is one or more of CD47, PD-1, CTLA-4, and ligands thereof. In specific embodiments, the neoplasia is non-Hodgkin's lymphoma, the antigen is CD19, and the polypeptide that has immunosuppressive activity is one or more of CD47, PD-1, CTLA-4, and their ligands. In specific embodiments, the neoplasia is ovarian cancer, the antigen is MUC16, and the polypeptide that has immunosuppressive activity is one or more of CD47, PD-1, CTLA-4, and their ligands.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the meanings that are generally understood by a person skilled in the art to which this invention belongs. The following references provide any person skilled in the art with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (191); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings assigned to them below, unless otherwise indicated.

By "activates an immunoreactive cell" is meant the induction of signal transduction or changes in the expression of proteins in the cell, leading to the initiation of an immune response. For example, when CD3 clusters in response to ligand binding and tyrosine-based immunoreceptor inhibitory motifs (ITAMs), a signal transduction cascade is generated. In certain embodiments, when an endogenous TCR or exogenous CAR binds an antigen, an immunological synapse is formed that includes the clustering of many molecules around the bound receptor (e.g., CD4 or CD8, CD3γ/δ/ε/ζ, etc.). This clustering of membrane-bound signaling molecules allows ITAM motifs contained in CD3 chains to become phosphorylated. This phosphorylation in turn initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce general T cell gene expression to increase IL-2 production for proliferation and expression of key T cell regulatory proteins in order to initiate a T cell immune response. By "stimulating an immunoreactive cell" is meant a signal that leads to a robust and sustained immune response. In various embodiments, this occurs after activation of an immune cell (eg, T cells) or is simultaneously mediated through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Without being bound by a particular theory, obtaining multiple stimulatory signals is important to establish a reliable and long-term T-cell immune response. Without receiving these stimulatory signals, T cells are quickly inhibited and do not respond to the antigen. While the effects of these co-stimulatory signals vary and remain partially understood, they generally result in an increase in gene expression in order to produce long-lived, proliferative, and anti-apoptotic T cells that respond vigorously to antigen for complete and sustained eradication.

The term "antigen-recognizing receptor" as used herein refers to a receptor that can activate an immune cell (eg, T cell) in response to antigen binding. Exemplary antigen recognition receptors can be natural or endogenous T cell receptors or chimeric antigen receptors in which a tumor antigen binding domain is fused to an intracellular signaling domain capable of activating an immune cell (eg, T cell).

As used here, the term "antibody" means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and have been used consistently both in vitro and in vivo. Accordingly, as used herein, the term "antibody" means not only intact immunoglobulin molecules, but also well-known active F(ab') ₂ and Fab fragments. F(ab') ₂ and Fab fragments that lack the Fe fragment of an intact antibody are more rapidly cleared from the circulation and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983) Antibodies of the invention include whole native antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', V region single chain fragments (scFv), fusion polypeptides, and unconventional antibodies.

As used herein, the term "single chain variable fragment" or "scFv" is a fusion protein of the heavy (VH) and light (VL) immunoglobulin variable regions covalently linked to form a VH::VL hexodimer. The heavy (VH) and light (VL) chains are either directly linked or attached by a peptide-coding linker (e.g., 30, 15, 20, 25 amino acids) that connects the N-terminus of the VH to the C-terminus of the VL, or the C- end VH with N-terminus VL. Typically, the linker is enriched in glycine for flexibility and serine or threonine for solubility. Despite the removal of constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain F _V polypeptide antibodies can be expressed from a nucleic acid comprising VH and VL coding sequences as described in Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and US Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, for example, Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcope ia Muscle 2012 August 12; Shieh et al., J Imunol2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 1 16(8):2252-61;Brocks et al., Immunotechnology 1997 3(3):173-84;Moosmayer et al., Ther Immunol 1995 2(10:31-40). have been described (see e.g. Peter et al., J Bioi Chern 2003 25278(38):36740-7; Xie et al, Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55 Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

By "affinity" is meant the degree of binding strength. Without being bound by theory, affinity depends on the accuracy of the stereochemical match between the combined antibody regions and antigenic determinants, on the size of the contact area between them, and on the distribution of charged and hydrophobic groups. Affinity also includes the term "avidity", which refers to the strength of the antigen-antibody bond after the formation of reversible complexes. Methods for calculating the affinity of an antibody for an antigen are known in the art, including the use of binding experiments to calculate affinity. Antibody activity in functional tests (eg, flow cytometric analysis) also reflects antibody affinity. Antibodies and affinities can be phenotypically characterized and compared using functional tests (eg, analysis by flow cytometry).

The term "chimeric antigen receptor" or "CAR", as used herein, refers to an antigen-binding domain that is fused to an intracellular signaling domain capable of activating or stimulating an immune cell. Most commonly, the CAR extracellular binding domain consists of a single chain variable fragment (scFv) derived from the fusion of the heavy and light variable regions of a mouse or humanized monoclonal antibody. In addition, scFvs that are derived from a Fab (rather than an antibody, eg derived from Fab libraries) can be used, in various embodiments, the scFv is fused to a transmembrane domain and then to an intracellular signaling domain. "First generation" CARs include those that exclusively provide CD3ζ signals upon antigen binding, "second generation" CARs include those that provide both costimulation (eg, CD28 or CD137) and activation (CD3Q). "Third generation" CARs include those that provide multiple costimulation (eg, CD28 and CD137) and activation (CO3Q). In various embodiments, the CAR is chosen to have high affinity or avidity for the antigen.

The term "immunosuppressive activity" refers to the induction of signal transduction or changes in the expression of proteins in a cell (eg, an activated immunoreactive cell), leading to a decrease in the immune response. Polypeptides known to suppress or reduce the immune response through their binding include CD47, PD-1, CTLA-4 and their respective ligands, including SIRPa, PD-L1, PD-L2, B7-1 and B7 -2. Such polypeptides are present in the tumor microenvironment and suppress the immune response to tumor cells. In various embodiments, inhibiting, blocking, or counteracting the interaction of immunosuppressive polypeptides and/or their ligands enhances the immune response of an immunoreactive cell.

The term "immunostimulatory activity" refers to the induction of signal transduction or changes in the expression of proteins in a cell (eg, an activated immunoreactive cell), leading to an increase in the immune response. Immunostimulatory activity may include pro-inflammatory activity. Polypeptides known to stimulate or enhance an immune response through their binding include CD28, OX-40, 4-1BB and their respective ligands including B7-1, B7-2, OX-40L and 4-1BBL . Such polypeptides are present in the tumor microenvironment and activate the immune response to tumor cells. In various embodiments, the activating, stimulatory, or agonistic pro-inflammatory polypeptides and/or ligands thereof enhance the immune response of an immunoreactive cell.

By "CD3ζ polypeptide" is meant a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with NCBI reference no: NP_932170 or a fragment thereof that has an activating or stimulatory activity. Typical CD3ζ; given below [SEQ ID NO: 1].

1 mkwkalftaa ilqaqlpite aqsfglldpk

61 apayqqgqnq lynelnlgrr eeydvldkrr grdpemggkp qrrknpqegl ynelqkdkma

121 eayseigmkg errrgkghdg lyqglstatk dtydalhmqa lppr

By "CD3ζ nucleic acid molecule" is meant a polynucleotide encoding a CD3ζ polypeptide.

By "CD28 polypeptide" is meant a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with NCBI reference no: NP_006130 or a fragment thereof which has stimulatory activity. A typical CD28 is shown below [SEQ ID NO: 2].

1 mlrlllalnl fpsiqvtgnk ilvkqspmlv aydnavnlsc kysynlfsre fraslhkgld

61 savevc nysqqlqvys ktgfiicdgkl gnesvtfylq nlyvnqtdiy fckievmypp

121 pyldneksng tiihvkgkhl cpsplfpgps kpfwvlvvvg gvlacysllv tvafiifwvr

181 skrsrllhsd ymnmtprrpg ptrkhyqpya pprdfaayrs

By "CD28 nucleic acid molecule" is meant a polynucleotide encoding a CD28 polypeptide.

By "CD40L polypeptide" is meant a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with NCBI Ref: NP_000065, GenBank Ref: GenBank Ref: AAH74950.1 or its fragment, which is a CD40 ligand, or a protein encoded by a nucleic acid PCR-amplified from PBMC isolated from healthy donors using the following primers (1) 5'-CACGTGCATGATCGAAACATACAACCAAACTTCTCCCCGATCTGC-'3 [SEQ ID NO: 3] and (2 ) 5'-CTCGAGGGATCCTCAGAGTTTGAGTAAGCCAAAGGA-3' [SEQ ID NO: 4] (Figure 22A).

By "CD40L nucleic acid molecule" is meant a polynucleotide encoding a CD40L polypeptide.

By "polypeptide 4-1BB" is meant a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with NCBI Ref. No: P41273 or NP_001552 or a fragment thereof that acts as a tumor necrosis (TNF). An exemplary 4-1BB is shown below [SEQ ID NO: 5].

1 mgnscyniva tlllvlnfer trslqdpcsn cpagtfcdnn rnqicspcpp nsfssaggqr

61 tcdicrqckg vfrtrkecss tsnaecdctp gfhclgagcs mceqdckqgq eltkkgckdc

121 cfgtfndqkr gicrpwtncs ldgksvlvng tkerdvvcgp spadlspgas svtppapare

181 pghspqiisf flaltstall fllffltlrf svvkrgrkkl lyifkqpfmr pvqttqeedg

241 cscrfpeeee ggcel

By "4-1BBL nucleic acid molecule" is meant a polynucleotide encoding a 4-1BBL polypeptide.

By "OX40L polypeptide" is meant a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with NCBI Ref: BAB18304 or NP_003317 or a fragment thereof which is a tumor necrosis factor (TNF) ligand ) [SEQ ID NO: 6].

1 mervqpleen vgnaarprfe rnklllvasv iqglglllcf tyiclhfsal qvshrypriq

61 sikvqfteyk kekgfiltsq kedeimkvqn nsviincdgf ylislkgyfs qevnislhyq

121 kdeeplfqlk kvrsvnslmv asltykdkvy Invttdntsl ddfhvnggel ilihqnpgef

181 cvl

By "OX40L nucleic acid molecule" is meant a polynucleotide encoding an OX40L polypeptide.

By "1928z" is meant a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence below, which includes the CDS leader sequence amino acids 1-18 and is capable of binding CD19 [SEQ ID NO: 7].

MALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNW

VKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSED

SAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQS

PKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDR

FTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKRAAAIEVM

YPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVT

VAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFS

RSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRRDPEMGGKPRRKNPQEGLY

NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRX

An exemplary nucleic acid sequence encoding a 1928z polypeptide comprising a CDS leader sequence is shown below [SEQ ID NO: 8].

ccatggctctcccagtgactgccctactgcttcccctagcgcttctcctgcatg

cagaggtgaagctgcagcagtctggggctgagctggtgaggcctgggtcctcag

tgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaact

gggtgaagcagaggcctggacagggtcttgagtggattggacagatttatcctg

gagatggtgatactaactacaatggaaagttcaagggtcaagccacactgactg

cagacaaatcctccagcacagcctacatgcagctcagcggcctaacatctgagg

actctgcggtctatttctgtgcaagaaagaccattagttcggtagtagatttct

actttgacactggggccaagggaccacggtcaccgtctcctcaggtggaggtg

gatcaggtggaggtggatctggtggaggtggatctgacattgagctcacccagt

ctccaaaattcatgtccacatcagtaggagacagggtcagcgtcacctgcaagg

ccagtcagaatgtgggtactaatgtagcctggtatcaacagaaaccaggacaat

ctcctaaaccactgatttactcggcaacctaccgggaacagtggagtccctgatc

gcttcacaggcagtggatctgggacagatttcactctcaccatcactaacgtgc

agtctaaagacttggcagactatttctgtcaacaatataacaggtatccgtaca

cgtccgggggggggaccaagctggagatcaaacgggcggccgcaattgaagtta

tgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatg

tgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccct

tttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaa

cagtggccttttattattttctggggtgaggagtaagaggagcaggctcctgcaca

gtgactacatgaacatgactccccgccgccccggggcccacccgcaagcattacc

agccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttca

gcaggagcgcagagccccccgcgtaccagcagggccagaaccagctctataacg

agctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggcc

gggaccctgagatggggggaaagccgagaaggaagaagaaccctcaggaaggcctgt

acaatgaactgcagaaagataagatggcgggaggcctacagtgagattgggatga

aaggcgagcgccgggaggggcaaggggcacgatggcctttaccagggtctcagta

cagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcg

By "4H1128z" is meant a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence below, which includes the CDS leader sequence from amino acids 1-18 and is capable of binding MUC [SEQ ID NO: 9].

MALPVTALLLPLALLLHAEVKLQESGGGFVKPGGSLKVSCAASGFTFSSYAMSW

VRLSPEMRLEWVATISSAGGYIFYSDSVQGRFTISRDNAKNTLHLQMGSLRSGD

TAMYYCARQGFGNYGDYYAMDY GQGTTVTVSSGGGGSGGGGSGGGGSDIELTQ

SPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNQLAWYQQKPGQSPELLIYWASTR

QSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQSYNLLTFGPGTKLEIKRA

AAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC

YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPPGPTRKHYQPYAPPRDFAAYR

SRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN

PQEGLYNELQ DC AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ

ALPPR

An exemplary nucleic acid sequence encoding a 4H1128z polypeptide comprising a kappa leader sequence is shown below [SEQ ID NO: 10].

ccatggctctcccagtgactgccctactgcttcccctagcgcttctcctgcatg

cagaggtgaagctgcaggagtcagggggaggcttcgtgaagcctggagggtccc

tcaaagtctcctgtgcagcctctggattcactttcagtagctatgccatgtcct

gggttcgcctgagtccggagatgaggctggagtgggtcgcaaccattagcagtg

ctggtggttacatcttctattctgacagtgtgcagggacgattcaccatttcca

gagacaatgccaagaacaccctgcacctgcaaatggggcagtctgaggtctgggg

acacggccatgtattactgtgcaaggcagggatttggtaactacggtgattact

atgctatggactactggggccaagggaccacggtcaccgtctcctcaggtggag

gtggatcaggtggaggtggatctggtggaggtggatctgacattgagctcaccc

agtctccatcctccctggctgtgtcagcaggagagaaggtcactatgagctgca

aatccagtcagagtctgctcaacagtagaacccgaaagaaccagttggcttggt

accagcaaaaaccaggacagtctcctgaactgctgatctactgggcatcacta

ggcaatctggagtccctgatcgcttcacaggcagtggatctgggacagatttca

ctctcaccatcagcagtgtgcaggctgaagacctggcagtttattactgccagc

aatcttataatctactcacgttcggtcctgggaccaagctggagatcaaacgggg

cggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagca

atggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttc

ccggaccttctaagcccttttgggtgctggtggtggttggtggagtcctggctt

gctatagcttgctagtaacagtggccttttattattttctgggtgaggagtaaga

ggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccggggc

ccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatc

gctccagagtgaagttcagcaggagcgcagagccccccgcgtaccagcaggggcc

agaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttt

tggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaaga

accctcaggaaggcctgtacaatgaactgcagaaagataagatggcgggaggcct

acagtgagattgggatgaaaggcgagcgccgggaggggcaaggggcacgatggcc

tttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgc

aggccctgccccctcgc

By "B6H12.2 scFv" is meant a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence below and capable of binding CD47 [SEQ ID NO: 11] .

EVQLVESGGDLVKPGGSLKLSCAASGFTFSGYGMSWVRQTPDKRLEWVAT

ITSGGTYTYYPDSVKGRFTISRDNAKNTLYLQIDSLKSEDTAIYFCARSL

AGNAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGD

RVSLSCRASQTISDYLHWYQQKSHESPRLLIKFASQSISGIPSRFSGSGSGS

GSDFTLSINSVEPEDVGVYYCQNGHGFPRTFGGGTKLEIKEQKLISEEDL

By "5C4 scFv" is meant a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence below and having the ability to bind human PD-1 [SEQ ID NO: 12].

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAV

IWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATND

DYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLS

CRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT

LTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK

By "J43 scFv" is meant a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence below, which includes a kappa leader sequence from amino acids 1-21 and is capable of binding Human PD-1 [SEQ ID NO: 13].

METDTLLLWVLLLWVPGSTGDMGLGLQWVFFVALLKGVHCEVRLLESGGGLVKP

EGSLKLSCVASGFTFSDYFMSWVRQAPGKGLEWVAHIYTKSYNYATYYSGSVKG

RFTISDDSRSMVYLQMNNLRTEDTATYYCTRDGSGYPSLDFWGQGTQVTVSSA

TTTAPSVYPLAPACDSTTKSGGGGSGGGGSGGGGSYELTQPPSASVNVGETVKI

TCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTI

RDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLGGPKSSPKVTVFPPSPEE

LRTNKATLVCLVNDFYPGSATVTWKANGATINDGVKTTKPSKQGQNYMTSSYLS

LTADQWKSHNRVSCQVTHEGETVEKSLSPAECLEQKLISEEDL*

An exemplary nucleic acid sequence encoding a scFv J43 polypeptide comprising a kappa leader sequence is shown below [SEQ ID NO: 14].

ccATGGAGACAGACACACTCCTGCTATGGGTACTGCCTGCTCTGGGTTCCAGGTT

CCACTGGTGACatgggattgggactgcagtgggttttctttgttgctcttttaa

aaggtgtccactgtgaggtgcggcttctggagtctggtggaggattagtgaagc

ctgaggggtcactgaaactctcctgtgtggcctctggattcaccttcagtgact

atttcatgagctgggtccgccaggctccagggaaggggctggagtgggttgctc

acatatacacgaaaagttataattatgcaacttattactcgggttcggtgaaag

gcagattcaccatctccagagatgattcccgaagcatggtctacctgcaaatga

acaacctgagaactgaggacacggccacttat actgtacaagagatggaagcg

gatatccctctctggatttctggggtcaagggacccaagtcactgtctcctcag

ccacaacaacagccccatctgtctatcccttggcccctgcctgtgacagcacaa

ccaaatcgggtggaggtggatcaggtggaggtggatctggtggaggtggatctt

atgagctgactcagccaccttcagcatcagtcaatgtaggagagactgtcaaaa

tcacctgctctggggaccaattgccgaaatattttgcagattggtttcatcaaa

ggtcagaccagaccattttgcaagtgatatatgatgataataagcgcccctcgg

ggatccctgaaagaatctctgggtccagctcagggacaacagccaccttgacca

tcagagatgtccgggctgaggatgaaggtgactattactgtttctcaggatatg

ttgatagtgatagcaaattgtatgttttttggcagcggaacccagctcaccgtcc

taggtggacccaagtcttctcccaaagtcacagtgtttccaccccttcacctgagg

agctccggacaaacaaagccacactggtgtgtctggttaatgacttctacccgg

gttctgcaacagtgacctggaaggcaaatggagcaactatcaatgatggggtga

agactacaaagccttccaaacagggccaaaactacatgaccagcagctacctaa

gtttgacagcagaccagtggaaatctcacaacagggtttcctgccaagttaccc

atgaaggggaaactgtggagaagagtttgtcccctgcagaatgtctcgaacaaa

aactcatctcagaagaggatctgTAActcgag

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but generally have significant identity. Polynucleotides having "significant identity" with an endogenous sequence are generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant a pair to form a double-stranded molecule between complementary polynucleotide sequences (eg, the gene described herein) or portions thereof, under varying stringency conditions. (See, for example, Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, the hard salt concentration is typically less than about 750 mM NaCl and 75 mM sodium citrate, preferably less than about 500 mM NaCl and 50 mM sodium citrate, and more preferably less than about 250 mM NaCl and 25 mM sodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters such as hybridization time, detergent concentration such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA are well known to those skilled in the art. Different levels of hardness are achieved by combining these different conditions as required. In a preferred embodiment, hybridization will occur at 30°C, 750 mm NaCl, 75 mm sodium citrate and 1% SDS. More preferably, hybridization occurs at 37° C. in 500 mM NaCl, 50 mM sodium citrate, 1% SDS, 35% formamide and 100 μg/ml denatured salmon sperm DNA (ssDNA). Most preferably, hybridization occurs at 42° C. in 250 mM NaCl, 25 mM sodium citrate, 1% SDS, 50% formamide, and 200 μg/ml slDNA. Suitable changes to these conditions should be obvious to a person skilled in the art.

For most applications, the wash steps that follow hybridization also vary in severity. The harshness conditions of the wash can be determined from the salt concentration and temperature. As stated above, the harshness of the wash can be increased by decreasing the salt concentration or by increasing the temperature. For example, the hard salt concentration for the wash step is preferably less than about 30 mM NaCl and 3 mM sodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM sodium citrate. Stringent temperature conditions for the stage of washing typically include a temperature of at least about 25°C, more preferably at least about 42°C and even more preferably at least about 68°C. In a preferred embodiment, the wash steps are carried out at 25° C. in 30 mM NaCl, 3 mM sodium citrate and 0.1% SDS. In a more preferred embodiment, the wash steps are carried out at 42° C. in 15 mM NaCl, 1.5 mM sodium citrate and 0.1% SDS. In a more preferred embodiment, the wash steps are carried out at 68° C. in 15 mM NaCl, 1.5 mM sodium citrate and 0.1% SDS. Additional variations on these conditions will be apparent to those skilled in the art. Hybridization methods are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Rogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al, Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By "substantially identical" is meant a polypeptide or nucleic acid molecule that shares at least 50% identity with an amino acid sequence (e.g., any of the amino acid sequences described herein) or a nucleic acid sequence (e.g., any of the nucleic acid sequences acids described here). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison. .

Sequence identity is typically measured using sequence analysis software (e.g., sequence analysis software package from the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705, BLAST, BESTFIT, GAP, or PILEUP/ PRETTYBOX). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In a typical approach, the BLAST program can be used to determine the degree of identity, with a hit probability of e-3 and e-100 showing closely related sequences.

By "analogue" is meant a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

The term "ligand" as used herein refers to a molecule that binds to a receptor. In particular, the ligand binds to a receptor on another cell, which makes it possible for one cell to recognize and/or interact with another.

The term "constitutive expression" as used herein refers to expression under any physiological conditions.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasia or pathogenic infection of a cell.

"Effective amount" means an amount sufficient to have a therapeutic effect. In one embodiment, an "effective amount" is an amount sufficient to delay, mitigate, or inhibit continued proliferation, growth, or metastasis (eg, invasion or migration) of the neoplasia.

By "endogenous" is meant a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

By "forced tolerance" is meant the prevention of the activity of autonomous cells or immunoreactive cells that target transplanted organs or tissues.

By "exogenous" is meant a nucleic acid molecule or polypeptide that is not endogenously present in the cell, or is not present at a level sufficient to achieve the functional effects obtained by overexpression. The term "exogenous" thus encompasses any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous and overexpressed nucleic acid molecules and polypeptides.

By "heterologous nucleic acid molecule or polypeptide" is meant a nucleic acid molecule (eg, cDNA, DNA, or RNA) or polypeptide that is not normally present in a cell or a sample derived from a cell. This nucleic acid may be from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

By "immunoreactive cell" is meant a cell that performs functions in an immune response, or a precursor or progeny thereof.

By "increase" is meant a positive change of at least 5%. The change can be 5%, 10%, 25%, 30%, 50%, 75% or even 100%.

By "isolated cell" is meant a cell that is separated from the molecular and/or cellular components that naturally accompany the cell.

The terms "isolated", "purified" or "biologically pure" refer to a material that is, to varying degrees, free from the components that normally accompany it, as found in its native state. "Isolate" refers to the degree of separation from the original source or environment. "Purity" refers to the degree of separation, which is higher than the selection. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not significantly affect the biological properties of the protein or cause other adverse effects. That is, a nucleic acid or peptide of the present invention is purified if it is substantially free of cellular material, viral material or culture medium when produced by recombinant DNA methods, or chemical precursors or other chemicals in the case of chemical synthesis. Purity and homogeneity are typically determined using analytical chemistry methods such as polyacrylamide gel electrophoresis and high performance liquid chromatography. The term "purified" may mean that the nucleic acid or protein results in the formation of essentially one band in the electrophoresis gel. For a protein that can be modified, such as phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be separately purified.

The term "tumor antigen-binding domain", as used herein, refers to a domain capable of specifically binding to a particular antigenic determinant or set of antigenic determinants presented on a tumor.

The term "obtainable", as in "obtainable agent", is intended to include purchased, synthesized or otherwise acquired agent (or specified substance or material).

"Linker", as used herein, means a functional group (eg, a chemical reagent or a polypeptide) that covalently links two or more polypeptides or nucleic acids so that they are connected to each other. As used herein, a "peptide linker" refers to one or more amino acids used to join two proteins together (eg, to connect the V _H and V _L domains). An exemplary linker sequence used in the invention is GGGGSGGGGSGGGGS [SEQ ID NO:51].

By "modulate" is meant to change positively or negatively. Typical modulations include 1%, 2%, 5%, 10%, 25%, 50%, 75% or 100% replacement.

By "neoplasia" is meant a disease characterized by the abnormal proliferation of a cell or tissue and its subsequent migration or invasion into other tissues or organs. The growth of neoplasia is usually uncontrolled and progressive, and occurs under conditions that would not cause or lead to the cessation of normal cell reproduction. Neoplasias can affect various types of cells, tissues, or organs, including, but not limited to, an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder , heart, intestines, kidney, liver, lung, lymph node, nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urinary tract, ureter , urethra, uterus and vagina, or tissue or cell type of it. Neoplasias include malignant tumors such as sarcomas, carcinomas or plasmacytomas (malignant tumor of plasma cells).

By "pathogenic microorganism" is meant a virus, bacteria, fungus, parasite or protozoan capable of causing disease.

Exemplary viruses include, but are not limited to, Retroviridae (e.g., human immunodeficiency viruses such as HIV-I (also referred to as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III; and other isolates such as HIV -LP; Picornaviridae (e.g. poliomyelitis viruses, hepatitis A virus; enteroviruses, human coxsackieviruses, rhinoviruses, ECHO viruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, virus mumps, Measles virus, respiratory syncytial virus); Orthomyxoviridae (eg, influenza viruses); Bungaviridae (eg, Hantaan viruses, Bunge viruses, phleboviruses and Naira viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (eg reoviruses, orbiviruses and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, chickenpox viruses); and Iridoviridae (e.g. African swine fever virus) and unclassified viruses (eg, delta hepatitis agent (believed to be a defective satellite of hepatitis B virus), hepatitis agents other than hepatitis A and hepatitis B (class 1 = internally transmitted; class 2 = parenterally transmitted (i.e. hepatitis C); Norwalk virus and related viruses, and astroviruses).

Representative bacteria include, but are not limited to, Pasteurella species, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae , Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp. , Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtheriae, corynebacterhim sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp. , Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia and Actinomyces israelii.

By "receptor" is meant a polypeptide, or portion thereof, present on a cell membrane that selectively binds to one or more ligands.

By "reduce" is meant to change negatively by at least 5%. The change can be 5%, 10%, 25%, 30%, 50%, 75% or even 100%.

"Recognizes" means selectively binds the target. The T cell that recognizes the virus usually expresses a receptor that binds the antigen expressed by the virus.

By "reference" or "control" is meant a standard for comparison. For example, the level of scFv antigen binding by a cell, expression of CAR and scFv, can be compared with the level of scFv antigen binding in a corresponding cell expressing only CAR.

By "secreted" is meant a polypeptide that is released from a cell via a secretory pathway through the endoplasmic reticulum, the Golgi apparatus, and as vesicles that fuse transiently with the cell's plasma membrane, releasing proteins outside the cell.

By "signal sequence" or "leader sequence" is meant a peptide sequence (5, 10, 15, 20, 25, 30 amino acids long) present at the N-terminus of the newly synthesized polypeptide chain of newly synthesized proteins that directs their entry into the secretory pathway. Exemplary leader sequences include the kappa leader sequence: METDTLLLWVLLLWVPGSTGD [SEQ ID NO: 15] (human), METDTLLLWVLLLWVPGSTGD [SEQ ID NO: 16] (mouse); and CDS leader sequence: MALPVTALLLPLALLLHAARP [SEQ ID NO: 17].

By "soluble" is meant a polypeptide that diffuses freely in an aqueous medium (eg, not bound to a membrane).

By "specifically binds" is meant a polypeptide or fragment thereof that recognizes and binds to a biological molecule of interest (e.g., a polypeptide), but which does not substantially recognize and bind other molecules in the sample, e.g., a biological sample that naturally includes the polypeptide according to the invention.

The term "tumor antigen" as used herein refers to an antigen (eg, polypeptide) that is uniquely or differentially expressed on tumor cells as compared to a normal or non-IS neoplastic cell. With respect to the invention, a tumor antigen includes any tumor-expressed polypeptide that is capable of activating or inducing an immune response via an antigen recognition receptor (e.g., CD19, MUCI) or is capable of suppressing an immune response via receptor-ligand binding (e.g., CD47, PD-L1 /L2, B7.1/2).

By "viral antigen" is meant a polypeptide expressed by a virus that is capable of inducing an immune response.

The terms "comprises", "comprising" and are intended to have the broad meaning given to them in US patent law and may mean "includes", "including", and the like.

As used herein, "treatment" refers to a clinical intervention in an attempt to alter the course of a disease in an individual or cell being treated, and may be done either for prophylaxis or in the course of clinical pathology. The therapeutic effects of treatment include, without limitation, prevention of the onset or recurrence of the disease, mitigation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or temporary relief of the disease state, and remission or improvement in prognosis. By preventing the progression of the disease or disorder, treatment may prevent the worsening of the condition caused by the disorder in an afflicted or diagnosed individual or an individual with a suspected disease, but also treatment may prevent the development of the disorder or symptom of the disorder in an individual at risk for or suspected of having the disorder.

The term "individual" as used herein refers to a vertebrate, preferably a mammal, more preferably a human.

The term "immune-compromised" as used herein refers to an individual who is immunocompromised. The individual is very vulnerable to opportunistic infections, infections caused by organisms that do not normally cause disease in a person with a healthy immune system, but which can affect people with a poorly functioning or suppressed immune system.

Other aspects of the invention are described in the following disclosures and are within the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The following detailed description, given by way of example but not intended to limit the invention to the particular embodiments described, may be understood in conjunction with the accompanying drawings.

Figure 1 illustrates T cells modified to express a chimeric antigen receptor (CAR), alone or in combination with a secreted scFv (eg, aPD-1, PD-Ll, CTLA-4, or aCD47). T cells modified to express the chimeric antigen receptor (CAR) itself are subject to suppression within an unfavorable tumor microenvironment. Without being bound by a particular theory, further modification of these cells to express secreted scFv to block immunosuppressive signaling improved antitumor function due to their ability to modulate the tumor microenvironment and resist suppressive factors.

Figures 2A and 2B illustrate the structure of secreted anti-CD47 scFv constructs. Figure 2A illustrates the structure of a secreted anti-CD47 scFv designed to include a kappa (κ) leader sequence to allow secretion of this protein. The variable heavy (V _H ) and light (V _L ) chains were linked with a serine-glycine linker (G4S) and a myc-tag peptide was included to allow scFv detection. Figure 2B illustrates a secreted scFv linked to a 1928z CAR construct with a P2A element as shown in the figure.

Figure 3 illustrates the sequence of B6H12.2 scFv operably linked to a Kappa leader sequence [SEQ ID NO: 18]. The variable heavy (V _H ) and variable light (V _L ) B6H12.2 hybridoma sequences were PCR amplified with a kappa leader sequence, a c-myc tag, and linked to a serine-glycine linker. The nucleic acid sequence and amino acid translation are shown.

Figure 4 illustrates the sequence of B6H12.2 scFv operably linked to the CDS leader sequence [SEQ ID NO:19]. The variable heavy (V _H ) and variable light (V _L ) B6H12.2 hybridoma sequences were PCR amplified with a CDS leader, c-myc tag, and linked to a serine-glycine linker. The nucleic acid sequence and amino acid translation are shown.

Figure 5 illustrates the nucleic acid sequence of construct 1928z-2A-B6H12.2 (kappa leader) [SEQ ID NO:20]. The B6H12.2 scFv sequence was cloned into an SFG expression vector for expression with the CD19-targeted 1928z CAR. The P2A element was used to connect two elements as shown.

Figure 6 illustrates the nucleic acid sequence of construct 4HH28z-2A-B6H12.2 (kappa leader) [SEQ ID NO:21]. The B6H12.2 scFv sequence was cloned into an SFG expression vector to express the MUC-CD targeted 4H1128z chimeric antigen receptor (CAR). A P2A element was used to connect two elements as shown.

Figures 7A and 7B illustrate the creation of 1928-2A-B6H12.2 293Glv9 packaging cells. Viral packaging cells were generated using the vector 1928z-2A-B6H12.2 or 1928z. Figure 7 illustrates the selection of two clones, clones 5 and 6, based on 1928z CAR expression that was comparable to control 1928z 293Glv9 cells. CAR expression was determined by flow cytometry with staining and antibody 12dll. Figure 7B illustrates an experiment in which a supernatant from 1928z or 1928z-2A-B6H12.2 packaging cells was incubated with CD47 ⁺ tumor cells, Nalm-6 and Raji, and the tumor cells were washed and stained with anti-CD47. Tumor cells incubated in 1928z-2A-B6H12.2 supernatant (Blue lines) had reduced anti-CD47 binding compared to incubation in 1928z supernatant (Black line). Supernatant from B6H12.2 hybridoma cell was used as control (Red line).

Figures 8A and 8B illustrate the generation of human peripheral blood 1928z-2A-B6H12.2 T cells. Human peripheral blood T cells were transduced with 1928z or 1928z-2A-B6H12.2 packaging cell supernatant. Figure 6A illustrates flow cytometric analysis of CAR expression using the 12d1l antibody, and bound anti-CD47 scFv was stained with fluorescently labeled c-myc tag antibody. Figure 6B illustrates the ability of anti-CD47 scFv to block CD47 as determined by staining T cells with an anti-CD47 antibody. 1928z-2A-B6H12.2 T cells had reduced anti-CD47 binding (Blue line) compared to 1928z T cells (Red line). 1928z T cells incubated in B6H12.2 hybridoma supernatant were used as controls (black line).

Figures 9A-9C illustrate human peripheral blood 1928z-2A T cells. Flow cytometry was performed to characterize the phenotype of 1928z and 1928z-2A-B6H12.2 T cells. Figure 9A illustrates that 1928z and 1928z-2A T cells had an equivalent ratio of CD4:CD8 T cells, and equivalent expression of the CD69 and CD25 activation markers. 1928z T cells were overexpressing CD62L compared to 1928Z-2A - B6H12.2 T cells. Figure 9b illustrates the ability of 1928z and 1928z-2A - B6H12.2 T cells to secrete cytokines as assessed by flow cytometry after incubation with 3T3 (CD19 ⁺ /B7.1 ⁺ ) aAPC cells and Golgi transport inhibitors, Golgi plug and Golgi Stop. 1928z and 1928z-2A-B6H12.2 T cells produced equivalent levels of Il-2 and IFNg after stimulation with 3T3(CD19 ⁺ /B7.1 ⁺ ) cells. Figure 9C illustrates that 1928z and 1928z-2A-B6H12.2 T cells have equivalent cytolytic potential as determined by a standard ⁵¹ chromium release assay using Raji tumor cells.

Figures 10A and 10b illustrate the antitumor efficacy of 1928z-2A-B6H12.2 T cells. The antitumor efficacy of 1928z-2A-B6H12.2 T cells in vivo was studied in a preclinical SCID-Beige mouse model. Mice were intravenously inoculated with 1 x 10 ⁶ Nalm-6-Firefly luciferase* tumor cells and then treated with 5.7 x 10 ⁶ CAR ⁺ 1928z, 1928z-2A-B6H12.2, or control 4H1128z ovarian cancer-targeting T cells -2A-B6H12.2, also inoculated intravenously. Figure 10A illustrates that mice treated with 1928z-2A-B6H12.2 T cells had increased survival compared to non-treated, 1928z or treated 4H1128z-2A-B6H12.2 mice. Figure 10B illustrates that 1928z-2A-B6H12.2 treated mice reduced tumor mass compared to non-treated, 1928z or 4H1128z-2A-B6H12.2 T-cell treated mice using bioluminescent imaging to monitor tumor progression.

Figure 11 illustrates a 5C4 scFv sequence operably linked to a kappa leader sequence [SEQ ID NO:22]. The variable heavy (V _H ) and variable light (V _L ) sequences of antibody clone 5C4 were designed with a kappa leader sequence, a c-myc tag, and linked with a serine-glycine linker. The nucleic acid sequence and amino acid translation are shown.

Figure 12 illustrates the nucleic acid sequence of construct 1928z-2A-5C4 (kappa leader) [SEQ ID NO:23j. The 5C4 scFv sequence was cloned into an SFG expression vector for expression from the CD19-targeted 1928z CAR. P2A element was used to connect two elements as shown.

Figure 13 illustrates the nucleic acid sequence of construct 4H1128z-2A-5C4 (kappa leader) [SEQ ID NO:24]. The 5C4 scFv was cloned into an SFG expression vector to be expressed with the MUC-CD-targeted 4H1128z CAR. The P2A element was used to combine the two elements as shown.

Figure 14 illustrates the establishment of 1928z-2A-5C4 (kappa leader) 293Giv9 cells. Viral packaging cells were generated using 1928z-2A-5C4. Two clones, clones A6 and B6, were selected based on the expression of 1928z CAR, which is comparable to control l928z 293Glv9 cells. CAR expression was determined by flow cytometry and staining with antibody 12d11.

Figure 15 illustrates the generation of a 1928z-2A-5C4 (kappa leader) human peripheral blood T cell. Human peripheral blood T cells were transduced with supernatant from 1928z or 1928z-2A-5C4 packaging cells. Flow cytometry was used to analyze CAR expression with the 12d11 antibody and bound anti-CD47 scFv by staining with a fluorescently labeled c-myc tag antibody.

Figure 16 illustrates PD-L1 expression on 3T3(CD19 ⁺ /B7.1 ⁺ ), Raji and Nalm-6 cells. Flow cytometry was used to determine PD-L1 expression on 3T3(CD19 ⁺ /B7.1 ⁺ ), Raji and Nalm-6 cells that were transduced to express PD-L1. Transduced cells expressing significant levels of PD-L1 compared to control non-transduced cells and circulating populations were sorted for use in experiments.

Figure 17 illustrates the spread of 1928z and 1928z-2A-5C4 T cells. 1928z and 1928z-2A-5C4 T cells were incubated with 3T3(CD19 ⁺ /B7.1 ⁺ ) or 3T3(CD19 ⁺ B7.1 ⁺ /PD-L1 ⁺ ), T cell expansion was monitored with trypan blue, and expression CAR was determined by flow cytometry. Growth and expression of CARs correlated with those of cells propagating on 3T3(CD19 ⁺ /B7.1 ⁺ ) cells.

Figure 18 illustrates the J43 scFv sequence operably linked to the mouse kappa leader sequence [SEQ ID NO:25]. The sequences of the variable heavy (V _H ) and variable light (V _L ) antibodies of clone J43 were designed with a mouse kappa leader, a c-myc tag, and linked with a serine-glycine linker. The sequence and amino acid translation are shown.

Figure 19 illustrates the nucleic acid sequence of construct 19m28mziRESJ43 (mouse kappa leader) [SEQ ID NO:26]. The J43 scFv was cloned into an SFG expression vector for expression from the CD19-targeted 19m28mz CAR. An internal ribosome entry site (IRES) element is used to connect two elements as shown.

Figure 20 illustrates the nucleic acid sequence of construct 4H11m28mziRESJ43 (mouse kappa leader) [SEQ ID NO:27]. The J43 scFv was cloned into an SFG expression vector to express the MUC-CD-targeted 4H11m28mz CAR. An internal ribosome entry site (IRES) element is used to connect two elements as shown.

Figure 21 illustrates strategies to genetically modify CART cells to express scFv molecules ("armored CAR T cells") to overcome the "hostile" tumor microenvironment. CAR* T cells can be modified to secrete antagonistic scFvs with immune regulatory functions. Upon activation of a CAR by a cognate antigen (1), armored CAR-modified T cells can be induced to secrete scFvs antagonistic to the inhibitory PD-1 T-cell receptor on both infused CAR-modified T cells and endogenous antitumor agents. T cells, enhancing the antitumor effector function (2) induced to secrete scFv antagonistic to the inhibitory CTLA-4 T cell receptor on both infused CAR modified T cells and endogenous antitumor T cells, enhancing the antitumor effector function (3) or induced to secrete scFvs antagonistic to the CD47 receptor expressed on the tumor cell, abolishing the tumor cell's masking from recognition by the host's innate antitumor immune response, leading to tumor recognition and eradication by host macrophages.

Figures 22A - 22D illustrate the constitutive expression of CD40L by human T cells. (A) Diagram of a retroviral construct encoding a human CD40L vector; LTR, long terminal repeat; SD, SA, splice donor and acceptor; Ψ, packing element. (B) Flow cytometry of CD4+ and CD8+ CD40L-modified T cells after retroviral gene transfer; x-axis APC-conjugated human anti-CD40L (CD154). (C) Enhanced proliferation of CD40L-modified T cells compared to sham-transduced T cells. (D) Increased secretion of soluble CD40L (sCD40L), IFN-γ, GM-CSF from CD40L-modified T cells compared to sham-transduced T cells. All results represent at least three separate experiments. (* denotes statistical significance)

Figures 23A and 23B illustrate the increased immunogenicity of CD40+ tumor cells by CD40L-modified T cells. A) Flow cytometry showing increased activity of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58 and CD70), HLA-class molecules (HLA class I and HLA-DR) and Fas death receptor (CD95) in the tumor line DOHH2 cells co-cultured with CD40L-modified T cells (solid line) versus sham-transduced T cells from the same donor (grey line). (B) CD40 tumor (NALM6 shown) showing no phenotypic change after co-culture with CD40L-modified T cells. All results represent at least three separate experiments.

Figures 24A and 24B illustrate the increased immunogenicity of CLL cells with autologous CD40L-modified T cells. (B) Flow cytometry of patient-derived CD40L-modified T cells after retroviral gene transfer with a CD40L-containing retroviral vector; x-axis APC-conjugated human anti-CD40L (CD154). (B) Flow cytometry showing increased activity of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58 and CD70), HLA-class molecules (HLA class I and HLA-DR), and Fas death receptor (CD95) on CLL α-cells after co-culture with autologous CD40L-modified T cells (solid line) compared to sham-transduced T cells from the same donor (gray line). All results represent at least three separate experiments.

Figures 25A and 25B illustrate IL-12 secretion and maturation of monocyte-derived dendritic cells (moDCs) with CD40L-modified T cells. (A) Cytokine analysis of culture medium for co-culture (24 hours) moDCs and CD40L-modified T cells from three separate donors, demonstrating increased secretion of IL-I2p70. (B) Flow cytometry of moDC showing maturation after co-culture with CD40L-modified T cells. All results represent at least three separate experiments.

Figures 26A-C illustrate the efficient transduction of human T cells with the CAR/CD40L vector, demonstrating increased cytotoxicity. (A) Diagram of a retroviral construct containing the 1928z-IRES-CD40L and PZ1-IRES-CD40L genes; LTR, long terminal repeat; CD, CA, splice donor and acceptor; Ψ, packaging element; CD8 shows the CD8 leader sequence; scFv, single chain variable fragment; VH and VL, variable heavy and light chains; TM, transmembrane domain. (B) FACS analysis of human T cells transduced to express 19-28z/CD40L vector (pre-stimulation) followed by upregulation of CAR/CD40L expression after co-culture on AAPC (NIH 3T3 fibroblasts expressing CD19 and CD80; 1928z/ are shown; CD40LT cells) used for in vivo experiments. x - axis, PE-conjugated 1928z CAR-specific antibody (19e3); Y-axis, APC-conjugated human anti-CD40L (CD154). (C) As determined in a standard ⁵¹ Cr release assay, 19-28z/40L T cells have a significantly increased ability to lyse DOHH2 tumor cells compared to 1928z T cells. All results represent at least three separate experiments. (* denotes statistical significance).

Figure 27 illustrates tumor eradication and long-term survival after infusion of 1928z CD40L T cells. Survival curve of SCID-Beige mice inoculated with DOHH2 tumor cells via intravenous (i.v.) injections 2 days before a single i.v. doses of CAR-modified T cells. An increase in long-term survival was demonstrated in mice treated with 1928z/CD40L T cells (n=10) compared to a panel of control T cells (1928z group, n=8; and Pz1/40L group n=5). The results represent at least two separate experiments. (* denotes statistical significance).

Figure 28 illustrates the increased immunogenicity of CD40+ tumor cells with sCD40L. A) Flow cytometry showing increased activity of co-stimulatory molecule (CD80), adhesion molecules (CD54, CD58 and CD70), HLA molecule (HLA class I and HLA-DR), and Fas death receptor on tumor cell line DOHH2 after co-cultivation with conditioned media (CD40L-modified T cell media) containing elevated levels of sCD40L (solid line) compared to media (sham-transduced T cell media) without elevated sCD40L levels (gray line).

Figure 29 illustrates 1928z/CD40L T cells showing increased cytotoxicity. As determined in a standard 51Cr release assay, 19-28z/40l T cells have a significantly increased ability to lyse Raji tumor cells compared to 19-28z T cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally provides cells, including genetically modified immunoreactive cells (e.g., T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL) cells), expressing at least an antigen-recognition receptor combination ( e.g. TCR or CAR) and either (i) a scFv that binds an immunosuppressive antigen (e.g. aPD-1, aPD-Ll, aCTLA-4 or aCD47)) (ii) a scFv that binds an immunostimulatory antigen (e.g. ocCD28, aOX- 40, aCD40 or α4-1BB) or (iii) CD40L, and methods of using such cells to treat neoplasia and other pathologies where an increase in an antigen-specific immune response is desired. The invention is based at least in part on the discovery that scFvs that bind an immunosuppressive antigen (eg CD47 and PD-L1 as shown herein) are useful in activating and stimulating an immunoreactive cell. In particular, the scFvs of the invention reduce or prevent suppression of the immune response of an activated immunoreactive cell in the tumor microenvironment. Malignant cells have evolved a number of mechanisms to protect themselves from immune recognition and elimination. The present approach provides immunogenicity in the tumor microenvironment for tumor eradication, and represents a significant advance over conventional adoptive T cell therapy.

Tumor microenvironment

Tumors have a microenvironment that is hostile to the immune response, including a number of cancer cell mechanisms to protect themselves from immune recognition and elimination. This "hostile tumor microenvironment" includes various immunosuppressive factors, including infiltrating regulatory CD4 ⁺ T cells (Treg), myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAMs), immunosuppressive cytokines, including IL-10 and TGF-β, and expression of ligands targeting immune suppressor receptors expressed by activated T cells (CTLA-4 and PD-1). These immune suppression mechanisms play a role in maintaining tolerance and suppressing inappropriate immune responses, however, in the tumor microenvironment, these mechanisms prevent an effective antitumor immune response. Together, these immunosuppressive factors can induce either marked anergy or apoptosis of adoptively transferred CAR modified T cells upon encountering tumor target cells.

Cytotoxic T lymphocyte antigen 4 (CTLA-4)

CTLA-4 is an inhibitory receptor expressed by activated T cells which, when contacted with its respective ligands (CD80 and CD86; B7-1 and B7-2, respectively), mediates inhibition of activated T cells or anergy. In both preclinical and clinical studies, CTLA-4 blockade by systemic antibody infusion enhanced the endogenous antitumor response, albeit in the clinical setting, with significant unforeseen toxicities. Without wishing to be bound by a particular theory, CTLA-4 targeted blockade by delivery of antagonistic scFvs by tumor-targeted CAR-modified T cells reduces toxicity and also provides a surrogate "endogenous" population of tumor-targeted T cells (CART cell population), protected from immune suppression. Preclinical studies (eg, human tumor xenograft models and mouse tumor models of B-cell malignancies and ovarian carcinomas) can be used to evaluate the effect of scFv secretion on both the CAR-modified T cell population as well as endogenous antitumor immune responses. Anti-CTLA-4 scFv can be derived from the 9D9 hybridoma, which secretes mouse anti-mouse CTLA-4 monoclonal antibodies, or from the 9H10 hybridoma, which secretes hamster anti-mouse CTLA-4 monoclonal antibodies.

Programmed cell death protein 1 (PD-1)

PD-1 is a negative immune regulator of activated T cells after interacting with the respective PD-L1 and PD-L2 ligands expressed on endogenous macrophages and dendritic cells. PD-L1 activation is one of the mechanisms by which a tumor cell can evade the host's immune system. Again, in both preclinical and recently published clinical trials, PD-1 blockade by antagonistic antibodies induced antitumor responses mediated through the host's endogenous immune system. Xenograft and syngeneic mouse tumor models can be used to show that antagonistic anti-PD-1 scFv secreted by tumor-targeted CAR-modified T cells enhance the antitumor efficacy of these scFv-secreting CAR-modified T cells.

CD47

CD47 is a membrane protein with a wide tissue distribution and has been shown in recent preclinical models to protect a wide range of tumor cells from recognition by macrophages. In these models, infusion of anti-CD47 monoclonal antibodies resulted in a reduction in sustained tumor progression. In other words, CD47 blockade of tumor cells exposes these tumor cells to recognition and phagocytosis by host macrophages. Given the fairly ubiquitous expression of this antigen, systemic infusion of a blocking antibody could potentially lead to non-target toxicity. Again, according to the targeting paradigm, secretion of a similarly blocking anti-CD47 scFv delivered directly to the tumor microenvironment by CAR-modified T cells produces/enhances the desired antitumor effect, in this case mediated by the innate rather than the adaptive immune system. host systems. In addition, this approach is not limited to the treatment of neoplasia, but is amenable to a wide range of applications where enhancement of an antigen-specific immune response is desired, such applications include not only the treatment of neoplasia, but are also applicable to enhance the immune response against a pathogenic infection or infectious disease and to enhance immune tolerance in regulatory T cells in the context of autoimmune diseases or allogeneic transplantation.

CD40L

The CD40 ligand (CD40L, CD-154), a type II transmembrane protein belonging to the tumor necrosis factor (TNF) gene superfamily, has the potential to enhance tumor-specific T-cell function. Initially identified on activated CD4+ T cells, CD40L expression is inducible in a wide range of immune, hematopoietic, epithelial, endothelial and smooth muscle cells. In activated T cells, CD40L is expressed within minutes, peaking at 6 hours, and then decreasing over the next 12-24 hours. CD40L binds to its recognition receptor, CD40, which is expressed constitutively on a variety of immune and non-immune cells, including B cells, macrophages, and dendritic cells (DCs). Significantly, CD40 is also expressed on several hematologic and non-hematologic malignancies, including chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, nasopharyngeal carcinoma, osteosarcoma, Ewing's sarcoma, melanoma, cancer breast, ovary, and cervix, demonstrating the application of CAR/CD40L T cells for a wide range of malignant neoplasms. See references 8-17 listed below in the bibliography in example 6.

Hematopoietic cell lines

Mammalian hematopoietic (blood) cells provide a wide range of physiological activities. Hematopoietic cells are divided into lymphoid, myeloid and erythroid lines. The lymphoid line, including B-, T-cells and natural killer (NK) cells, provides for the production of antibodies, the regulation of cellular immunity, the detection of foreign agents in the blood, the detection of cells foreign to the host, and the like. The term "T cells" as used herein refers to lymphocytes that mature in the thymus and are primarily responsible for cellular immunity. T cells are involved in the adaptive immune system. The term "natural killer (NK) cells" as used herein refers to lymphocytes that are part of cell-mediated immunity and act during the innate immune response. They do not require prior activation to carry out their cytotoxic effect on target cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells.

Cells used in the methods of the invention

The present invention provides cells expressing an antigen recognition receptor that activates an immunoreactive cell (e.g., TCR, CAR) and a scFv that binds an immunosuppressive antigen (e.g., αPD-1, αPD-L1, αCTLA-4, or CD47) and methods the use of such cells to treat a disease that requires an enhanced immune response. In one approach, tumor antigen-specific T cells, NK cells, CTL cells, or other immunoreactive cells are used to express scFv that binds an immunosuppressive antigen to treat or prevent neoplasia. For example, T cells expressing a chimeric 1928z antigen receptor that recognizes CD19 are co-expressed in a T cell that expresses scFv that binds CD47. Such cells are administered to a human subject in need thereof for the treatment or prevention of blood cancers (eg leukemias, lymphomas and myelomas). In another approach, viral antigen-specific T cells, NK cells, CTL cells can be used to treat viral diseases. For example, a chimeric co-stimulatory antigen receptor that recognizes the first CMV antigen and scFv that binds PD-1 are co-expressed in cytotoxic T lymphocytes for the treatment of CMV.

A patient's own T cells can be genetically modified for tumor targeting by introducing genes that code for technical T cell receptors in the region in question, called chimeric antigen receptors (CARs). First generation CARs typically consist of an antibody-derived antigen recognition domain, a single chain antibody fragment (scFv), fused to a transmembrane variable domain fused to the cytoplasmic signaling domain of the T cell receptor chain. Additional inclusion of one or two costimulatory receptor signaling domains, including CD28, 4-1BB, and OX-40 proximal to the C chain, enhances CAR signaling, leading to second and third generation CARs, respectively.

Tumor antigen-specific T lymphocytes (and NK cells)

Types of tumor antigen-specific human lymphocytes that can be used in the methods of the invention include, without limitation, donor peripheral lymphocytes genetically modified to express chimeric antigen receptors (CAR) (Sadelain, M., et ah 2003 Nat Rev Cancer 3: 35-45), peripheral lymphocytes of donors genetically modified to express the full-length tumor antigen-recognition T-cell receptor complex, consisting of α- and β-hexodimer (Morgan, R.A., et al, 2006 Science 314: 126-129), cultures lymphocytes derived from tumor infiltrating lymphocytes (TIL) in tumor biopsies (Panelli, M.C., et al 2000 J Immunol 164:495-504; Panelli, M.C., et al. 2000 J Immunol 164:4382-4392), and selectively in in vitro disseminated antigen-specific peripheral blood leukocytes using artificial antigen presenting cells (AAPC) or pulsed dendritic cells (Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, G.A., et al. 2003 Blood 102:2498-2505). T cells can be autologous, allogeneic, or derived in vitro from engineered progenitors or stem cells. Any suitable tumor antigen (antigenic peptide) is suitable for use in tumor related embodiments described herein. Antigen sources include, but are not limited to, cancer proteins. The antigen can be expressed as a peptide or as an intact protein or portion thereof. The intact protein, or portion thereof, may be naturally occurring or mutagenic.

Suitable antigens include prostate-specific membrane antigen (PCMA) and prostate stem cell antigen (PCSA).

Viral antigen-specific T lymphocytes (and NK cells)

Antigens suitable for use in the treatment of pathogenic infection or other infectious diseases, for example, in an immunocompromised individual include, without limitation, viral antigens present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV) and influenza virus.

The crude source of CTL can be any source known in the art, such as bone marrow, fetus, neonate, or adult, or other source of hematopoietic cells, such as fetal liver, peripheral blood, or cord blood. Various methods may be used to separate the cells. For example, using negative selection methods, CTLs can be initially removed. Monoclonal antibodies (mAbs) are particularly useful for identifying markers associated with certain cell lines and/or stages of differentiation for both positive and negative selections.

A significant proportion of terminally differentiated cells can be initially removed by relatively rough separation. For example, magnetic bead separations can be used initially to remove large amounts of extraneous cells. Preferably at least about 80%, typically at least 70% of the total hematopoietic cells are removed prior to cell isolation.

Separation procedures include, but are not limited to, density gradient centrifugation; resetting; connection with particles that change the density of cells; magnetic separation using antibody-coated magnetic beads; affinity chromatography; cytotoxic agents attached to or used in combination with the mAb, including, but not limited to, complement and cytotoxins; and panning with antibodies attached to a solid matrix, such as a plate, chip, wash, or any other suitable technique.

Separation and analysis methods include, but are not limited to, flow cytometry, which may have varying degrees of complexity, such as multiple color channels, low angle detection and muted light scattering channels, impedance channels.

Cells can be selected for dead cells using dead cell-binding dyes such as propidium iodide (PI). Preferably, cells are harvested in media containing 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA), or any other suitable, preferably sterile, isotonic medium.

Accordingly, the invention generally provides an immunoreactive cell, such as a virus-specific or tumor-specific T cell, comprising a receptor that binds a first antigen and activates an immunoreactive cell, and a receptor that binds a second antigen and stimulates an immunoreactive cell.

Vectors

Genetic modification of immunoreactive cells (eg, T cells, CTL cells, NK cells) can be achieved by transducing a substantially homogeneous cell composition using a recombinant DNA construct. Preferably, a retroviral (either gamma-retroviral or lentiviral) vector is used to introduce the DNA construct into the cell. For example, a polynucleotide encoding a receptor that binds an antigen (e.g., a tumor antigen, or a variant or fragment thereof) can be cloned into a retroviral vector and expression can be directed from its endogenous promoter, from a retroviral long terminal repeat, or from a promoter specific for the type of target cell of interest. Non-viral vectors may also be used.

For initial genetic modification of cells to provide tumor antigen or viral antigen specific cells, a retroviral vector is typically used for transduction, however any other suitable viral vector or non-viral delivery system may be used. For subsequent genetic modification of cells to provide cells containing an antigen-presenting complex comprising at least two co-stimulatory ligands, retroviral gene transfer (transduction) also appears to be effective. Retroviral vector combinations and an appropriate packaging line are also suitable where the capsid proteins would be functional to infect human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA 12 (Miller, et al. (1985) Mol Cell Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al (1988) Proc, Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are also suitable, for example particles pseudotyped with VSVG, RD114 or GALV and any others known in the art.

Possible transduction methods also include direct co-cultivation of cells with producer cells, for example, the method of Bregni, et al. (1992) Blood 80: 1418-1422, or culturing with viral supernatant alone or concentrated vector stock with or without appropriate growth factors and polycations, for example, according to the method of Xu, et al. (1994) Exp. hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

Other viral vectors may be used to express the co-stimulatory ligands of the invention in an immunoreactive cell. Preferably, the selected vector exhibits high infection efficiency and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al, Current Eye Research 15:833-844, 1996; Bloomer et al, Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319, 1997) . Other viral vectors that may be used include, for example, adenovirus, lentiviral, and adena-associated viral vectors, vaccinia virus, bovine papillomavirus, or herpes virus such as Epstein-Barr virus (also see, for example, vectors Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61 , 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al, Nucleic Acid Research and Molecular Biology 36:31 1-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17 :407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al, Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and used in the clinical setting (Rosenberg et al, N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).

Non-viral approaches can also be used to express the protein in the cell. For example, a nucleic acid molecule can be introduced into a cell by introducing the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et ah, Am. J. Med. Sci. 298:278, 1989; Staubinger et ah, Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263: 14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection in a surgical setting (Wolff et al., Science 247: 1465, 1990). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes may also be potentially useful for delivering DNA into the cell. Transplantation of normal genes in diseased tissues of an individual can also be achieved by transferring the normal nucleic acid into a cultureable ex vivo cell type (e.g., an autologous or heterologous primary cell or its progeny), after which the cell (or its progeny) is introduced into the target tissue. or administered systemically. Recombinant receptors can also be derived from or made using transposases or targeted nucleases (eg, zincfinger nucleases, meganucleases, or TALE nucleases). Transient expression can be obtained by RNA electroporation.

cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein) promoters, and regulated by any appropriate mammalian regulatory element or intron (e.g., enhancer/promoter structure). /intron elongation factor 1a). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct nucleic acid expression. Useful enhancers may include, but are not limited to, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation may be mediated by related regulatory sequences or, if desired, by regulatory sequences derived from heterologous sources, including any of the promoters or regulatory elements described above.

The resulting cells can be grown under the same conditions as those of the unmodified cells, whereby the unmodified cells can be propagated and used for various purposes.

Polypeptides and analogues

Also included in the invention are the aCD19, aCD19, CD28, CD3ζ, 4H1128z, B6H12.2 scFv, scFv 5C4, and J43 scFv polypeptides, or fragments thereof, which are modified to enhance their anti-tumor activity (e.g., a humanized monoclonal antibody) when expression in an immunoreactive cell. The invention provides methods for optimizing an amino acid or nucleic acid sequence by making a change in the sequence. Such changes may include certain mutations, deletions, insertions, or post-translational modifications. The invention further includes analogues of any naturally occurring polypeptide of the invention. Analogues may differ from the naturally occurring polypeptide of the invention by amino acid sequence differences, post-translational modifications, or both. Analogues of the invention typically exhibit at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with all or part of the occurring the naturally occurring amino acid sequence of the invention. The length of the comparison sequence is at least 5, 10, 15 or 20 amino acid residues, preferably at least 25, 50 or 75 amino acid residues, and more preferably more than 100 amino acid residues. Again, in a typical approach, the BLAST program can be used to determine the degree of identity, with a hit probability of e ^-3 to e ^-100 indicating a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of polypeptides, for example, acetylation, carboxylation, phosphorylation or glycosylation; such modifications may occur during polypeptide synthesis or processing, or after treatment with isolated modifying enzymes. Analogues may also differ from the naturally occurring polypeptides of the invention by changes in the primary sequence. These include genetic variants, both natural and induced (for example, by random mutagenesis by radiation or exposure to ethane methyl sulfate, or by site-directed mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.) , CSH Press, 1989, or Ausubel et al, supra). Also included are cyclized peptides, molecules, and analogs that contain residues other than L-amino acids, such as D-amino acids, or non-natural or synthetic amino acids, such as beta (β) and gamma (γ) amino acids.

In addition to full length polypeptides, the invention also provides fragments of any of the polypeptides or peptide domains of the invention. As used herein, the term "fragment" means at least 5, 10, 13 or 15 amino acids. In other embodiments, the fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids, and in other embodiments, at least 60 to 80, 100, 200, 300 or more contiguous amino acids. Fragments of the invention may be created using methods known to those skilled in the art, or may result from normal protein processing (e.g., removal of amino acids from a growing polypeptide that are not necessary for biological activity, or removal of amino acids by alternative mRNA splicing events or alternative protein processing).

Non-protein analogues have a chemical structure designed to mimic the functional activity of the protein of the invention. Such analogs are administered in accordance with the methods of the invention. Such analogs may have physiological activity greater than that of the parent polypeptide. Methods for designing analogs are well known in the art, and analog synthesis can be accomplished using these methods by altering chemical structures such that the resulting analogs are superior in antitumor activity to the parent polypeptide when expressed in an immunoreactive cell. These chemical modifications include, but are not limited to, substitution of alternative R groups and varying the degree of saturation at particular carbon atoms of the reference polypeptide. Preferably, the protein analogs have a relatively high resistance to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Functional activity tests include, but are not limited to, those described in the examples below.

Costimulatory ligands

Interaction with at least one co-stimulatory ligand provides a non-antigen specific signal important for full activation of immune cells (eg, T cells). Costimulatory ligands include, but are not limited to, tumor necrosis factor (TNF) ligands, cytokines (such as IL-2, IL-12, IL-1, or IL21), and immunoglobulin (Ig) superfamily ligands. Tumor necrosis factor (TNF) is a cytokine involved in systemic inflammation and stimulates acute phase reactions. Its main role is to regulate immunocompetent cells. Tumor necrosis factor (TNF) ligands share a number of common features. Most ligands are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. TNF ligands include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L)/CD154, CD137L/4-1BBL, tumor necrosis factor alpha (TNFα), CD134L/OX40L/CD252, CD27L/CD70, FAS -ligand (FasL), CD30L/CD153, tumor necrosis factor beta (ΤΝΡβ)/lymphotoxin-alpha (LTα), lymphotoxin-beta (LTβ), CD257/B-cell-activating factor (BAFF)/Blys/THANK/Tall- 1, glucocorticoid-inducible TNF receptor ligand (GITRL), and TNF-associated apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins that are involved in recognition, binding, or adhesion processes in cells. These proteins share structural features with immunoglobulins - they have an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86 as ligands for CD28.

Introduction

Compositions containing genetically modified immunoreactive cells of the invention (eg, T cells, NK cells, CTL cells, or progenitors thereof) may be administered systemically or directly to an individual for the treatment of neoplasia, a pathogenic infection, or an infectious disease. In one embodiment, the cells of the invention are injected directly into an organ of interest (eg, an organ affected by neoplasia). Alternatively, compositions containing genetically modified immunoreactive cells are provided indirectly to the organ of interest, eg, by administration to the circulatory system (eg, tumors of the vascular bed). Extension and differentiation agents may be provided before, during, or after cell administration to increase the production of T cells, NK cells, or CTL cells in vitro or in vivo.

The modified cells can be introduced in any physiologically acceptable vehicle, usually intravascularly, although they can also be introduced into the bone or other convenient site where the cells can find a suitable site for regeneration and differentiation (eg, thymus). Typically, at least 1×10 ⁵ cells are injected, eventually reaching 1×10 ¹⁰ or more. Genetically modified immunoreactive cells according to the invention may include a purified population of cells. Those skilled in the art can easily determine the percentage of genetically modified immunoreactive cells in a population using various well known methods such as fluorescence excited cell sorter (FACS). Preferred purity ranges in populations containing genetically modified immunoreactive cells are about 50% to about 55%, about 55% to about 60%, and about 65% to about 70%. More preferably, the purity is from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%; and even more preferably, the purity is from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100%. Dosages can be easily adjusted with the help of a person skilled in the art (for example, if the purity decreases, an increase in the dose of the drug may be required). The cells may be administered by injection, catheter, or the like. Factors may also be included if desired, including, but not limited to, interleukins such as IL-2, IL-3, IL-6, IL-11, IL7, IL12, ILIS, IL21, as well as other interleukins, colony -stimulating factors such as G-, M- and GM-CSF, interferons such as interferon gamma and erythropoietin.

Compositions of the invention include pharmaceutical compositions containing genetically modified immunoreactive cells or their precursors and a pharmaceutically acceptable carrier. Administration may be autologous or heterologous. For example, immunoreactive cells or progenitors can be obtained from one individual and administered to the same individual, or another compatible individual. The peripheral blood-derived immunoreactive cells of the invention or their progeny (e.g., in vivo, ex vivo, or in vitro derivatives) can be administered by localized injection, including catheter, systemic, localized injection, intravenous injection, or parenteral administration. When a therapeutic composition of the present invention (eg, a pharmaceutical composition containing genetically modified immunoreactive cells) is administered, it will generally be formulated into a single dose injection (solution, suspension, emulsion) dosage form.

Lineups

Compositions of the invention comprising immunoreactive genetically modified cells can be readily formulated as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which can be buffered to provide a selected pH level. Liquid formulations are generally easier to prepare than gels, other viscous formulations, and solid formulations. In addition, liquid formulations are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated in an appropriate viscosity range to provide longer periods of contact with particular tissues. Liquid or viscous compositions may contain carriers, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, a polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the genetically modified immunoreactive cells used in the practice of the present invention in the required amount of an appropriate solvent with varying amounts of other ingredients as required. Such compositions may be admixed with a suitable carrier, diluent or excipient such as sterile water, saline, glucose, dextrose, or the like. The compositions may also be lyophilized. The compositions may contain adjuvants such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing agents, preservatives, flavoring agents, coloring agents, and the like, depending on the route of administration and formulation desired. Standard guidelines such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, can be consulted to prepare suitable formulations without undue experimentation.

Various additives that enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added. Prevention of the action of microorganisms can be provided by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be achieved by the use of absorption delaying agents such as aluminum monostearate and gelatin. In accordance with the present invention, however, any carrier, diluent or additive used must be compatible with genetically modified immunoreactive cells or their progenitors.

The compositions may be isotonic, ie. they may have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of the present invention can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is preferred, especially for buffers containing sodium ions.

The viscosity of the composition, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickener. Methylcellulose is preferred because it is physically and economically available and easy to handle.

Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener depends on the agent chosen. The important point is to use an amount that will achieve the required viscosity. Obviously, the choice of suitable carriers and other additives will depend upon the exact route of administration and the nature of the particular dosage form, e.g. liquid form).

Those skilled in the art will recognize that the components of the compositions should be chosen to be chemically inert and not affect the viability or efficacy of the genetically modified immunoreactive cells as described in the present invention. This presents no problem to those skilled in chemical and pharmaceutical matters, or problems can be easily avoided by reference to standard guidelines or by simple experimentation (without involving undue experimentation) from this disclosure and the documents cited herein.

One consideration regarding the therapeutic use of the genetically modified immunoreactive cells of the invention is the number of cells required to achieve an optimal effect. The number of cells administered varies depending on the individual being treated. In one embodiment, 10 ⁴ to 10 ¹⁰ , 10 ⁵ to 10 ⁹ , or 10 ⁶ to 10 ⁸ genetically modified immunoreactive cells of the invention are administered to a human subject. More efficient cells can be administered even in smaller amounts. In some embodiments, at least about 1x10 ⁸ , 2x10 ⁸ , 3x10 ⁸ , 4x10 ⁸ , and 5x10 ⁸ genetically modified immunoreactive cells of the invention are administered to a human subject. The precise determination of what is considered an effective dose may be based on factors specific to each individual, including their size, age, sex, weight, and the condition of the particular individual. Dosages can be readily determined by one of ordinary skill in the art from this disclosure and knowledge in the art.

One skilled in the art can easily determine the amount of cells and optional additives, delivery vehicles and/or carriers in the compositions and administered in the methods of the invention. Typically, any additives (in addition to the active cell(s) and/or agent(s)) are present in an amount of from 0.001 to 50% (w/w) of a solution in phosphate buffered saline and the active ingredient is present in an amount on the order of micrograms to milligrams, for example, from about 0.0001 to about 5 wt.%, preferably from about 0.0001 to about 1 wt.%, even more preferably from about 0.0001 to about 0.05 wt.%, or from about 0.001 to about 20 wt.%, preferably from about 0.01 to about 10 wt. % and even more preferably, from about 0.05 to about 5 wt.%. Of course, for any composition administered to an animal or human, and for any particular route of administration, it is therefore preferable to determine: toxicity, for example, by determining the lethal dose (LD) and LD50 in a suitable animal model, for example, rodents such as mice; and, the dosage of the composition(s), the concentration of the components therein, and the timing of administration of the composition(s) that elicit an appropriate response. Such definitions do not require undue experimentation based on the skill of the art, this disclosure, and the documents cited herein. And the time for successive injections can be set without undue experimentation.

Methods of treatment

Provided herein are methods for treating neoplasia in an individual. Also provided herein are methods of treating a pathogenic infection or other infectious disease in an individual, such as an immunocompromised human individual. The methods include administering the T cells, NK cells, or CTL cells of the invention in an amount effective to achieve the desired effect, whether temporary alleviation of an existing condition or prevention of relapse. For treatment, the amount administered is the amount effective to produce the desired effect. An effective amount may be provided in one or more administrations. An effective amount may be provided by bolus or by continuous perfusion.

An "effective amount" (or "therapeutically effective amount") is an amount sufficient to effect a beneficial or desired clinical result in treatment. An effective amount may be administered to an individual in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to alleviate, improve, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount will generally be determined by the physician on an individual basis and within the skill of the art. Several factors are generally considered in determining an adequate dose to achieve an effective amount. These factors include the age, sex, and weight of the individual, the condition being treated, the severity of the condition, and the form and effective concentration of the antigen-binding moiety administered.

For adoptive immunotherapy using antigen-specific T cells, cell doses are infused, typically in the range of 10 ⁶ -10 ¹⁰ (eg, 10 ⁹ ). Upon introduction of genetically modified cells into the host and subsequent differentiation, T cells are induced that are specifically directed against a specific antigen. "Induction" of T cells may include the inactivation of antigen-specific T cells, for example, by deletion or anergy. Inactivation is particularly useful for establishing or restoring tolerance, such as autoimmune disorders. The modified cells can be administered by any method known to those skilled in the art, including, but not limited to, intravenous, subcutaneous, internodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and directly into the thymus.

Therapeutic methods

The invention provides methods for enhancing an immune response in an individual in need thereof. In one embodiment, the invention provides methods for treating or preventing neoplasia in an individual. The invention provides methods of treatment that are particularly useful for treating individuals with blood cancers (eg leukemias, lymphomas and myelomas) or ovarian cancers that are not amenable to conventional therapeutic interventions. Suitable human subjects for therapy generally comprise two treatment groups that can be distinguished by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are those carrying clinically measurable tumors. Clinically measurable tumors can be detected based on tumor mass (eg, palpation, tomography, ultrasound, mammography, or x-ray; positive biochemical or histopathological markers alone are not sufficient to identify this population). The pharmaceutical composition embodied in this invention is administered to these individuals in order to achieve an antitumor response in order to alleviate their condition. Ideally, the result is a reduction in tumor mass, but any clinical improvement is favorable. Clinical improvement includes a reduction in the risk or rate of progression or a reduction in the pathological consequences of a tumor.

The second group of suitable individuals is known in the art as the "auxiliary group". These are individuals who had a history of neoplasia but were responsive to a different therapy regimen. Prior therapy includes, but is not limited to, surgical resection, radiation therapy, and conventional chemotherapy. As a consequence, these individuals do not have a clinically measurable tumor. However, they are suspected of being at risk of disease progression, either close to the original tumor site or by metastasis. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is made based on features observed before or after the initial treatment. These features are known in the clinical field, and are quite specific for each of the various neoplasias. Signs characteristic of high-risk subgroups are those in which the tumor has invaded adjacent tissues, or which demonstrate involvement of the lymph nodes.

Another group has a genetic predisposition to neoplasia, but which has not yet been confirmed by the clinical signs of neoplasia. For example, women who test positive for a breast cancer-associated genetic mutation but are still of childbearing age may wish to receive one or more of the antigen-binding fragments described herein in prophylactic treatment to prevent the occurrence of neoplasia, until it is fit for a preventive operation.

Neoplasia human individuals having any of the following neoplasias: glioblastoma, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate cancer and small cell lung cancer) are particularly suitable individuals. Suitable carcinomas further include any known in the art of oncology, including, but not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuronal ectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma. glands, small and large cells of lung adenocarcinoma, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphangiosarcoma, lymphangioendothelial sarcoma, hepatoma, cholangiocarcinoma, synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, cancer colon , basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, fetal carcinoma, Wilms tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia and heavy chain disease, breast tumors such as ductal and lobular adenocarcinomas, squamous and adeno cervical carcinomas , epithelial carcinomas of the uterus and ovaries, adenocarcinomas of the prostate, transitional squamous cell carcinoma of the bladder, B-T-cell lymphomas (nodular and diffuse), plasmacytoma, acute and chronic leukemias, malignant melanoma, soft tissue sarcomas and leiomyosarcomas.

Individuals may have an advanced form of the disease, in which case the goal of treatment may include mitigating or neutralizing the progression of the disease, and/or reducing side effects. Individuals may have a history of a condition for which they have already been treated, in which case the therapeutic goals typically include reducing or delaying the risk of relapse.

Accordingly, the invention provides a method for treating or preventing neoplasia in an individual, comprising administering an effective amount of an immunoreactive cell comprising a receptor that binds to a tumor antigen and activates the immunoreactive cell (e.g., TCR, CAR) and a vector encoding a single chain variable fragment (scFv), which binds an antigen having immunosuppressive activity (eg CD47, PD-1, CTLA-4 and their ligands). In one embodiment, the neoplasia is selected from the group consisting of blood cancers (e.g., leukemias, lymphomas, and myelomas), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, bowel cancer, liver cancer. , lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, and throat cancer. In another embodiment, the tumor antigen is one or more of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD7, CDIO, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, cytomegalovirus (CMV) infected cell antigen (eg, cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM) ), tyrosine protein kinase receptor erb-B2,3,4, folate binding protein (FBP), embryonic acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), epidermal growth factor receptor 2 human (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor alpha-2 subunit (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis Y (LeY), molecules cell adhesion L1 (L1CAM), melanoma antigen family A, (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), NKG2D ligands, NY-ESO-1 testicular cancer antigen, oncofetal antigen ( h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), or Wilms tumor protein (WT-1) .

As a consequence of the surface expression of a receptor that binds a tumor antigen and activates an immunoreactive cell (eg, TC, CAR) and a vector encoding a single chain variable fragment (scFv) that binds an antigen with immunosuppressive activity (eg, CD47, PD-1, CTLA -4 and their ligands), adaptively transmitted human Tor NK cells are endowed with augmented and selective cytolytic activity in the tumor site. In addition, after localization in a tumor or viral infection and their proliferation, costimulatory ligand-expressing T cells turn the site of a tumor or viral infection into a highly conductive environment for a wide range of immunocompetent cells involved in the physiological antitumor or antiviral response (tumor-invading lymphocytes , NK-, NKT-cells, dendritic cells and macrophages).

In other embodiments, the invention provides methods for treating individuals with a pathogenic infection (eg, viral infection, bacterial infection, fungal infection, parasitic infection, or protozoan infection). The invention is particularly useful for enhancing an immune response in an immunocompromised individual. Typical viral infections treatable with the method of the invention include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus infections.

Accordingly, the invention provides a method for treating or preventing a pathogenic infection in an individual, comprising administering an effective amount of an immunoreactive cell as described herein.

Sets

The invention provides kits for the treatment or prevention of neoplasia, pathogenic infection, immune disorder or allogeneic transplantation. In one embodiment, the kit comprises a therapeutic or prophylactic composition comprising an effective amount of an immunoreactive cell comprising an activating antigen receptor and a single chain variable fragment (scFv) that binds an antigen having immunosuppressive activity in a unit dosage form. In specific embodiments, the cells further include a co-stimulatory ligand. In some embodiments, the kit includes a sterile container that contains a therapeutic or prophylactic vaccine; such containers may be boxes, ampoules, bottles, vials, test tubes, pouches, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for storing medicines.

If desired, the immunoreactive cell is supplied with instructions for administering the cell to an individual having or at risk of developing neoplasia, pathogenic infection, immune disorder, or allogeneic transplantation. The instructions typically contain information about the use of the composition for the treatment or prevention of neoplasia, pathogenic infection, immune disorder or allogeneic transplantation. In other embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosage and administration regimen for the treatment or prevention of neoplasia, pathogenic infection, immune disorder or allogeneic transplantation or symptoms thereof; precautionary measures; warnings; indications; contraindications; overdose information; adverse reactions; pharmacology in animals; clinical researches; and/or links. The instructions may be printed directly on the container (if any), or as a sticker attached to the container, or as a separate sheet, brochure, card, or folder supplied in or with the container.

EXAMPLES

The practice of the present invention utilizes, unless otherwise indicated, the conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such methods are described in detail in the literature, for example, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These methods are applicable to the production of polynucleotides and polypeptides according to the invention, and, as such, may be considered in the manufacture and practice of the invention. Particularly useful techniques for specific embodiments will be discussed in the following sections.

The following examples are offered to provide any person skilled in the art with a full disclosure and description of how to prepare and apply the testing, screening and treatment methods of the invention and are not intended to limit the scope of what the inventors consider their invention.

Example 1 T cells co-expressing chimeric antigen receptor (CAR) and anti-SCD47 scFv killed tumors.

A scFv that specifically binds to human CD47 has been generated, and human peripheral blood T cells modified with this scFv and a tumor antigen (CD19) recognizing CAR have demonstrated in vitro antitumor efficacy as well as enhanced antitumor efficacy in a preclinical model.

Constructs were obtained including 1928z-2A-B6H12.2 (Figures 1-5), which was confirmed by sequencing of the CAR and scFv sequences. In addition, control constructs were generated with a CAR specific for ovarian cancer antigen, MUC-CD, called 4H1128z (Figure 6). Stable producing cell lines were generated for constructs using the kappa leader sequence and validated by flow cytometry (FIG. 7A). The packaging cell line supernatant containing secreted anti-CD47 scFV was able to block the CD47 antibody from binding to Nalm-6 and Raji tumor cells in a flow cytometric assay. Tumor cells incubated with packaging cell supernatant were also stained with c-myc tag antibody to demonstrate scFv binding (Figure 7B).

The packaging cells were used to transduce human peripheral blood T cells, where the transduction efficiency was assessed by analysis of CAR expression by flow cytometry (FIG. 8A). The secreted scFv was able to function in an autocrine manner, with the anti-CD47 antibody decreasing binding to 1928z-2A-B6H12.2 T cells compared to I928z T cells. Positive staining with antibody to the c-myc tag indicated bound scFv (Figure 8B). The transduced T cell phenotype was examined by flow cytometry and showed similarity between 1928z-2A-B6H12.2 and 1928z T cells, except for CD62L, which was found to be downregulated in 1928z-2A-B6H12.2 T cells ( figure 9A). The function of T cells producing anti-CD47 scFv was examined using multivariable flow cytometry and standard 51Cr release assay. 1928z-2A-B6H12.2 T cells were shown to have equivalent cytokine production and cytotoxic function compared to 1928z T cells (Figures 9B and 9C).

The ability of 1928z-2A-B6H12.2 T cells to respond to a tumor in vivo was investigated using the SCID-Beige preclinical mouse model. SCID-Beige mice were intravenously injected with 1×10 ⁶ Nalm-6 tumor cells modified to express Firefly luciferase, 3 days later mice were treated with 5.7×10 ⁶ CAR ⁺ I928z or 1928z-2A-B6H12.2 T cells or control T-cells 4H1128z-2A-B6Hl2.2, also administered intravenously. Tumor growth was monitored clinically and by bioluminescent imaging. Treatment of tumor-bearing mice with 1928z-2A-B6HI2.2 T cells reduced tumor burden and increased survival of tumor-bearing mice compared to treatment with 1928z T cells (Figures 10A and 10B).

Example 2 T cells co-expressing chimeric antigen receptor (CAR) and anti-human PD-1 scFv had increased proliferation and retained CAR expression.

scFv against human PD-1 was created based on the V _H and V _L chains of the anti-PD-1 antibody (clone 5C4) (US patent No. 8008449). The 5C4 scFv was designed to include a kappa leader sequence, a serine-glycine linker and a c-myc tag (Figure 11). This scFv construct was cloned into the SFG retrovirus framework to generate 1928z-2A-5C4 and 4H1128z-2A-5C4 (Figures 12 and 13). To develop a high affinity scFv that binds to human PD-1 (e.g., for expression in 1928z/4H1128z CART cells), a human antibody phage display library is screened to identify scFvs that specifically bind human (and potentially mouse PD-1) PD-1. ).

Stable 293Glv9 packaging cell lines were generated and expression of the 1928z CAR and 4H1128z CAR was assessed by flow cytometry (FIG. 14). The supernatant from these packaging cells was used to transduce human peripheral blood T cells and the transduction efficiency was assessed by flow cytometry to detect CAR expression (Figure 15).

The ability of this anti-human PD-1 scFv to increase T cell proliferation in response to artificial antigen presenting cells (aAPC) was investigated. PD-L1 positive 3T3 tumor cells and aAPC were generated for study (Figure 16). After co-culture of transduced T cells with 3T3 aAPC expressing human CD19, human B7.1 and human PD-L1, 1928z-2A-C4 T cells increased proliferation and retained CAR expression compared to 1928z T cells (Figure 17) . The phenotype and antitumor function of T cells co-expressing 1928z CAR and anti-PD1 scFV can be determined using flow cytometry, Luminex cytokine assays, ⁵¹ chromium release assays, and the SCID-Beige preclinical model for in vivo antitumor function.

Example 3 Co-expression of chimeric antigen receptor (CAR) and anti-mouse PD-1 scFv stimulates mouse T cells.

The anti-mouse PD-1 scFv was generated from the VH and VL from the anti-PD-1 antibody clone J43 (US Pat. No. 7,858,746 to Honjo et al.). The J43 scFv was designed to include a mouse kappa chain leader sequence, a serine-glycine linker, and a c-myc tag (Figure 18). This scFv construct was cloned into a SFG retrovirus framework expressing CAR targeting human CD19 or human MUC-CD, which signals through mouse C28 and mouse CD3zeta, thereby stimulating mouse T cells. These constructs 19m28mz-IRES-J43 (Figure 19) and 4H11m28mz-IRES-J43 (Figure 20) are used to generate stable Phoenix packaging lines and genetically modified primary murine T cells as previously described (Lee et al., Cancer Res 2011, 71(8):2871). Mouse T cells 19m28mz and 19m28mz-IRES-J43 are cultured with thymoma EL4 tumor cells that have been modified to express human CD19 and mouse PD-L1, proliferation of mouse T cells to monitor viable cell count, and CFSE labeling.

For mouse T cells expressing the 4H11m28mz CAR that targets the MUC-CD antigen, function can be assessed in response to IDS tumor cells modified to express mouse MUC-CD and PD-L1 (Chekmasova et al., Clin Cancer Res, 2010 6:3594). A human scFv that binds mouse PD-1 as described above is cloned into SFG-19m28mz and 4H11m28mz vector constructs and used to modify mouse T cells. Syngeneic models are available to evaluate the in vivo antitumor effects of T cells modified to express human scFv that binds murine PD-1: ovarian cancer models using ID8-MUC-CD tumor cells that are inoculated intraperitoneally into C57BL/6 mice; and transgenic mice that express human CD19 instead of mouse CD19 inoculated with thymoma EL4 tumor cells modified to express human CD19 (Pegram et al, Blood 2012, 1 19(18):4133). Thus, the antitumor effect can be assessed in an immunocompetent model, which makes it possible to evaluate the effect of anti-PD-1 scFv on the tumor microenvironment.

Example 4 Co-expression of a chimeric antigen receptor (CAR) and an agonistic scFv in immune cells.

In one embodiment, the invention provides an immune cell that expresses an antigen-binding receptor (e.g., CAR or TCR) and a single-chain variable fragment (scFv) that binds an antigen having immunostimulatory agonistic activity (e.g., CD28, OX-40, 4- 1BB and its ligands). To generate agonistic scFvs targeting the costimulatory molecule 4-1BB, the 3H3 hybridoma cell line was developed (Shuford et al., J Exp Med 1997, 186:47-55; provided by Prof. Mick Croft (La Jolla Institute of Allergology and Immunology)). To generate agonist scFvs targeting the costimulatory molecule OX-40, the hybridoma cell line OX-86 was developed (al-Shamkhani et al., Eur J Immunoll996, 26(8): 1695-9; European Cell Culture Collection (Cat. No. 96110601 )). Hybridoma mRNA was isolated from cells using the QIAgen RNAeasy kit, according to the manufacturer's instructions (QIAgen, CA, USA), and cDNA was then prepared using the New England Biolabs Protoscript AMV First strand cDNA synthesis kit, according to the manufacturer's instructions (New England Biolabs, Massachusetts, USA). The variable heavy (V _H ) and light (V _L ) chains were then PCR amplified using the following degenerate primers:

Primers Orlandi (Orlandi et al., Proc. Natl. Acad. Sci. 1989, 86:3833-37)

VHFOR: 5'- tga gga gac ggt gac cgt ggt ccc tt gcc cca g -3' [SEQ ID NO:28]

VH1BACK: 5'- agg tsm arc tgc ags agt cwg g -3' [SEQ ID NO:29]

VKFOR: 5'- gtt aga tct cca gcttgg tcc c -3' [SEQ ID NO:30]

VK1BACK: 5'- gac att cag ctg acc cag tct cca -3' [SEQ ID NO:31]

Primers Cooper (Wang et al., Blood 2002, 99:2459-2467)

Vk 5'-GGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC-3' [SEQ ID NO:32],

Ck 5'-CTCATTCCTGTTGAAGCTCTTGACAATGGG-3' [SEQ ID O:33];

RACE PRIMERS (Kettleborough et al., Eur. J Imrnunol 1993, 23:206-211)

VKfrla: Ata tcc atg gca gac gtc cag atg atc cag tct cca [SEQ ID NO:34]

Vkfrlb: ata tcc atg gca gac att gtg ctg act cag tct cc [SEQ ID NO:35]

Vkfrlc: ata tcc atg gca gat gtt gtg atg ace caa act cca [SEQ ID NO: 36]

Vkfrld: ata tcc atg gca caa att gtt ctc acc cag tct cc [SEQ ID NO:37]

Vkfrle: ata tcc atg gca gac att gtg atg aca cag tct cca [SEQ ID NO:38]

Vkfrlf: ata tcc atg gca gat att gtg atg acg cag gct gca [SEQ ID NO:39]

Vkfrl g: ata tcc atg gca gac att gtg atg acc cag tct c [SEQ ID NO:40]

Reverse Kappa: gct tca aca gga atg agt gtt aac teg agg tag [SEQ ID NO:41]

To assemble V _H and V _L into scFv, a serine-glycine linker is added during PCR of the V _H and V _L chains, as well as the c-myc tag and mouse kappa Ig chain or CDS leader sequence. The resulting polynucleotide is cloned into an existing retroviral expression vector (SFG scaffold) encoding a 1928z chimeric antigen receptor (CAR) to generate SFG-1928z-2A-3H3 or 1928z-2A-OX86.

Stable packaging cell lines are generated as described for mouse anti-PD-1 J43 scFv and tested in a similar mouse model of adaptive T cell transfer.

The results presented herein indicate that genetically engineered CAR T cells expressing scFv molecules ("armored CART cells") are immunoreactive and can overcome a "hostile" tumor microenvironment, and thus are effective in treating neoplasia. . CAR ⁺ T cells are modified to secrete antagonistic scFvs with immune regulatory functions (Figure 21). Upon activation of a CAR by a cognate antigen (1), armored CAR-modified T cells can be induced to secrete scFvs antagonistic to the inhibitory PD-1 T-cell receptor on both infused CAR-modified T cells and endogenous antitumor agents. T cells, enhancing antitumor effector functions (2), induced to secrete scFv antagonistic to inhibitory CTLA-4 T cell receptor on both infused CAR modified T cells and endogenous antitumor T cells, enhancing antitumor effector functions (3), or induced to secrete scFv antagonistic to the CD47 receptor expressed on the tumor cell, abolishing the tumor cell's masking from recognition by the host's innate antitumor immune response, leading to tumor recognition and eradication by host macrophages.

The results described below were obtained using the following methods and materials unless otherwise noted.

Creation of anti-CD47 B6H12.2 scFv

The hybridoma cell line B6H12.2 was obtained from the American Tissue Culture Collection (ATCC, VA, USA; catalog number HB-9771). B6H32.2 mRNA was isolated from hybridoma cells using the QIAgen RNAeasy kit, according to the manufacturer's instructions (QIAgen, CA, USA), and cDNA was prepared using the New England Biolabs Protoscript AMV First strand cDNA synthesis kit, according to the manufacturer's instructions (New England Biolabs, Massachusetts, USA). The variable heavy (V _H ) and light (V _L ) chains were PCR amplified using primers designed to include a kappa leader sequence, a serine-glycine linker and a c-myc tag (see Figure 1) as follows:

Primer 1 Forward primer B6H12.2 VH [SEQ ID NO:42]:

5'- CCA TGG AGA CAG ACA CAC TCC TGC TAT GGG TAC TGC TGC TCT GGG TTC CAG GTT CCA CTG GTG ACG AGG TGC TGC AGC TGG TGG AGT CCG GGG -3'

Primer 2 Reverse primer B6H12.2 VH [SEQ ID NO:43]:

5'- AGA TCC ACC TCC ACC AGA TCC ACC TCC ACC TGA TCC ACC TCC ACC TGA GGA GAC GGT GAC TGA GGT TCC TTG ACC -3'

Primer 3 Forward primer B6H12.2 VL [SEQ ID NO:44]:

5'- GGT GGA GGT GGA TCA GGT GGA GGT GGA TCT GGT GGA GGT GGA TCT GAC ATT GTG ATG ACT CAG TCT CCA GCC ACC -3'

Primer 4 Reverse primer VL B6H12.2 [SEQ ID NO:45]:

5'- CTC GAG TTA CAG ATC CTC TTC TGA GAT GAG TTT TTG TTG TTT GAT TTC CAG CTT GGT GCC TCC ACC GAA CG -3'

In addition to the above design, a CD8L sequence scFv was generated to determine an effective leader sequence for scFv export from T cells using the following alternative forward primer:

Primer 5. B6H12.2 VH CD8L forward [SEQ ID NO:46]:

5'- TAT ACC ATG GCC TTA CCA GTG ACC GCC TTG CTC CTG CCG CTG GCC TTG CTG CTC CAC GCC GCC AGG CCG GAG GTG CAG CTG GTG GAG TCC GGG -3'

VH and VL PCR products were cloned into pCR2.1TOPO according to the manufacturer's instructions (Invitrogen, NY, USA). Sequencing using primers M13F2 and M13R2 (Invitrogen) was performed at MSKCC's lead DNA sequencing laboratory to confirm the sequence of both V _H and V _L products. Overlapping PCR was performed using PCR products V _H and V _L and primers 1 or 5 and 4 to create anti-CD47 scFv (see figure 1).

The anti-CD47 scFv construct was cloned into an existing retroviral expression vector (SFG scaffold) encoding the 1928z chimeric antigen receptor (CAR) to generate SFG-1928z-2A-B6H12.2. SFG-1928z-2A-B6H12.2 DNA was sequenced for sequence confirmation.

Obtaining a stable packaging cell line for human T cells

To establish stable packaging cell lines, H29 cells were transiently transfected with 10 µg of SFG-1928z-2A-B6HI2.2 DNA using the Promega calcium phosphate transfection kit, according to the manufacturer's instructions (Promega). The supernatant from the H29 supernatant was used to transduce 293Glv9 cells, which were subsequently sub-cloned to obtain stable packaging cells. The selection of two sub-clones (clone 5 and clone 6) was based on the expression of both the 1928z CAR and the ability of the 293Glv9 supernatant to transduce human peripheral blood T cells (as determined by flow cytometry after staining with 12dll antibody). Transduction of human peripheral blood T cells was performed as previously described (Brentjens et al., Clin Cancer Res 2007, 13(18PtI):5426).

Assessment of anti-CD47 scFv production/function

Production of anti-CD47 scFv from 1928z-2A-B6H12.2 293Glv9 and transduction of human peripheral blood T cells was determined by incubation of CD47 ⁺ tumor cells (Raji and Nalm-6) in the supernatant from these cells. Tumor cells were then washed with water and stained with fluorescently conjugated anti-c-myc tag antibodies (Cell Signaling, Massachusetts, USA) to detect supernatant derived protein associated with tumor cells. Tumor cells were also stained with fluorescently conjugated anti-CD47 (clone B6H12.2, eBioscience) to detect the ability of B6H12 scFv to block CD47.

In vivo adaptive transmission model

Mice were injected intravenously with 1×10 ⁶ Nalm-6 modified to express Firefly luciferase (day 0). On day 3, mice were treated with 5.7×10 ⁶ CAR ⁺ T cells, also inoculated intravenously. Tumor growth was monitored clinically and by bioluminescent imaging as previously described (Santos et al., Nature Medicine 2009, 15(3):338).

Generation of 5C4 scFv against human PD-1

The sequence of an antibody that specifically binds to human PD-1, clone 5C4, was obtained as described above. This sequence was modified to include a kappa leader sequence, a serine-glycine linker and a c-myc tag and purchased from GeneArt (Invitrogen, Figure 9). Cloning of this scFv into an SFG retroviral scaffold, production of stable packaging cells, transduction of human peripheral blood T cells, and evaluation of transduction efficiency were performed as described above.

Assessment of human anti-PD-1 function

PD-1 ligand, PD-L1 was PCR amplified from SKOV3 tumor cells (ATCC) that were incubated with 200 ng/ml recombinant human interferon-gamma (RnD systems, Minnesota, USA). The primers used to amplify human PD-L1 are listed below:

Primer 6 Forward primer to human PD-L1 [SEQ ID NO:47]

5'- CACGTGCCATGGATGAGGATAT TTGCTGTCTT TATAT -3'

Primer 7 Reverse primer to human PD-L1 [SEQ ID NO:48]

5'CTCGAGTTACGTCTCCTCCAAATGTGTATCACTTT3'

The human PD-L1 sequence was cloned into an SFG retroviral scaffold and transduced into the 3T3, Raji and Nalm-6 cell lines as previously described (Brentjens et al., Clin Cancer Res 2007, 13(18 Pt 1):5426). Cells were stained with anti-PD-L1 (clone MIH1, BD Pharmingen, CA, USA) and FACS sorted to provide a total cell population expressing PD-L1 (Figure 14).

Human T cells 1928z-2A-5C4 and 1928z were cultured with 3T3 (CD19 B7.1/PD-L1) aAPC and viable cell count was performed using trypan blue exclusion and flow cytometry was performed to determine CAR expression. This correlated with T cell expansion when cultured with 3T3 (CD19/B7.1) aAPC.

Generation of scFv against mouse PD-1

An antibody sequence was generated that specifically binds to mouse PD-1, a clone named J43, as described above. This sequence was modified to include the kappa chain leader and c-myc tag sequence, with a serine-glycine linker, for scFv formation and purchase from GeneArt (Invitrogen, Figure 16). It was cloned into an existing retroviral expression vector (SFG) encoding mouse CAR, where signaling is mediated through mouse CD28 and CD3 zeta molecules. 19m28mz-IRES-J43 and 4H11m28mz-IRES-J43 were generated for B-cell and ovarian tumor targets, respectively (Figures 17 and 18).

Mouse Anti-PD-1 Function Evaluation

PD-1 ligand, PD-L1 was PCR amplified from Renca tumor cells (ATCC), the primers used to amplify mouse PD-L1 are given below:

Primer 8 Mouse PD-L1 forward primer [SEQ ID NO:49]

5'- TAT TAC ACG TGT TAC ATG AGG ATA TTT GCT GTC TTT -3'

Primer 9 Mouse PD-L1 Reverse Primer [SEQ ID NO: 50]

5' TAT AGG ATC CTC GAG GAT GTT ACG TCT CCT CCA AAT GTG TA 3'

mouse anti-PD-1 scFv was cloned into an SFG retroviral scaffold and transduced into 3T3 aAPC, IDS and EL4 cell lines. Cells stained with anti-PD-L1 scFv (clone MIH1 BD Pharmingen) were FACS sorted to provide a total cell population expressing PD-L1.

Chromium release CTL kill test

Target cells expressing the desired antigen were labeled with ⁵¹ Cr and co-cultured with T cells at decreasing effector:target ratios. After 4 hours of cultivation, the supernatant was collected and used to measure the radioactivity released from chromium. Specific lysis was determined by subtracting the background radioactivity from target cells not cultured with 25T cells and dividing by the radioactivity measured from target cells completely lysed with 0.2% Triton X-100.

Example 5 Blocking CD47 improves CAR T cell therapy.

T cells can be genetically modified for targeted tumor antigens by expression of a chimeric antigen receptor (CAR). Adoptive transfer of CD19-specific CAR T cells has shown clinical benefit in some patients with hematopoietic malignancies, but patients with chronic lymphocytic leukemia with extensive lymphadenopathy have had suboptimal responses to CAR T cell therapy. In addition, CAR T cell therapy has not been shown to be effective against solid tumors in clinical trials. In this example, the clinical efficacy of CAR T cells was enhanced by inducing an innate antitumor immune response through the secretion of CD47-blocking single chain variable fragments (scFv) from CAR T cells. Previous studies show that blocking the interaction between CD47 on tumor cells and SIRPa on macrophages leads to tumor cell phagocytosis. To exploit this effect, T cells were modified to express a CD19-specific CAR (1928z) and secrete scFv specific for human CD47 cloned from a B6H12.2 hybridoma (1928z/B6H12.2 T cells). 1928z B6H12.2 T cells have been shown to secrete a functional scFv specific for human CD47 that does not affect CAR-mediated cytokine secretion or in vitro cytotoxicity. Supernatant from 1928z/B6H12.2 T cells but not from 1928z T cells stimulated macrophages to phagocytose tumor cells in vitro. Adoptive transfer of 1928z/B6H12.2 T cells mediated enhanced antitumor effects and eradicated Nalm6 tumors in a preclinical mouse model. This new strategy combines CAR T cell-mediated effects and natural immune cell-mediated tumor cell killing to increase the antitumor efficacy of CAR T cell therapy.

Example 6 Enhancement of antitumor efficacy of modified T cell chimeric antigen receptor through constitutive expression of CD40L.

Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-ALL. However, in most clinical trials, CAR-modified T cells have failed to demonstrate a significant therapeutic effect, especially in the context of B-cell malignancies and low-grade solid tumors. In the experiments presented in this section of examples, we further enhance the antitumor efficacy of CAR-modified T cells by constructing T cells constitutively expressing the CD40 ligand (CD40L, CD-154). T cells modified to constitutively express CD40L (CD40L-modified T cells) increased the proliferation and secretion of pro-inflammatory TH1 cytokines. Further, CD40L-modified T cells enhanced the immunogenicity of CD40+ tumor cells by activating costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58 and CD70), HLA molecules (class I and HLA-DR) and the FAS death receptor ( CD95) on the surface of the tumor cell. In addition, CD40L-modified T cells induced maturation and stimulated the secretion of the pro-inflammatory cytokine IL-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CAR/CD40L T cells increased cytotoxicity against CD40+ tumors and prolonged survival of tumor-bearing mice in a systemic lymphoma xenograft model. These preclinical data support the clinical utility of CAR T cells further modified to constitutively express CD40L, with expected increased antitumor efficacy and improved clinical outcomes.

Materials and methods

Cell cultures

Tumor cell lines DoHH2, Raji, and NALM-6 (American Type Culture Collection) were maintained in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), non-essential amino acids, sodium pyruvate, HEPES buffer (N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 2-mercaptoethanol (Invitrogen). Retroviral producing cell lines 293GP-GLV9 have been previously described and cultured in DMEM (Invitrogen) supplemented with 10% FBS ²⁹ . NIH-3T3 artificial antigen presenting cells (AAPCs) were cultured in DMEM supplemented with 10% heat-inactivated donor calf serum (MS) as previously described ³⁰ . Human T cells were isolated from the peripheral blood of healthy donors according to Memorial Sloan-Kettering Cancer Center (MSKCC) IRB-approved protocol 95-054, using BD vacuum container CPT tubes (Beckton Dickinson) according to the manufacturer's instructions. Patient T cells and CLL cells were obtained from patients treated according to the IRB-approved protocol 06-138 MSKCC and isolated using Dynabeads ClinExVivo CD3/CD28 beads (Invitrogen). T cells were cultured in RPMI 1640 supplemented with 10% FBS and 20 U/ml IL-2 (R&D Systems). Monocyte-derived dendritic cells (moDCs) were obtained from a tissue culture of plastic-adhering peripheral blood mononuclear cells (PBMC) from healthy donors and cultured in RPMI 1640 medium supplemented with 1% pooled human A/B serum, HEPES buffer, 2-mercaptoethanol (Invitrogen ), interleukin-4 (IL-4; 500 IU/ml - R&D Systems) and granulocyte-monocyte colony stimulating factor (GM-CSF; 1000 IU/ml - R&D Systems) as previously described. ³¹ All media were supplemented with 2 mmol/L L-glutamine (Invitrogen), 100 units/mL penicillin, and 100 g/mL streptomycin (Invitrogen).

Construction of retroviral constructs

Human CD40L cDNA was PCR amplified from PBMC isolated from healthy donors using the following primers (1) 5'-CACGTGCATGATCGAAACATACAACCAAACTTCTCCCCGATCTGC-'3 [SEQ ID NO: 3] and (2) 5'-CTCGAGGGATCCTCAGAGTTTGAGTAAGCCAAAGGA-3' [SEQ ID NO:4 ] (figure 22A). A gamma retroviral vector encoding human CD40L was constructed using the SFG vector framework ³² . The 1928z and Pz1 (anti-prostate specific membrane antigen CAR; anti-PSMA) SFG vector constructs have already been described previously ^33,34 . The 1928z-IRES-40L and Pz1 IRES-40L gamma-retroviral vector construct was generated by overlapping PCR (Figure 26A).

Retroviral transduction of human T-lymphocytes

The generation of stable 293GP-GLV9 retroviral producing cell lines and the genetic modification of human T cells have been described previously ^29,36 . For T-cell transduction, healthy-donor isolated PBMCs were activated with 2 μg/mL phytohemagglutinin (PHA) (Sigma), while patient-derived T cells were isolated, activated, and expanded with Dynabeads ClinExVivo CD3/CD28 beads according to with manufacturer's recommendations. Activated T cells were retrovirally transduced on retronectin-coated, non-tissue culture-treated plates as described above ³⁶ . Gene transfer was assessed on day 7 by flow cytometry. Control sham-transduced T cells were obtained in the same manner, except that the supernatant was obtained from empty 293GP-GLV9 cell cultures. The proliferation of CD40L-modified T cells was assessed using the guava®easyCyte™ cell counter with the guava®ViaCount reagent (EMD Miliipore) according to the manufacturer's instructions. Propagation of modified T cells for in vivo experiments was performed using AAPC derived from NIH-3T3 mouse fibroblasts engineered to express target antigens (CD19 or PSMA) together with costimulation (CD80) as previously described.

Co-cultivation assays

Tumor cells (DOHH2, Raji, Ph ⁺ , ALL 3.1, NALM-6) were co-cultured at a 5:1 ratio with CD40L-modified T cells and sham-transduced T cells. Flow cytometry was performed three days later to determine the phenotype of tumor cells. moDC (2.5×10 ⁵ ) were co-cultured with autologous CD40L-modified T cells or mock-transduced T cells at a ratio of 1:5, and the tissue culture supernatant was analyzed after 24 hours for IL-12p70 in the Luminex IS100 system (see . below). moDCs were also co-cultured at a 5:1 ratio with CD40L-modified T cells and mock-transduced T cells, and the moDC phenotype was analyzed by flow cytometry after 24 hours.

Cytotoxicity assay

The cytolytic capacity of the transduced T cells was determined using a standard ⁵¹ Cr release assay as previously described ³⁴ .

Cytokine detection testing

Cytokine detection in tissue culture supernatant was assessed using the MILLIPLEX human cytokine detection system (Miliipore Corporation) in conjunction with the Luminex IS100 system and IS 2.3 software (Luminex Corporation) following the manufacturer's instructions.

flow cytometry

Flow cytometry was performed using a FACScan cytometer and data was analyzed using FlowJo software version 9.2 (Tree Star). CAR expression was detected with the Armenian hamster CAR specific monoclonal antibody 19E3 (1928z) and 12D11 (1928z and Pz1, MSKCC monoclonal antibody laboratory). CD40L expression was detected with human mouse anti-CD154 (BD Biosciences). Human T cells were also stained with mouse anti-human CD3 (BD Biosciences), CD4 and CD8 (Invitrogen). moDC were stained using mouse anti-human CD11b, HLA-DR, CD83 and CD86 (Invitrogen). The phenotype of tumor cells DOHH2, Raji and NALM6 was detected using mouse antibodies to human CD19, CD40, CD54, CD80, CD86, HLA-I class and HLA-DR (Invitrogen), CD58, CD70 and CD95 (BD Biosciences).

In vivo studies of CAR T cells

We inoculated 8-12 week old SCID-Beige (CB17.Cg-Prkdc ^scid Lyst ^bg-J /Cr1) mice (Charles River Laboratories) with DOHH2 tumor cells (5×10 ⁵ cells) by intravenous injection. Two days later, mice were injected intravenously with transduced T cells (1×10 ⁷ CAR T cells). Tumor growth was monitored clinically and mice were euthanized when the disease became clinically apparent (development of hind limb paralysis or reduced response to stimuli). All studies in mice were performed in accordance with the approved protocol (00-05-065) of the Institutional Animal Care and Use Committee of the Memorial Sloan-Kettering Cancer Center.

Statistical analysis

All analyzes were calculated using Graphpad Prism 5.0 software, survival data were assessed using log-rank analysis and all other analyzes were performed using the Mann Whitney test (one way).

results

Constitutive expression of CD40L by human T cells

We initially transduced T cells from healthy donors with the CD40L retroviral vector (Figure 22A). Retroviral transduction of T cells with the CD40L gene typically resulted in >40% gene transfer with stable CD40L expression in both CD4+ and cd8+ T cell subpopulations (Figure 22B). The proliferation of CD40L-modified T cells was significantly increased compared to sham-transduced T cells obtained from the same three donors (Figure 22C). Tissue culture medium from CD40L-modified T cells was analyzed and showed a significant increase in soluble CD40L (sCD40L), as expected, as well as a significant increase in the secretion of the pro-inflammatory cytokines IFN-γ and GM-CSF compared to sham-transduced T cells (Figure 22E).

CD40L-modified T cells alter the phenotype of both CD40+ tumor cell lines and patient-derived CLL cells

To investigate the ability of the CD40L/CD40 pathway to modify the phenotype of tumor cells, co-cultivation of CD40+ B-cell tumor cells and CD40L-modified T cells or sham-transduced T cells was performed. Cultivation with CD40L-modified T cells, but not with mock-transduced T cells, resulted in an increase in the activity of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58 and CD70), HLA-class molecules (HLA class I and HLA -DR) and FAS death receptor (CD95) on the surface of tumor cells DOHH2 (Figure 23A). Phenotypic changes are also apparent when DOHH2 tumor cells are cultured in conditioned media from a CD40L-modified T cell that contains elevated levels of sCD40L (Figure 28). To determine whether CD40 expression on a tumor cell is a necessary condition for changing the tumor cell phenotype, a CD40 tumor cell line (NALM6) was co-cultured with CD40L-modified T cells and mock-transduced T cells. These studies showed no change in phenotype, demonstrating the need for CD40 expression in tumors to induce CD40L-mediated changes in tumor cell phenotype (Figure 23B).

To further test this effect in a clinically relevant setting, we co-cultured CD40L-modified T cells derived from CLL patients with autologous CLL tumor cells. Retroviral transduction of T cells derived from CLL patients resulted in >40% gene transfer with stable expression of the CD40L gene (Figure 24A). In this setting, patient-derived CD40L-modified T cells, but not sham-transduced T cells, demonstrated the ability to increase the activity of costimulatory molecules, cell adhesion molecules, HLA class molecules, and Fas death receptors on the surface of autologous CLL cells (Figure 24B ).

CD40L-modified T cells induce IL-12p70 secretion and mediate moDC maturation

Considering the role of CD40L in DC maturation and secretion of the pro-inflammatory cytokine IL-12, we next investigated whether CD40L-modified T cells could produce the same effect when co-cultured with autologous moDCs. Significantly, we found CD40L-modified T cell-induced Il-12p70 secretion in co-cultures containing moDCs and autologous CD40L-modified T cells from three separate donors (Figure 25A). MoDC maturation, as measured by increased activity of surface co-stimulatory molecules (HLA-DR, CD86 and CD83), was also noted after co-culturing with CD40L-modified T cells, but not after co-culturing with sham-transduced T cells (Figure 25B).

Expression of both CAR and CD40L in T cells leads to increased cytotoxicity in vitro and in vivo

Next, we assessed the ability of T cells to express both anti-CD19 CAR (1928z) and CD40L using a bicistronic retroviral vector (1928z/CD40L; FIG. 26A). Transduction of T cells typically resulted in >40% expression of both 1928z and CD40L (1928z/CD40L T cells; FIG. 26B). Control retroviral vectors were also created containing anti-CD19 CAR (1928z) and anti-PSMA CAR (Pz1 and Pz1/CD40L; figure 26B). To evaluate the in vitro antitumor activity of 1928z CD40L T cells, a standard 4 hour ⁵¹ Cr release assay was performed. Constitutive expression of CD40L statistically increased the lytic capacity of 1928z T cells against CD19+ tumor cells compared to a group of control T cells, including T cells modified to express only the 1928z CAR (Figure 26C). Enhanced cytotoxicity is also demonstrated against other CD19+/CD40+ tumor cell lines (Figure 29).

To study the in vivo antitumor activity of 1928z/CD40L T cells, we used a xenograft model of systemic DOHH2 lymphoma. We have previously noted that systemic DOHH2 tumor cells are markedly resistant to CD19-targeted CAR T cell therapy in SCID-Beige mice. To assess whether further modification of CAR T cells with CD40L could enhance antitumor efficacy in this model, we vaccinated and SCID-Beige treated mice bearing a systemic DOHH2 tumor with CAR/CD40L T cells. Significantly, treatment with 1928z/CD40L T cells compared to treatment with 1928z T cells or control T cells (Pz1 and Pz1/CD40L T cells) showed increased survival and resulted in long-term survival in 30% of mice treated with 1928z/ 40L T cells (Figure 27).

Discussion

Adoptive therapy using CAR T cells has demonstrated promising clinical responses in patients with B-cell malignancies ^2-4 . These studies have demonstrated the effectiveness of CAR T cells as a single antitumor effector cell. However, this approach may have limited success against tumors with a robust immunosuppressive tumor microenvironment ⁵ . In addition, in their current form, CAR T cells have not demonstrated the ability to respond to tumor escape after loss of the target antigen. ⁶ One possible way to overcome these limitations is to further engineer CAR T cells by constitutively expressing CD40L in an effort to improve T cell cytolytic potential/proliferation, enhance tumor immunogenicity, and improve DC antigen presentation/function. Modification of CAR T cells through constitutive expression of CD40L may also further activate the endogenous immune response, enhancing antitumor efficacy.

To evaluate the role of constitutive CD40L expression by T cells, we first developed a retroviral vector containing the CD40L gene only. When transduced into T cells, constitutive expression was demonstrated in both CD4 ⁺ and CD8 ⁺ T cell subsets (Figure 22B). Although more commonly associated with CD4 ⁺ T cells, CD40L expression and helper function have recently been reported in memory CD8 ⁺ T cells ³⁷ . CD40L expression is also known to increase T cell proliferation and secretion of pro-inflammatory TH1 cytokines (IFN-γ, GM-CSF). ^21-22 CD40L-modified T cells show the ability to secrete pro-inflammatory cytokines and increase proliferation compared to similarly activated but sham-transduced T cells from the same donor (Figures 22C and 22D). Equipping T cells by constitutively expressing CD40L has the potential to enhance their antitumor function/activation.

Suppression of cell surface proteins, including HLA class I, co-stimulatory molecules and/or adhesion molecules, is often used by tumors to avoid immune recognition. ^5,38,39 Apoptotic resistance may also occur with loss of the Fas death receptor on the surface of malignant cells ⁴⁰ . To avoid this, CD40L can interact with CD40 on malignant cells to mediate an increase in the activity of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58 and CD70), HLA class molecules (HLA class I and HLA-D) and promote apoptosis. via the Fas/FasL pathway on malignant B-cell tumors ^41,42 . CD40L-modified T cells altered the CD40+ tumor cell phenotype, resulting in an increase in the activity of these critical surface proteins, thereby counteracting the immune evasion capacity of the tumor cells (Figure 2). This effect depends on the expression of CD40 by the tumor cells, since there was no phenotypic change when CD40 tumor cells were co-cultured with CD40L-modified T cells (Figures 23A-B). This effect was also seen in a more clinically relevant setting in which co-cultured CD40L-modified T cells derived from CLL patients enhanced the immunogenicity of autologous CLL cells (Figures 24A-B). This finding demonstrates the conserved ability of T cells to enhance the immunogenicity of autologous tumor cells through the constitutive expression of CD40L. Importantly, cell-to-cell contact is not necessary to change the phenotype of tumor cells, as environment containing elevated levels of sCD40L resulted in similar phenotypic changes (Figure 28). Increasing tumor cell immunogenicity via the CD40L/CD40 pathway has been shown to induce an endogenous antitumor response in previously published vaccine studies by infusion of autologous CLL tumor cells transduced with an adenoviral vector encoding CD40L (ad-CD40L CLL cells). ^27,28 Infusion of tumor-specific T cells further modified to constitutively express CD40L could also have a similar ability to induce an endogenous antitumor response. This can lead to the spread of the epitope through the recruitment of endogenous antitumor T or NK cells, thereby limiting the ability of tumors to escape through suppression of a particular target antigen.

Dendritic cell (DC) function is impeded in the tumor microenvironment. Typically, DCs are matured, translocated, and present antigen in the lymph nodes, thereby stimulating the adaptive branch of the immune system for the presence of malignancy or pathogen.⁵. At the same time, DCs exposed to the overwhelming tumor microenvironment have a paradoxical function of inducing T_regs and tolerogenic tumor-specific T cells⁴³. To avoid this, the CD40L/CD40 pathway may increase DC antigen presentation, production of the pro-inflammatory cytokine IL-12, and promote cytotoxic function of cd8+ T cells.^19.20. CD40 agonist antibodies have previously been shown to activate DC and increase CD8⁺ T cell response, thus replacing the need for CD4⁺ T-cell help.²⁶ In addition, CD40L-modified tumor-specific CD8⁺ T cells have been shown to stimulate DC maturation and enhance antitumor responses of adoptively transferred CD8s.⁺ T cells in tumor-bearing mice⁴⁴. To test the ability of CD40L-modified T cells to enhance human DC function, an in vitro co-culture experiment with autologous moDCs was used. Significantly, CD40L-modified T cells stimulated the secretion of IL-12p70 from moDC (Figures 25A-B). IL-12 is a pleiotropic cytokine with several immunostimulatory functions, including the ability to enhance T cell proliferation, cytotoxic capability, and mediate resistance to T_reg suppression, as previously shown by us and others^7.45. The ability of CAR/40L T cells to stimulate IL-12 production from DC may translate into improved antitumor activity of adoptively transferred CAR T cells, as well as recruitment and activation of endogenous tumor-specific T cells and natural killer (NK) cells. By stimulating IL-12 production in the immediate vicinity of the tumor, we expect minimal IL-12 related toxicity, in contrast to previous studies showing severe toxicosis following systemic IL-12 administration. In addition to stimulating IL-12 production, CD40L-modified T cells stimulate DC maturation, which in the context of CAR T cell cytotoxicity should further enhance DC uptake of tumor antigen and presentation, resulting in recruitment/activation of the endogenous antitumor response by effector T cells and NK cells (Figures 25A-B). Taken together, enhanced DC function should lead to increased antitumor efficacy of GM tumor-specific T cells through the recruitment of an endogenous antitumor immune response.

The ability of CAR T cells to redirect T cell specificity has been demonstrated in a number of preclinical and clinical reports. ¹ We have developed a retroviral vector containing the anti-CD19 CAR (1928z) and the CD40L gene (Figure 28A). Constitutive expression of both 1928z and CD40L by T cells is readily achievable (Figure 28B). Significantly, when testing the cytotoxic potential of 1928z/40L T cells against a panel of CD19+ targets, we noted increased cytotoxicity compared to T cells modified with 1928z CAR alone (FIG. 28C). Recently, Lauren and colleagues reported an increased cytotoxicity of CAR T cells against tumor cell lines after CD40/IL-4 dependent enhancement of surface adhesion molecule activity, which could also explain the increased cytotoxicity observed in our experiments ⁴⁶ . A xenograft model using the aggressive transformed follicular lymphoma DOHH2 cell line was used to test the in vivo potential of CAR/CD40L T cells. This model was historically resistant to radiation due to 1928z T cells (figure 27). However, with additional CD40L modification, our 1928z/CD40L T cells increased the survival of tumor-bearing mice compared to mice treated with 1928z T cells alone, and resulted in a 30% prolongation of survival in the 1928z/CD40L T-treated group. cells (figure 27). Although this model shows a difference in survival, the lack of a competent immune system in SCID-Beige mice makes this model unsuitable for investigating the maximum benefit that constitutive expression of CD40L in CAR T cells can have in cancer eradication. Although we observe enhanced antitumor efficacy in our model, this is likely due to an increase in CAR T cell cytotoxicity via an autocrine/paracrine CD40/CD40L pathway rather than via recruitment/activation of an endogenous immune response via CD40L-modified CARs. T cells. An immune-competent syngeneic tumor model can be used to investigate the full effect of constitutive CD40L CAR T-cell expression in the tumor microenvironment and recruit endogenous antitumor immune responses. The Immune Competent Syngeneic Model of Human CD19+ B Cell Malignancy has recently been developed and is being used to evaluate 1928z/CD40L T cells in the context of a competent immune system.

Constitutive expression of CD40L by bone marrow or thymus cells has been shown to result in T-lymphoproliferative disorders after infusion into CD40L-deficient mice ⁴⁷ . Clonal populations that have arisen within the thymus following continuous CD40L stimulation of thymocytes can lead to malignant transformation (rather than insertional oncogenesis of CD40L-modified cells). Although we have already noted minimal toxicity and no malignant transformation following CAR/CD40L T cell infusion, given concerns about malignant T cell transformation, an effective "suicide" gene such as iCasp9 could, if desired, be included in the retroviral vector ⁴⁸ .

Links for Example 6

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16. Ciaravino G, Bhat M, Manbeian CA, Teng NN. Differential expression of CD40 and CD95 in ovarian carcinoma. Eur J Gynaecol Oncol. 2004; 25(l):27-32.

17. Altenburg A, Baldus SE, Smola H, Pfister H, Hess S. CD40 ligand-CD40 interaction induces chemokines in cervical carcinoma cells in synergism with IFN-gamma. J Immunol. 199; 12(7):4140-4147.

18 Grewal IS, Flavell RA. CD40 and CD 154 in cell-mediated immunity. Annu Rev Immunol. 1998; 16:111-135.

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30. Brentjens RJ, Latouche JB, Santos E, et al. Eradication of systemic B-cell tuniors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-1. Nat Med. 2003;9(3):279-286.

31. Ratzinger G, Reagan JL, Heller G, Busam KJ, Young JW. Differential CD52 expression by distinct myeloid dendritic ceil subsets: implications for alemtuzumab activity at the level of antigen presentation in allogeneic graft-host interactions in transplantation. Blood. 2003; 101(4): 1422-1429.

32. Riviere I, Brose K, Mulligan RC. Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells. Proc Natl Acad Sci U S A. 1995;92(15):6733-6737.

33. Brentjens RJ, Santos E, Nikhamin Y, et al. Genetically targeted T cells eradicate systemic acute lymphoblastic leukemia xenografts. Clinic Cancer Res. 2007;13(18 Pt 1):5426-5435.

34. Gong MC, Latouche JB, rause A, Heston WD, Bander NH, Sadelain M. Cancer patient T cells genetically targeted to prostate-specific membrane antigen specifically lyse prostate cancer cells and release cytokines in response to prostate-specific membrane antigen. neoplasia. 1999;1(2):123-127.

35. Santos EB, Yeh R, Lee J, et al. Sensitive in vivo imaging of T cells using a membrane-bound Gaussia rinceps luciferase. Nat Med. 2009;15(3):338-344.

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37 Frentsch M, Stark R, Matzmohr N, et al. CD40L expression permits CD8+ T cells to execute immunological helper functions. Blood. 2013; 122(3):405-412.

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42 Schattner EJ, Mascarenhas J, Bishop J, et al. CD4+ T-cell induction of Fas-mediated apoptosis in Burkitt's lymphoma B cells. Blood. 1996; 88(4): 1375-1382.

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Embodiments of the invention

From the foregoing description it is obvious that variations and modifications can be made to the invention described herein in order to adapt it to different applications and conditions. Such embodiments are also within the scope of the following claims.

The listing of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The listing of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or parts thereof.

Some of the subject matter of this appendix may be related to U.S. Patent Application No. 12/593751, which is a U.S. national phase application under 35 U.S.C. §371, International Patent Application No: PCT/US2008/004251, filed March 8, 2010, which asserts the benefit of US Provisional Application Ser. No. 60/921144, filed March 30, 2007, the descriptions of which are hereby incorporated herein in their entirety by reference.

All patents and publications referenced in this specification are incorporated herein by reference to the same extent as if each independent patent and publication were specifically and individually cited for inclusion by reference.

--->

SEQUENCE LIST

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caggtggagg tggatcaggt ggaggtggat ctggtggagg tggatctgac attgagctca 480

cccagtctcc aaaattcatg tccacatcag taggagacag ggtcagcgtc acctgcaagg 540

ccagtcagaa tgtgggtact aatgtagcct ggtatcaaca gaaaccagga caatctccta 600

660

gtggatctgg gacagatttc actctcacca tcactaacgt gcagtctaaa gacttggcag 720

actatttctg tcaacaatat aacaggtatc cgtacacgtc cggagggggg accaagctgg 780

agatcaaacg ggcggccgca attgaagtta tgtatcctcc tccttaccta gacaatgaga 840

agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt cccctatttc 900

ccggaccttc taagcccttt tgggtgctgg tggtggttgg tggagtcctg gcttgctata 960

gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg agcaggctcc 1020

tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc aagcattacc 1080

agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag ttcagcagga 1140

gcgcagagcc ccccgcgtac cagcagggcc agaaccagct ctataacgag ctcaatctag 1200

gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct gagatggggg 1260

gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag aaagataaga 1320

1380

atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc cttcacatgc 1440

aggccctgcc ccctcgcg 1458

<210> 9

<211> 491

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 9

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Glu Val Lys Leu Gln Glu Ser Gly Gly Gly Phe Val Lys Pro

20 25 30

Gly Gly Ser Leu Lys Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

35 40 45

Ser Tyr Ala Met Ser Trp Val Arg Leu Ser Pro Glu Met Arg Leu Glu

50 55 60

Trp Val Ala Thr Ile Ser Ser Ala Gly Gly Tyr Ile Phe Tyr Ser Asp

65 70 75 80

Ser Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr

85 90 95

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

100 105 110

Tyr Cys Ala Arg Gln Gly Phe Gly Asn Tyr Gly Asp Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu

145 150 155 160

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

165 170 175

Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys

180 185 190

Asn Gln Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Glu Leu

195 200 205

Leu Ile Tyr Trp Ala Ser Thr Arg Gln Ser Gly Val Pro Asp Arg Phe

210 215 220

Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val

225 230 235 240

Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Asn Leu

245 250 255

Leu Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala

260 265 270

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

275 280 285

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

290 295 300

Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly

305 310 315 320

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

325 330 335

Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 10

<211> 1475

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 10

ccatggctct cccagtgact gccctactgc ttcccctagc gcttctcctg catgcagagg 60

tgaagctgca ggagtcaggg ggaggcttcg tgaagcctgg agggtccctc aaagtctcct 120

gtgcagcctc tggattcact ttcagtagct atgccatgtc ctgggttcgc ctgagtccgg 180

agatgaggct ggagtgggtc gcaaccatta gcagtgctgg tggttacatc ttctattctg 240

acagtgtgca gggacgattc accatttcca gagacaatgc caagaacacc ctgcacctgc 300

360

gtaactacgg tgattactat gctatggact actggggcca agggaccacg gtcaccgtct 420

cctcaggtgg aggtggatca ggtggaggtg gatctggtgg aggtggatct gacattgagc 480

tcacccagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact atgagctgca 540

aatccagtca gagtctgctc aacagtagaa cccgaaagaa ccagttggct tggtaccagc 600

aaaaaccagg acagtctcct gaactgctga tctactgggc atccactagg caatctggag 660

tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc atcagcagtg 720

tgcaggctga agacctggca gtttattact gccagcaatc ttataatcta ctcacgttcg 780

gtcctgggac caagctggag atcaaacggg cggccgcaat tgaagttatg tatcctcctc 840

cttacctaga caatgagaag agcaatggaa ccattatcca tgtgaaaggg aaacaccttt 900

gtccaagtcc cctatttccc ggaccttcta agcccttttg ggtgctggtg gtggttggtg 960

gagtcctggc ttgctatagc ttgctagtaa cagtggcctt tattattttc tgggtgagga 1020

gtaagaggag caggctcctg cacagtgact acatgaacat gactccccgc cgccccgggc 1080

ccacccgcaa gcattaccag ccctatgccc caccacgcga cttcgcagcc tatcgctcca 1140

gagtgaagtt cagcaggagc gcagagcccc ccgcgtacca gcagggccag aaccagctct 1200

ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag agacgtggcc 1260

gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg 1320

aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc 1380

ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc aaggacacct 1440

acgacgccct tcacatgcag gccctgcccc ctcgc 1475

<210> 11

<211> 250

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Thr Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Ile Asp Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Phe Cys

85 90 95

Ala Arg Ser Leu Ala Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu

130 135 140

Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln

145 150 155 160

Thr Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser

165 170 175

Pro Arg Leu Leu Ile Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro

180 185 190

Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile

195 200 205

Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly

210 215 220

His Gly Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

225 230 235 240

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

245 250

<210> 12

<211> 235

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 12

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

130 135 140

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

145 150 155 160

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

165 170 175

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

180 185 190

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

195 200 205

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

210 215 220

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

225 230 235

<210> 13

<211> 421

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 13

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Met Gly Leu Gly Leu Gln Trp Val Phe Phe Val

20 25 30

Ala Leu Leu Lys Gly Val His Cys Glu Val Arg Leu Leu Glu Ser Gly

35 40 45

Gly Gly Leu Val Lys Pro Glu Gly Ser Leu Lys Leu Ser Cys Val Ala

50 55 60

Ser Gly Phe Thr Phe Ser Asp Tyr Phe Met Ser Trp Val Arg Gln Ala

65 70 75 80

Pro Gly Lys Gly Leu Glu Trp Val Ala His Ile Tyr Thr Lys Ser Tyr

85 90 95

Asn Tyr Ala Thr Tyr Tyr Ser Gly Ser Val Lys Gly Arg Phe Thr Ile

100 105 110

Ser Arg Asp Asp Ser Arg Ser Met Val Tyr Leu Gln Met Asn Asn Leu

115 120 125

Arg Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Thr Arg Asp Gly Ser Gly

130 135 140

Tyr Pro Ser Leu Asp Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser

145 150 155 160

Ser Ala Thr Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Ala Cys

165 170 175

Asp Ser Thr Thr Lys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

180 185 190

Gly Gly Gly Gly Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Val

195 200 205

Asn Val Gly Glu Thr Val Lys Ile Thr Cys Ser Gly Asp Gln Leu Pro

210 215 220

Lys Tyr Phe Ala Asp Gln Phe His Gln Arg Ser Asp Gln Thr Ile Leu

225 230 235 240

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

245 250 255

Ile Ser Gly Ser Ser Ser Gly Thr Thr Ala Thr Leu Thr Ile Arg Asp

260 265 270

Val Arg Ala Glu Asp Glu Gly Asp Tyr Tyr Cys Phe Ser Gly Tyr Val

275 280 285

Asp Ser Asp Ser Lys Leu Tyr Val Phe Gly Ser Gly Thr Gln Leu Thr

290 295 300

Val Leu Gly Gly Pro Lys Ser Ser Pro Lys Val Thr Val Phe Pro Pro

305 310 315 320

Ser Pro Glu Glu Leu Arg Thr Asn Lys Ala Thr Leu Val Cys Leu Val

325 330 335

Asn Asp Phe Tyr Pro Gly Ser Ala Thr Val Thr Trp Lys Ala Asn Gly

340 345 350

Ala Thr Ile Asn Asp Gly Val Lys Thr Thr Lys Pro Ser Lys Gln Gly

355 360 365

Gln Asn Tyr Met Thr Ser Ser Tyr Leu Ser Leu Thr Ala Asp Gln Trp

370 375 380

Lys Ser His Asn Arg Val Ser Cys Gln Val Thr His Glu Gly Glu Thr

385 390 395 400

Val Glu Lys Ser Leu Ser Pro Ala Glu Cys Leu Glu Gln Lys Leu Ile

405 410 415

Ser Glu Glu Asp Leu

420

<210> 14

<211> 1274

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 14

ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca ggttccactg 60

gtgacatggg attgggactg cagtgggttt tctttgttgc tcttttaaaa ggtgtccact

gtgaggtgcg gcttctggag tctggtggag gattagtgaa gcctgagggg tcactgaaac 180

tctcctgtgt ggcctctgga ttcaccttca gtgactattt catgagctgg gtccgccagg 240

ctccagggaa ggggctggag tgggttgctc acatatacac gaaaagttat aattatgcaa 300

cttattactc gggttcggtg aaaggcagat tcaccatctc cagagatgat tcccgaagca 360

tggtctacct gcaaatgaac aacctgagaa ctgaggacac ggccacttat tactgtacaa 420

gagatggaag cggatatccc tctctggatt tctggggtca agggacccaa gtcactgtct 480

cctcagccac aacaacagcc ccatctgtct atcccttggc ccctgcctgt gacagcacaa 540

ccaaatcggg tggaggtgga tcaggtggag gtggatctgg tggaggtgga tcttatgagc 600

tgactcagcc accttcagca tcagtcaatg taggagagac tgtcaaaatc acctgctctg 660

gggaccaatt gccgaaatat tttgcagatt ggtttcatca aaggtcagac cagaccattt 720

tgcaagtgat atatgatgat aataagcgcc cctcggggat ccctgaaaga atctctgggt 780

ccagctcagg gacaacagcc accttgacca tcagagatgt ccgggctgag gatgaaggtg 840

actattactg tttctcagga tatgttgata gtgatagcaa attgtatgtt tttggcagcg 900

gaacccagct caccgtccta ggtggaccca agtcttctcc caaagtcaca gtgtttccac 960

cttcacctga ggagctccgg acaaacaaag ccacactggt gtgtctggtt aatgacttct 1020

acccgggttc tgcaacagtg acctggaagg caaatggagc aactatcaat gatggggtga 1080

agactacaaa gccttccaaa cagggccaaa actacatgac cagcagctac ctaagtttga 1140

cagcagacca gtggaaatct cacaacaggg tttcctgcca agttacccat gaaggggaaa 1200

ctgtggagaa gagtttgtcc cctgcagaat gtctcgaaca aaaactcatc tcagaagagg 1260

atctgtaact cgag 1274

<210> 15

<211> 21

<212> PRT

<213> Homo sapiens

<400> 15

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp

20

<210> 16

<211> 21

<212> PRT

<213> Mus sp.

<400> 16

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp

20

<210> 17

<211> 21

<212> PRT

<213> Unknown

<220>

<223> Description of unknown: CDS leader peptide

<400> 17

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 18

<211> 824

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 18

ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca ggttccactg 60

gtgacgaggt gcagctggtg gagtccgggg gagacttagt gaagcctgga gggtccctga 120

aactctcctg tgcagcctct ggattcactt tcagtggcta tggcatgtct tgggttcgcc 180

agactccaga caagaggctg gagtgggtcg caaccattac tagtggtggt acttacacct 240

actatccaga cagtgtgaag gggcgattca ccatctccag agacaatgcc aagaacaccc 300

tgtacctgca aatagacagt ctgaagtctg aggatacagc catatatttc tgtgcaagat 360

ccctcgcggg aaatgctatg gactactggg gtcaaggaac ctcagtcacc gtctcctcag 420

gtggaggtgg atcaggtgga ggtggatctg gtggaggtgg atctgacatt gtgatgactc 480

agtctccagc caccctgtct gtgactccag gagatagagt ctctctttcc tgcagggcca 540

gccagactat tagcgactac ttacactggt atcaacaaaa atcacatgag tctccaaggc 600

ttctcatcaa atttgcttcc caatccattt ctgggatccc ctccaggttc agtggcagtg 660

gatcaggctc agatttcact ctcagtatca acagtgtgga acctgaagat gttggagtgt 720

attactgtca aaatggtcac ggctttcctc ggacgttcgg tggaggcacc aagctggaaa 780

tcaaagaaca aaaactcatc tcagaagagg atctgtaact cgag 824

<210> 19

<211> 824

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 19

ccatggcctt accagtgacc gccttgctcc tgccgctggc cttgctgctc cacgccgcca 60

ggccgggaggt gcagctggtg gagtccgggg gagacttagt gaagcctgga gggtccctga 120

aactctcctg tgcagcctct ggattcactt tcagtggcta tggcatgtct tgggttcgcc 180

agactccaga caagaggctg gagtgggtcg caaccattac tagtggtggt acttacacct 240

actatccaga cagtgtgaag gggcgattca ccatctccag agacaatgcc aagaacaccc 300

tgtacctgca aatagacagt ctgaagtctg aggatacagc catatatttc tgtgcaagat 360

ccctcgcggg aaatgctatg gactactggg gtcaaggaac ctcagtcacc gtctcctcag 420

gtggaggtgg atcaggtgga ggtggatctg gtggaggtgg atctgacatt gtgatgactc 480

agtctccagc caccctgtct gtgactccag gagatagagt ctctctttcc tgcagggcca 540

gccagactat tagcgactac ttacactggt atcaacaaaa atcacatgag tctccaaggc 600

ttctcatcaa atttgcttcc caatccattt ctgggatccc ctccaggttc agtggcagtg 660

gatcaggctc agatttcact ctcagtatca acagtgtgga acctgaagat gttggagtgt 720

attactgtca aaatggtcac ggctttcctc ggacgttcgg tggaggcacc aagctggaaa 780

tcaaagaaca aaaactcatc tcagaagagg atctgtaact cgag 824

<210> 20

<211> 2337

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 20

atggctctcc cagtgactgc cctactgctt cccctagcgc ttctcctgca tgcagaggtg 60

aagctgcagc agtctggggc tgagctggtg aggcctgggt cctcagtgaa gatttcctgc 120

aaggcttctg gctatgcatt cagtagctac tggatgaact gggtgaagca gaggcctgga 180

cagggtcttg agtggattgg acagatttat cctggagatg gtgatactaa ctacaatgga 240

aagttcaagg gtcaagccac actgactgca gacaaatcct ccagcacagc ctacatgcag 300

ctcagcggcc taacatctga ggactctgcg gtctatttct gtgcaagaaa gaccattagt 360

tcggtagtag atttctactt tgactactgg ggccaaggga ccacggtcac cgtctcctca 420

ggtggaggtg gatcaggtgg aggtggatct ggtggaggtg gatctgacat tgagctcacc 480

cagtctccaa aattcatgtc cacatcagta ggagacaggg tcagcgtcac ctgcaaggcc 540

agtcagaatg tgggtactaa tgtagcctgg tatcaacaga aaccaggaca atctcctaaa 600

ccactgattt actcggcaac ctaccggaac agtggagtcc ctgatcgctt cacaggcagt 660

ggatctggga cagatttcac tctcaccatc actaacgtgc agtctaaaga cttggcagac 720

tatttctgtc aacaatataa caggtatccg tacacgtccg gaggggggac caagctggag 780

atcaaacggg cggccgcaat tgaagttatg tatcctcctc cttacctaga caatgagaag 840

agcaatggaa ccattatcca tgtgaaaggg aaacaccttt gtccaagtcc cctatttccc 900

ggaccttcta agcccttttg ggtgctggtg gtggttggtg gagtcctggc ttgctatagc 960

ttgctagtaa cagtggcctt tattattttc tgggtgagga gtaagaggag caggctcctg 1020

cacagtgact acatgaacat gactccccgc cgccccggggc cccccgcaa gcattaccag 1080

ccctatgccc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc 1140

gcagagcccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1200

1260

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgaa 1380

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440

gccctgcccc ctcgcggatc tggagcaaca aacttctcac tactcaaaca agcaggtgac 1500

gtggagaggaga atcccggacc catggagaca gacacactcc tgctatggggt actgctgctc 1560

tgggttccag gttccactgg tgacgaggtg cagctggtgg agtccggggg agacttagtg 1620

aagcctggag ggtccctgaa actctcctgt gcagcctctg gattcacttt cagtggctat 1680

ggcatgtctt gggttcgcca gactccagac aagaggctgg agtgggtcgc aaccattact 1740

agtggtggta cttacaccta ctatccagac agtgtgaagg ggcgattcac catctccaga 1800

gacaatgcca agaacaccct gtacctgcaa atagacagtc tgaagtctga ggatacagcc 1860

atatatttct gtgcaagatc cctcgcggga aatgctatgg actactgggg tcaaggaacc 1920

tcagtcaccg tctcctcagg tggaggtgga tcaggtggag gtggatctgg tggaggtgga 1980

tctgacattg tgatgactca gtctccagcc accctgtctg tgactccagg agatagagtc 2040

2100

tcacatgagt ctccaaggct tctcatcaaa tttgcttccc aatccatttc tgggatcccc 2160

tccaggttca gtggcagagg atcaggctca gatttcactc tcagtatcaa cagtgtggaa 2220

cctgaagatg ttggagtgta ttactgtcaa aatggtcacg gctttcctcg gacgttcggt 2280

ggaggcacca agctggaaat caaagaacaa aaactcatct cagaagagga tctgtaa 2337

<210> 21

<211> 1644

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 21

catggctctc ccagtgactg ccctactgct tcccctagcg cttctcctgc atgcagaggt 60

gaagctgcag gagtcagggg gaggcttcgt gaagcctgga gggtccctca aagtctcctg 120

tgcagcctct ggattcactt tcagtagcta tgccatgtcc tgggttcgcc tgagtccgga 180

gatgaggctg gagtgggtcg caaccattag cagtgctggt ggttacatct tctattctga 240

cagtgtgcag ggacgattca ccatttccag agacaatgcc aagaacaccc tgcacctgca 300

aatgggcagt ctgaggtctg gggacacggc catgtattac tgtgcaaggc agggatttgg 360

taactacggt gattactatg ctatggacta ctggggccaa gggaccacgg tcaccgtctc 420

ctcaggtgga ggtggatcag gtggaggtgg atctggtgga ggtggatctg acattgagct 480

cacccagtct ccatcctccc tggctgtgtc agcaggagag aaggtcacta tgagctgcaa 540

atccagtcag agtctgctca acagtagaac ccgaaagaac cagttggctt ggtaccagca 600

aaaaccagga cagtctcctg aactgctgat ctactgggca tccactaggc aatctggagt 660

ccctgatcgc ttcacaggca gtggatctgg gacagatttc actctcacca tcagcagtgt 720

gcaggctgaa gacctggcag tttattactg ccagcaatct tataatctac tgcaacaaac 780

ttctcactac tcaaacaagc aggtgacgtg gaggagaatc ccggacccat ggagacagac 840

acactcctgc tatgggtact gctgctctgg gttccaggtt ccactggtga cgaggtgcag 900

ctggtggagt ccgggggaga cttagtgaag cctggagggt ccctgaaact ctcctgtgca 960

gcctctggat tcactttcag tggctatggc atgtcttggg ttcgccagac tccagacaag 1020

aggctggagt gggtcgcaac cattactagt ggtggtactt acacctacta tccagacagt 1080

gtgaaggggc gattcaccat ctccagagac aatgccaaga acaccctgta cctgcaaata 1140

gacagtctga agtctgagga tacagccata tatttctgtg caagatccct cgcgggaaat 1200

gctatggact actggggtca aggaacctca gtcaccgtct cctcaggtgg aggtggatca 1260

ggtggaggtg gatctggtgg aggtggatct gacattgtga tgactcagtc tccagccacc 1320

ctgtctgtga ctccaggaga tagagtctct ctttcctgca gggccagcca gactattagc 1380

gactacttac acaggtatca acaaaaatca catgagtctc caaggcttct catcaaattt 1440

gcttcccaat ccatttctgg gatcccctcc aggttcagtg gcagtggatc aggctcagat 1500

ttcactctca gtatcaacag tgtggaacct gaagatgttg gagtgtatta ctgtcaaaat 1560

ggtcacggct ttcctcggac gttcggtgga ggcaccaagc tggaaatcaa agaacaaaaa 1620

ctcatctcag aagaggatct gtaa 1644

<210> 22

<211> 801

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 22

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60

gaccaggtgc agctggtgga gtctggggga ggcgtggtcc agcctgggag gtccctgaga 120

ctcgactgta aagcgtctgg aatcaccttc agtaactctg gcatgcactg ggtccgccag 180

gctccaggca aggggctgga gtgggtggca gttatttggt atgatggaag taaaagatac 240

tatgcagact ccgtgaaggg ccgattcacc atctccagag acaattccaa gaacacgctg 300

tttctgcaaa tgaacagcct gagagccgag gacacggctg tgtattactg tgcgacaaac 360

gacgactact ggggccaggg aaccctggtc accgtctcct caggtggagg tggatcaggt 420

ggaggtggat ctggtgggagg tggatctgaa attgtgttga cacagtctcc agccaccctg 480

tctttgtctc caggggaaag agccaccctc tcctgcaggg ccagtcagag tgttagtagt 540

tacttagcct ggtaccaaca gaaacctggc caggctccca ggctcctcat ctatgatgca 600

tccaacaggg ccactggcat cccagccagg ttcagtggca gtgggtctgg gacagacttc 660

actctcacca tcagcagcct agagcctgaa gattttgcag tttattactg tcagcagagt 720

agcaactggc ctcggacgtt cggccaaggg accaaggtgg aaatcaaaga acaaaaactc 780

atctcagaag aggatctgta a 801

<210> 23

<211> 2332

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 23

atggctctcc cagtgactgc cctactgctt cccctagcgc ttctcctgca tgcagaggtg 60

aagctgcagc agtctggggc tgagctggtg aggcctgggt cctcagtgaa gatttcctgc 120

aaggcttctg gctatgcatt cagtagctac tggatgaact gggtgaagca gaggcctgga 180

cagggtctag agtggattgg acagatttat cctggagatg gtgatactaa ctacaatgga 240

aagttcaagg gtcaagccac actgactgca gacaaatcct ccagcacagc ctacatgcag 300

ctcagcggcc taacatctga ggactctgcg gtctatttct gtgcaagaaa gaccattagt 360

tcggtagtag atttctactt tgactactgg ggccaaggga ccacggtcac cgtctcctca 420

ggtggaggtg gatcaggtgg aggtggatct ggtggaggtg gatctgacat cgagctcacc 480

cagtctccaa aattcatgtc cacatcagta ggagacaggg tcagcgtcac ctgcaaggcc 540

agtcagaatg tgggtactaa tgtagcctgg tatcaacaga aaccaggaca atctcctaaa 600

ccactgattt actcggcaac ctaccggaac agtggagtcc ctgatcgctt cacaggcagt 660

ggatctggga cagattccac tctcaccatc actaacgtgc agtctaaaga cttggcagac 720

tatttctgtc aacaatataa caggtatccg tacacgtccg gaggggggac caagctggag 780

atcaaacggg cggccgcaat tgaagttatg tatcctcctc cttacctaga caatgagaag 840

agcaatggaa ccattatcca tgtgaaaggg aaacaccttt gtcccaagtc ccctatttcc 900

cggaccttct aagccctttg ggtgctggtg gtggttggtg gagtcctggc ttgctatagc 960

cgctagtaac agtggccttt attattttct gggtgaggag taagaggagc aggctcctgc 1020

acagtgacta catgaacatg actccccgcc gccccggggcc cacccgcaag cattaccagc 1080

accacgcgac ttcgcagcct atcgctccag agtgaagttc agcaggagcg 1140

cagagccccc cgcgtaccag cagggccaga accagctcta taacgagctc aatctaggac 1200

gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag atggggggaa 1260

agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa gataagatgg 1320

cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag gggcacgatg 1380

gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt cacatgcagg 1440

ccctgccccc tcgcggatct ggagcaacaa acttctcact actcaaacaa gcaggtgacg 1500

tggaggagaa tcccggaccc atggagacag acacactcct gctatgggta ctgctgctct 1560

gggttccagg ttccactggt gaccaggtgc agctggtgga gtctggggga ggcgtggtcc 1620

agcctgggag gtccctgaga ctcgactgta aagcgtctgg aatcaccttc agtaactctg 1680

gcatgcactg ggtccgccag gctccaggca aggggctgga gtgggtggca gttatttggt 1740

atgatggaag taaaagatac tatgcagact ccgtgaaggg ccgattcacc atctccagag 1800

acaattccaa gaacacgctg tttctgcaaa tgaacagcct gagagccgag gacacggctg 1860

tgtattactg tgcgacaaac gacgactact ggggccaggg aaccctggtc accgtctcct 1920

caggtggagg tggatcaggt ggaggtggat ctggtggagg tggatctgaa attgtgttga 1980

cacagtctcc agccaccctg tctttgtctc caggggaaag agccaccctc tcctgcaggg 2040

ccagtcagag tgttagtagt tacttagcct ggtaccaaca gaaacctggc caggctccca 2100

ggctcctcat ctatgatgca tccaacaggg ccactggcat cccagccagg ttcagtggca 2160

gtgggtctgg gacagacttc actctcacca tcagcagcct agagcctgaa gattttgcag 2220

tttattactg tcagcagagt agcaactggc ctcggacgtt cggccaaggg accaaggtgg 2280

aaatcaaaga acaaaaactc atctcagaag aggatctgta actcgaggat cc 2332

<210> 24

<211> 1640

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 24

catggctctc ccagtgactg ccctactgct tcccctagcg cttctcctgc atgcagaggt 60

gaagctgcag gagtcagggg gaggcttcgt gaagcctgga gggtccctca aagtctcctg 120

tgcagcctct ggattcactt tcagtagcta tgccatgtcc tgggttcgcc tgagtccgga 180

gatgaggctg gagtgggtcg caaccattag cagtgctggt ggttacatct tctattctga 240

cagtgtgcag ggacgattca ccatttccag agacaatgcc aagaacaccc tgcacctgca 300

aatgggcagt ctgaggtctg gggacacggc catgtattac tgtgcaaggc agggatttgg 360

taactacggt gattactatg ctatggacta ctggggccaa gggaccacgg tcaccgtctc 420

ctcaggtgga ggtggatcag gtggaggtgg atctggtgga ggtggatctg acattgagct 480

cacccagtct ccatcctccc tggctgtgtc agcaggagag aaggtcacta tgagctgcaa 540

atccagtcag agtctgctca acagtagaac ccgaaagaac cagttggctt ggtaccagca 600

aaaaccagga cagtctcctg aactgctgat ctactgggca tccactaggc aatctggagt 660

ccctgatcgc ttcacaggca gtggatctgg gacagatttc actctcacca tcagcagtgt 720

gcaggctgaa gacctggcag tttattactg ccagcaatct tataatctac tgcaacaaac 780

ttctcactac tcaaacaagc aggtgacgtg gaggagaatc ccggacccat ggagacagac 840

acactcctgc tatgggtact gctgctctgg gttccaggtt ccactggtga ccaggtgcag 900

ctggtggagt ctgggggagg cgtggtccag cctgggaggt ccctgagact cgactgtaaa 960

gcgtctggaa tcaccttcag taactctggc atgcactggg tccgccaggc tccaggcaag 1020

gggctggagt gggtggcagt tatttggtat gatggaagta aaagatacta tgcagactcc 1080

gtgaagggcc gattcaccat ctccagagac aattccaaga acacgctgtt tctgcaaatg 1140

aacagcctga gagccgagga cacggctgtg tattactgtg cgacaaacga cgactactgg 1200

ggccagggaa ccctggtcac cgtctcctca ggtggaggtg gatcaggtgg aggtggatct 1260

ggtgggaggtg gatctgaaat tgtgttgaca cagtctccag ccaccctgtc tttgtctcca 1320

ggggaaagag ccaccctctc ctgcagggcc agtcagagtg ttagtagtta cttagcctgg 1380

taccaacaga aacctggcca ggctcccagg ctcctcatct atgatgcatc caacagggcc 1440

actggcatcc cagccaggtt cagtggcagt gggtctggga cagacttcac tctcaccatc 1500

agcagcctag agcctgaaga ttttgcagtt tattactgtc agcagagtag caactggcct 1560

cggacgttcg gccaagggac caaggtggaa atcaaagaac aaaaactcat ctcagaagag 1620

gatctgtaac tcgaggatcc 1640

<210> 25

<211> 1266

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 25

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60

120

gaggtgcggc ttctggagtc tggtggagga ttagtgaagc ctgaggggtc actgaaactc 180

tcctgtgtgg cctctggatt caccttcagt gactatttca tgagctgggt ccgccaggct 240

ccagggaagg ggctggagtg ggttgctcac atatacacga aaagttataa ttatgcaact 300

tattactcgg gttcggtgaa aggcagattc accatctcca gagatgattc ccgaagcatg 360

gtctacctgc aaatgaacaa cctgagaact gaggacacgg ccacttatta ctgtacaaga 420

gatggaagcg gatatccctc tctggatttc tggggtcaag ggacccaagt cactgtctcc 480

tcagccacaa caacagcccc atctgtctat cccttggccc ctgcctgtga cagcacaacc 540

aaatcgggtg gaggtggatc aggtggaggt ggatctggtg gaggtggatc ttatgagctg 600

actcagccac cttcagcatc agtcaatgta ggagagactg tcaaaatcac ctgctctggg 660

gaccaattgc cgaaatattt tgcagattgg tttcatcaaa ggtcagacca gaccattttg 720

caagtgatat atgatgataa taagcgcccc tcggggatcc ctgaaagaat ctctgggtcc 780

agctcaggga caacagccac cttgaccatc agagatgtcc gggctgagga tgaaggtgac 840

tattactgtt tctcaggata tgttgatagt gatagcaaat tgtatgtttt tggcagcgga 900

acccagctca ccgtcctagg tggacccaag tcttctccca aagtcacagt gtttccacct 960

tcacctgagg agctccggac aaacaaagcc acactggtgt gtctggttaa tgacttctac 1020

ccgggttctg caacagtgac ctggaaggca aatggagcaa ctatcaatga tggggtgaag 1080

actacaaagc cttccaaaca gggccaaaac tacatgacca gcagctacct aagtttgaca 1140

gcagaccagt ggaaatctca caacagggtt tcctgccaag ttacccatga aggggaaact 1200

gtggagaaga gtttgtcccc tgcagaatgt ctcgaacaaa aactcatctc agaagaggat 1260

ctgtaa 1266

<210> 26

<211> 3325

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 26

catggctctc ccagtgactc ccctactgct acccctagcg ttctcctgca tgcagaggtg 60

aagctgcagc agtctggggc tgagctggtg aggcctgggt cctcagtgaa gatttcctgc 120

aaggcttctg gctatgcatt cagtagctac tggatgaact gggtgaagca gaggcctgga 180

cagggtcttg agtggattgg acagatttat cctggagatg gtgatactaa ctacaatgga 240

aagttcaagg gtcaagccac actgactgca gacaaatcct ccagcacagc ctacatgcag 300

ctcagcggcc taacatctga ggactctgcg gtctatttct gtgcaagaaa gaccattagt 360

tcggtagtag atttctactt tgactactgc ggccaaggga ccacggtcac cgtctcctca 420

ggtggaggtg ggcaggggag gtggatctgg ggaggtggat ctgacattga gctcacccag 480

tctccaaaat tcatgtccac atcagtagga cacagggtca gcgtcacctg caaggccagt 540

cagaatgtgg gtactaatgt agcctggtat caacagaaac caggacaatc tcctaaacca 600

ctgatttact cggcaaccta cgggaacagt ggagtccctg agcgcttcac aggcagtgga 660

tctgggcag atttcactct caccatcact aacgtgcagt ctaaagactt ggcagactat 720

ttctgtcaac aatataacag gtagccgtac acgtccggag gggggaccaa gctggagatc 780

aaacgggcgg ccgcaattga gttcatgtac cctccgcctt acctagacaa cgagaggagc 840

aatggaacta ttattcacat aaaagagaaa catctttgtc atactcagtc atctcctaag 900

ctgttttggg cactggtcgt ggttgtggag tcctgttttg ttatggcttg ctagtgacag 960

tggctcttgt gttatctgga caaatagtag aaggaacaga ctccttcaaa gtgactacat 1020

gaacatgact ccccggaggg cagggctcac tcgaaagcct taccagccct acgcccctgc 1080

cagagacttt gcagcgtacc gccccagagc aaaattcagc aggagtgcag agactgctgc 1140

caacctgcag gaccccaacc agctctacaa tgagctcaat ctagggcgaa gagaggaata 1200

tgacgtcttg gagaagaagc cggctcggga tccagagatg gcagccaaac agcagaggag 1260

caggaacccc caggaaggcg tatacaatgc actgcagaaa gacaagatgg cagaagccta 1320

cagtgagatc ggcacaaaag gcgagaggcg gagaggcaag gggcacgatg gcctttacca 1380

gggtctcagc actgccacca aggacaccta tgatgggctg catatgcaga ccctggcccc 1440

tcgctaacag ccactcgagg atccgcccct ctccctcccc cccccctaac gttactggcc 1500

gaagccgctt ggaataaggc cggtgtgcgt ttgactatat gttatttttcc accatattgc 1560

cgtcttttgg caatgtgagg gccgggaaac ctggccctgt cttcttgacg agcattccta 1620

ggggtctttc ccctctcgcc aaaggaatgc agggtctgtt gaatgtcgtg aaggaagcag 1680

ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga 1740

accccccacc tggcgacagg tgcctcagcg acccaaaggcc acgtgtataa gatacaccag 1800

caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat 1860

ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta 1920

tgggatctga tctggggcct cggtcacatg ctttacatgt gtttagtcga ggttaaaaaa 1980

acgtctagggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataata 2040

tggccacaaa ctgccatgga gacagacaca ctcctgctaa gggtactgct gctctgggtt 2100

ccaggttcca ctggagacat gggattggga ctgcagaggg ttttctttgt tgctctttta 2160

aaaggtgtcc actgtgaggt gcggcttctg gagtctggtg gaggattagt gaagcctgag 2220

gggtcactga aatctcctgt gtggcctcag gattcacctt cagagactat ttcatgagct 2280

gggtccgcca ggctcgggg aaggggctgg agtgggttgc tcacatatac acgaaaagtt 2340

ataattatgc aacttattac tcgggttcgg tgaaaggcag attcaccatc tccagagatg 2400

attcccgaag catggtctac cgcaaatgaa caacctgaga actgaggaca cggccactta 2460

ttactgtaca agagatggaa gcggatatcc ctctctggat ttctggggtc aagggaccca 2520

agtcactgtc tcctcagcca caacaacagc cccatctgtc tatcccttgg cccctgcctg 2580

tgacagcaca accaaatcgg gtggaggtgg atcaggtgga ggtggatctg gtggaggtgg 2640

atcttatgag ctgactcagc caccttcagc atcagtcaat gtaggagaga ctgtcaaaat 2700

cacctgctct ggggaccaat tgccgaaata ttttgcagat tggtttcatc aaaggtcaga 2760

ccagaccatt ttgcaagtga tatatgatga taataagcgc ccctcgggga tccctgaaag 2820

aatctctggg tccagctcag ggacaacagc caccttgacc atcagagatg tccgggctga 2880

ggatgaaggt gactattact gtttctcagg atatgttgat agtgatagca aattgtatgt 2940

ttttggcagc ggaacccagc tcaccgtcct aggtggaccc aagtcttctc ccaaagtcac 3000

3060

3120

tgatggggtg aagactacaa agccttccaa acagggccaa aactacatga ccagcagcta 3180

3240

tgaaggggaa actgtggaga agagtttgtc ccctgcagaa tgtctcgaac aaaaactcat 3300

ctcagaagag gatctgtaac tggag 3325

<210> 27

<211> 3333

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 27

ctctcccagt gactgcccta ctgcttcccc tagcgctact cctgcatgca gaggtgaagc 60

tgcaggagtc agggggaggc ttcgtgaagc ctggagggtc cctcaaagtc tcctgtgcag 120

cctctggatt cactttcagt agctatgcca tgtcctgggt tcgcctgagt ccggagatga 180

ggctggagtg ggtcgcaacc attagcagtg ctggtggtta catcttctat tctgacagtg 240

tgcagggacg attcaccatt tccagagaca atgccaagaa caccctgcac ctgcaaatgg 300

gcagtctgag gtctggggac agggccatgt attactgtgc aaggcaggga tttggtaact 360

acggtgatta ctatgctatg gactactggg gccaagggac cacggtcacc gtctcctcag 420

gtggaggtgg atcaggtgga ggtggatctg gtggaggtgg atctgacatt gagctcaccc 480

agtctccatc ctccctggct gtgtcagcag gagagaaggt cactatgagc tgcaaatcca 540

gtcagagtct gctcaacagt agaacccgaa agaaccagtt ggcttggtac cagcaaaaac 600

caggacagtc tcctgaactg ctgatctact gggcatccac taggcaatct ggagtccctg 660

atcgcttcac aggcagtgga tctgggacag atttcactct caccatcagc agtgtgcagg 720

ctgaagacct ggcagtttat tactgccagc aatcttataa tctactggga ccaagctgga 780

gatcaaacgg gcggccgcaa ttgagttcat gtaccctccg ccttacctag acaacgagag 840

gagcaatgga actattattc acataaaaga gaaacatctt tgtcatactc agtcatctcc 900

taagctgttt tgggcactgg tcgtggttgc tggagtccag ttttgttatg gcttgctagt 960

gacagtggct ctttgtgtta tctggacaaa tagtagaagg aacagactcc ttcaaagtga 1020

ctacatgaac atgactcccc ggaggcctgg gctcactcga aagccttacc agccctacgc 1080

ccctgccaga gactttgcag cgtaccggcc cagagcaaaa ttcagcagga gtgcagagac 1140

tgctgccaac ctgcaggacc ccaaccagct ctacaatgag ctcaatctag ggcgaagaga 1200

ggaatatgac gtcttggaga agaagcggcc tcgggatcca gagatgggag gcaaacagca 1260

gaggaggagc aacccccagg aaggcgtata caatgcactg cagaaagaca agatggcaga 1320

agcctacagt gagatcggca caaaaggcga gaggcggaga ggcaaggggc acgatggcct 1380

ttaccagggt ctcagcactg ccaccaagga cacctatgat gccctgcata tgcagaccct 1440

ggcccctcgc taacagccac tcgaggatcc gcccctctcc ctcccccccc cctaacgtta 1500

ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca 1560

tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca 1620

ttcctagggg tctttcccct ctcgccaaag gaatgcaggg tctgttgaat gtcgtgaagg 1680

aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc 1740

agcggaaccc cccacctggc gacaggtgcc tctgcgacca aaggccacgt gtataagata 1800

cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag 1860

tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc 1920

attgtatggg atctgatctg gggcctcggt cacatgcttt acatgtgttt agtcgaggtt 1980

aaaaaaacgt ctaggcccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg 2040

ataatatggc cacaaactgc catggagaca gacacactcc tgctatgggt actgctgctc 2100

tgggttccag gttccactgg tgacatggga ttgggactgc agtgggtttt ctttgttgct 2160

cttttaaaag gtgtccactg tgaggtgcgg cttctggagt ctggtggagg attagtgaag 2220

cctgaggggt cactgaaact ctcctgtgtg gcctctggat tcaccttcag tgactatttc 2280

atgagctggg tccgccaggc tccagggaag gggctggagt gggttgctca catatacacg 2340

aaaagttata attatgcaac ttattactcg ggttcggtga aaggcagatt caccatctcc 2400

agagatgatt cccgaagcat ggtctacctg caaatgaaca acctgagaac tgaggacacg 2460

gccacttatt actgtacaag agatggaagc ggatatccct ctctggattt ctggggtcaa 2520

gggacccaag tcactgtctc ctcagccaca acaacagccc catctgtcta tcccttggcc 2580

cctgcctgtg acagcacaac caaatcgggt ggaggtggat caggtggagg tggatctggt 2640

ggaggtggat cttatgagct gactcagcca ccttcagcat cagtcaatgt aggagagact 2700

gtcaaaatca cctgctctgg ggaccaattg ccgaaatatt ttgcagattg gtttcatcaa 2760

aggtcagacc agaccatttt gcaagtgata tatgatgata ataagcgccc ctcggggatc 2820

cctgaaagaa tctctgggtc cagctcaggg acaacagcca ccttgaccat cagagatgtc 2880

cgggctgagg atgaaggtga ctattactgt ttctcaggat atgttgatag tgatagcaaa 2940

ttgtatgttt ttggcagcgg aacccagctc accgtcctag gtggacccaa gtcttctccc 3000

3060

tgtctggtta atgacttcta cccgggttct gcaacagtga cctggaaggc aaatggagca 3120

actatcaatg atggcgtgaa gactacaaag ccttccaaac agggccaaaa ctacatgacc 3180

agcagctacc taagtttgac agcagaccag tggaaatctc acaacagggt ttcctgccaa 3240

gttacccatg aaggggaaac tgtggagaag agtttgtccc ctgcagaatg tctcgaacaa 3300

aaactcatct cagaagagga tctgtaactc gag 3333

<210> 28

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic

primer

<400> 28

tgaggagacg gtgaccgtgg tcccttggcc ccag 34

<210> 29

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 29

aggtsmarct gcagsagtcw gg 22

<210> 30

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 30

gttagatctc cagcttggtc cc 22

<210> 31

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 31

gacattcagc tgacccagtc tcca 24

<210> 32

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 32

ggctgcagst tcagtggcag tggrtcwggr ac 32

<210> 33

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 33

ctcattcctg ttgaagctct tgacaatggg 30

<210> 34

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 34

atatccatgg cagacgtcca gatgatccag tctcca 36

<210> 35

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 35

atatccatgg cagacattgt gctgactcag tctcc 35

<210> 36

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 36

atatccatgg cagatgttgt gatgacccaa actcca 36

<210> 37

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 37

atatccatgg cacaaattgt tctcacccag tctcc 35

<210> 38

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 38

atatccatgg cagacattgt gatgacacag tctcca 36

<210> 39

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 39

atatccatgg cagatattgt gatgacgcag gctgca 36

<210> 40

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 40

atatccatgg cagacattgt gatgacccag tctc 34

<210> 41

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 41

gcttcaacag gaatgagtgt taactcgagg tag 33

<210> 42

<211> 93

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 42

ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca ggttccactg 60

gtgacgaggt gctgcagctg gtggagtccg ggg 93

<210> 43

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 43

agatccacct ccaccagatc cacctccacc tgatccacct ccacctgagg agacggtgac 60

tgaggttcct tgacc 75

<210> 44

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 44

ggtggaggtg gatcaggtgg aggtggatct ggtggaggtg gatctgacat tgtgatgact 60

cagtctccag ccacc 75

<210> 45

<211> 71

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 45

ctcgagttac agatcctctt ctgagatgag tttttgttgt ttgatttcca gcttggtgcc 60

tccaccgaac g 71

<210> 46

<211> 93

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 46

tataccatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct gctccacgcc 60

gccaggccgg aggtgcagct ggtggagtcc ggg 93

<210> 47

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 47

cacgtgccat ggatgaggat atttgctgtc tttatat 37

<210> 48

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 48

ctcgagttac gtctcctcca aatgtgtatc acttt 35

<210> 49

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 49

tattacacgt gttacatgag gatatttgct gtcttt 36

<210> 50

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 50

tataggatcc tcgaggatgt tacgtctcct ccaaatgtgt a 41

<210> 51

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 51

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 52

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic primer

<400> 52

Gly Gly Gly Gly Ser

15

<210> 53

<211> 271

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 53

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu

20 25 30

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

35 40 45

Thr Phe Ser Gly Tyr Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys

50 55 60

Arg Leu Glu Trp Val Ala Thr Ile Thr Ser Gly Gly Thr Tyr Thr Tyr

65 70 75 80

Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Ile Asp Ser Leu Lys Ser Glu Asp Thr

100 105 110

Ala Ile Tyr Phe Cys Ala Arg Ser Leu Ala Gly Asn Ala Met Asp Tyr

115 120 125

Trp Gly Glyn Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln

145 150 155 160

Ser Pro Ala Thr Leu Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser

165 170 175

Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln

180 185 190

Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys Phe Ala Ser Gln Ser

195 200 205

Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp

210 215 220

Phe Thr Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr

225 230 235 240

Tyr Cys Gln Asn Gly His Gly Phe Pro Arg Thr Phe Gly Gly Gly Thr

245 250 255

Lys Leu Glu Ile Lys Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

260 265 270

<210> 54

<211> 271

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 54

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu

20 25 30

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

35 40 45

Thr Phe Ser Gly Tyr Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys

50 55 60

Arg Leu Glu Trp Val Ala Thr Ile Thr Ser Gly Gly Thr Tyr Thr Tyr

65 70 75 80

Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Ile Asp Ser Leu Lys Ser Glu Asp Thr

100 105 110

Ala Ile Tyr Phe Cys Ala Arg Ser Leu Ala Gly Asn Ala Met Asp Tyr

115 120 125

Trp Gly Glyn Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln

145 150 155 160

Ser Pro Ala Thr Leu Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser

165 170 175

Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln

180 185 190

Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys Phe Ala Ser Gln Ser

195 200 205

Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp

210 215 220

Phe Thr Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr

225 230 235 240

Tyr Cys Gln Asn Gly His Gly Phe Pro Arg Thr Phe Gly Gly Gly Thr

245 250 255

Lys Leu Glu Ile Lys Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

260 265 270

<210> 55

<211> 266

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 55

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val

20 25 30

Val Gln Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile

35 40 45

Thr Phe Ser Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu

145 150 155 160

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

165 170 175

Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala

180 185 190

Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro

195 200 205

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

210 215 220

Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser

225 230 235 240

Ser Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

245 250 255

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

260 265

<210> 56

<211> 422

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 56

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Met Gly Leu Gly Leu Gln Trp Val Phe Phe Val

20 25 30

Ala Leu Leu Lys Gly Val His Cys Glu Val Arg Leu Leu Glu Ser Gly

35 40 45

Gly Gly Leu Val Lys Pro Glu Gly Ser Leu Lys Leu Ser Cys Val Ala

50 55 60

Ser Gly Phe Thr Phe Ser Asp Tyr Phe Met Ser Trp Val Arg Gln Ala

65 70 75 80

Pro Gly Lys Gly Leu Glu Trp Val Ala His Ile Tyr Thr Lys Ser Tyr

85 90 95

Asn Tyr Ala Thr Tyr Tyr Ser Gly Ser Val Lys Gly Arg Phe Thr Ile

100 105 110

Ser Arg Asp Asp Ser Arg Ser Met Val Tyr Leu Gln Met Asn Asn Leu

115 120 125

Arg Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Thr Arg Asp Gly Ser Gly

130 135 140

Tyr Pro Ser Leu Asp Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser

145 150 155 160

Ser Ala Thr Thr Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Ala

165 170 175

Cys Asp Ser Thr Thr Lys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

180 185 190

Ser Gly Gly Gly Gly Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser

195 200 205

Val Asn Val Gly Glu Thr Val Lys Ile Thr Cys Ser Gly Asp Gln Leu

210 215 220

Pro Lys Tyr Phe Ala Asp Trp Phe His Gln Arg Ser Asp Gln Thr Ile

225 230 235 240

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

245 250 255

Arg Ile Ser Gly Ser Ser Ser Gly Thr Thr Ala Thr Leu Thr Ile Arg

260 265 270

Asp Val Arg Ala Glu Asp Glu Gly Asp Tyr Tyr Cys Phe Ser Gly Tyr

275 280 285

Val Asp Ser Asp Ser Lys Leu Tyr Val Phe Gly Ser Gly Thr Gln Leu

290 295 300

Thr Val Leu Gly Gly Pro Lys Ser Ser Pro Lys Val Thr Val Phe Pro

305 310 315 320

Pro Ser Pro Glu Glu Leu Arg Thr Asn Lys Ala Thr Leu Val Cys Leu

325 330 335

Val Asn Asp Phe Tyr Pro Gly Ser Ala Thr Val Thr Trp Lys Ala Asn

340 345 350

Gly Ala Thr Ile Asp Gly Val Lys Thr Thr Lys Pro Ser Lys Gln

355 360 365

Gly Gln Asn Tyr Met Thr Ser Ser Tyr Leu Ser Leu Thr Ala Asp Gln

370 375 380

Trp Lys Ser His Asn Arg Val Ser Cys Gln Val Thr His Glu Gly Glu

385 390 395 400

Thr Val Glu Lys Ser Leu Ser Pro Ala Glu Cys Leu Glu Gln Lys Leu

405 410 415

Ile Ser Glu Glu Asp Leu

420

<---

Claims (40)

1. An immunoreactive cell for immunotherapy, comprising: a) an antigen recognition receptor that binds an antigen, wherein binding of the antigen recognition receptor to the antigen activates the immunoreactive cell, and b) a soluble single chain variable fragment (scFv) that binds to CD47, PD-1, CTLA-4, CD28, OX-40, 4-1BB and their ligands, wherein the immunoreactive cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, and a pluripotent stem cell from which lymphoid cells can be differentiated. 2. The immunoreactive cell of claim 1, wherein the antigen is a tumor or pathogen antigen. 3. An immunoreactive cell according to claim 1 or 2, wherein the soluble scFv is secreted. 4. Immunoreactive cell according to any one of paragraphs. 1-3, wherein said antigen recognition receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). 5. Immunoreactive cell according to any one of paragraphs. 1-4, wherein said antigen recognition receptor is exogenous or endogenous. 6. Immunoreactive cell according to any one of paragraphs. 1-5, wherein said antigen recognition receptor is recombinantly expressed. 7. Immunoreactive cell according to any one of paragraphs. 1-6, in which the antigen recognition receptor is expressed from the vector. 8. Immunoreactive cell according to any one of paragraphs. 1-7 in which the scFv is expressed from the vector. 9. Immunoreactive cell according to any one of paragraphs. 1-8, wherein said immunoreactive cell is autologous. 10. Immunoreactive cell according to any one of paragraphs. 1-9, wherein the antigen is a tumor antigen selected from the group consisting of CD 19, MUC16, MUC1, CA1X, CEA, CDS, CD7, CD 10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, cytomegalovirus (CMV) infected cell antigen, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor α, GD2 , GD3, HER-2, hTERT, IL-13R-a2, k-light chain, KDR, LeY, cell adhesion molecules L1, MAGE-A1, mesothelin, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2 and WT-1. 11. Immunoreactive cell according to any one of paragraphs. 1-10, wherein said antigen is CD19 or MUC16. 12. Immunoreactive cell according to any one of paragraphs. 1-11, wherein the intracellular signaling domain of said antigen-recognizing receptor is CD3ζ chain, CD97, CD11a-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, CD28 signaling domain, or combinations thereof. 13. Immunoreactive cell according to any one of paragraphs. 1-12, in which the cell expresses a recombinant or endogenous antigen receptor that is 1928z or 4H1128Z. 14. Immunoreactive cell according to any one of paragraphs. 1-13, in which the soluble scFv enhances the immune response of the immunoreactive cell. 15. Pharmaceutical composition for immunotherapy, containing an effective amount of immunoreactive cells according to any one of paragraphs. 1-14 and a pharmaceutically acceptable excipient. 16. The use of immunoreactive cells according to any one of paragraphs. 1-14 or a pharmaceutical composition according to claim 15 for reducing tumor burden in an individual. 17. The use of immunoreactive cells according to any one of paragraphs. 1-14 or a pharmaceutical composition according to claim 15 to improve the survival of an individual with neoplasia. 18. The use of immunoreactive cells according to any one of paragraphs. 1-14 or a pharmaceutical composition according to claim 15 to reduce the tumor mass and increase the survival of an individual with neoplasia. 19. Application according to any one of paragraphs. 16-18, wherein the cell or composition reduces the number of tumor cells, the size of the tumor, and/or kills the tumor in the individual. 20. Application according to any one of paragraphs. 16-19, wherein the neoplasia is selected from the group consisting of blood cancer, B-cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma, and ovarian cancer. 21. Application according to any one of paragraphs. 16-20, wherein the neoplasia is B-cell leukemia, the antigen is CD19, and the scFv binds to one or more of CD47, PD-1, CTLA-4, and their ligands. 22. Application according to any one of paragraphs. 16-20, wherein the neoplasia is multiple myeloma, the antigen is CD19, and the scFv binds to one or more of CD47, PD-1, CTLA-4, and their ligands. 23. Application according to any one of paragraphs. 16-20, wherein the neoplasia is acute lymphoblastic leukemia (ALL), the antigen is CD19, and the scFv binds to one or more of CD47, PD-1, CTLA-4, and their ligands. 24. Application according to any one of paragraphs. 16-20, wherein the neoplasia is chronic lymphocytic leukemia, the antigen is CD19, and the scFv binds to one or more of CD47, PD-1, CTLA-4, and their ligands. 25. Application according to any one of paragraphs. 16-20, wherein the neoplasia is non-Hodgkin's lymphoma, the antigen is CD19, and the scFv binds to one or more of CD47, PD-1, CTLA-4, and their ligands. 26. Application according to any one of paragraphs. 16-20, wherein the neoplasia is ovarian neoplasia, the antigen is MUC16, and the scFv binds to one or more of CD47, PD-1, CTLA-4, and their ligands. 27. The method of obtaining antigen-specific immunoreactive cells according to any one of paragraphs. 1-14, which includes introducing into the immunoreactive cell a nucleic acid sequence that encodes a single-chain variable fragment (scFv) that binds to CD47, PD-1, CTLA-4, CD28, OX-40, 4-1BB and their ligands, where the immunoreactive the cell contains an antigen-recognizing receptor that binds the antigen, and the immunoreactive cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, and a pluripotent stem cell, from which lymphoid cells can be differentiated. 28. The method of claim 27 wherein the soluble scFv is secreted. 29. The method of claim 27 or 28, wherein the scFv is expressed from the vector. 30. The method according to any one of paragraphs. 27-29, wherein the soluble scFv enhances the immune response of the immunoreactive cell. 31. The method according to any one of paragraphs. 27-30, wherein said antigen recognition receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). 32. The method according to any one of paragraphs. 27-31, wherein said antigen recognition receptor is exogenous or endogenous. 33. The method according to any one of paragraphs. 27-32, wherein said antigen recognition receptor is recombinantly expressed. 34. The method according to any one of paragraphs. 27-33, wherein said antigen recognition receptor is expressed from a vector. 35. The method according to any one of paragraphs. 27-34, wherein the intracellular signaling domain of said antigen-binding receptor is CD3ζ chain, CD97, CD11a-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, CD28 signaling domain, or combinations thereof. 36. The use of immunoreactive cells according to any one of paragraphs. 1-14 or the pharmaceutical composition according to claim 15 in the treatment of blood cancer in an individual in need thereof, where the immunoreactive cell is a T cell containing an antigen recognition receptor that binds CD19, and binding of the antigen recognition receptor to CD19 activates the immunoreactive cell, and where the soluble single chain variable fragment (scFv) binds to one or more of CD47, PD-1, CTLA-4 and their ligands, thereby providing treatment for blood cancer in the individual. 37. Use according to claim 36, wherein the blood cancer is selected from the group consisting of B-cell leukemia, myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.

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