DESCRIPTION

COMPOSITIONS AND METHODS RELATED TO A HUMAN CD19-SPECIFIC CHIMERIC ANTIGEN RECEPTOR (h-CAR)

BACKGROUND OF THE INVENTION [0001] The present invention claims priority to U.S. Provisional Patent Application Serial No. 61/020,991, filed January 14, 2008, which is incorporated by reference herein in its entirety.

I. FIELD OF THE INVENTION

[0002] Embodiments of this invention are directed generally to cell biology, immunology, molecular biology, and cancer therapy. More particularly, it concerns genetically engineered, redirected T cells and hematopoietic cells for cellular immunotherapy of B-cell proliferative disorders such as non-Hodgkin lymphoma and leukemia malignancies.

II. BACKGROUND

[0003] The immune system of vertebrates consists of a number of organs and cell types which have evolved to accurately recognize foreign antigens, specifically bind to, and eliminate/destroy such foreign antigens. Lymphocytes, among other cell types, are critical to the immune system. Lymphocytes are divided into three major sub-populations, T cells, NK cells, and B cells. Although inter-dependent, T cells and NK cells are largely responsible for cell-mediated immunity and B cells are largely responsible for antibody production (humoral immunity).

[0004] In humans, each B cell can produce an enormous number of antibody molecules. Such antibody production typically ceases (or substantially decreases) when a foreign antigen has been neutralized. Occasionally, however, proliferation of a particular B cell will continue unabated and may result in a cancer known as a B cell leukemias and lymphomas. B-cell lymphomas, such as the B-cell subtype of NHL, are significant contributors to cancer mortality. The response of B-cell malignancies to various forms of treatment is mixed. For example, in cases in which adequate clinical staging of NHL is possible, field radiation therapy can provide satisfactory treatment. Still, about one-half of the patients die from the disease. (Devesa et al, J. Nat'l Cancer Inst. 79:701 (1987))

[0005] The majority of acute and chronic lymphocytic leukemias are of the B-cell lineage (Freedman, Hematol. Oncol. Clin. North Am. 4:405 (1990)). Chronic lymphocytic leukemias are the most common leukemia in the Western world (Goodman et al., Leukemia and Lymphoma 22:1 (1996)). Acute leukemias are aggressive in nature and difficult to cure. Due to the very low rate of cellular proliferation, chronic lymphocytic leukemia is resistant to cytotoxic drug treatment. Over 30,000 new cases of Non-Hodgkin's lymphoma are diagnosed each year in the United States alone. (Shipp et al., Cancer: Principles and Practice of Oncology, Lippincott-Raven Publishers, Philadelphia, 1997, p2165).

[0006] Traditional methods of treating B-cell malignancies, including chemotherapy and radiotherapy, and hematopoietic stem-cell transplantation, have limited utility due to toxic side effects. While current therapies have produced significant complete response rates, a large percentage of patients remain at a significant risk for disease relapse (Glass et al.,

Cancer 80:2311, 1997). Immune -based strategies for targeting minimal residual disease are under development and may provide additional modalities for consolidating standard chemotherapy and radiotherapy regimens.

SUMMARY OF THE INVENTION

[0007] In one aspect compositions of the invention include a human CD19-specifϊc chimeric T cell receptor (or chimeric antigen receptor, CAR) polypeptide (designated hCD19CAR) comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human CD 19 binding region. In another aspect, the CD 19 binding region is an F(ab')2, Fab', Fab, Fv or scFv. The binding region may comprise an amino acid sequence that is at least, at most or about 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence of SEQ ID NO:2. The intracellular domain may comprise an intracellular signaling domain of human CD3ζ and may further comprise human CD28 intracellular segment. In certain aspects the transmembrane domain is a CD28 transmembrane domain.

[0008] In a further aspect compositions of the invention include a nucleic acid encoding the polypeptide described above. In certain aspects the nucleic acid sequence is optimized for human codon usage.

[0009] In still a further aspect compositions of the invention include cells expressing the polypeptide described above. The cell may comprise an expression cassette encoding

hCD19CAR polypeptide. The expression cassette can be comprised in a non- viral vector, such as a transposon, or a human transposon, or recombinant variant thereof. The expression cassette can be comprised in a viral vector or recombinant variant thereof. The expression cassette can be genomically integrated or episomally maintained or expressed from mRNA.

[0010] In yet a further aspect the invention includes a method of making a cell (such as a T cell, a NK cell, or a stem cell) expressing a human CD19-specific CAR comprising introducing an expression cassette into the cell, wherein the expression cassette encodes a polypeptide comprising a human extracelluar CD 19 binding domain, a transmembrane domain, and one or more intracellular signaling domain(s). The method may further comprise stimulating the cells with CD19 ⁺ cells, recombinant CD 19, or an antibody to the receptor to cause the cells to proliferate, kill, and/or make cytokines; for example, the cells may be stimulated to proliferate or expand with CD19 ⁺ artificial antigen presenting cells.

[0011] The invention also includes recombinant CD19-specific immune cells expressing and/or bear on the cell surface membrane a CD19-specific chimeric T cell receptor human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-CD 19 monoclonal antibody or antigen binding fragment thereof. The CD19-specific immune cell can be selected from the group consisting of T-cells, natural killer cells, macrophage, neutrophils and bone marrow (hematopoietic) stem cells.

[0012] In certain aspects the invention includes methods of treating a human disease condition associated with a cell expressing endogenous CD 19 comprising infusing a patient with an amount of a recombinant cell expressing a human CD19-specific CAR sufficient to treat the condition, wherein the human CD19-specific CAR comprises a human CD 19 extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. The condition can be lymphoma, leukemia, Non-Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, or B cell associated autoimmune diseases.

[0013] The invention relates to the generation of a human CD19-specific chimeric antigen receptor (hCD19RCD28 or hCAR). In certain aspects recombinant cells expressing hCAR have improved in vivo persistence and anti- tumor efficacy. The human hCAR has a reduced immunogenicity compared to murine hCAR, which comprises a scFv segment derived from a

murine CD19-specifϊc monoclonal antibody (mAb). Anti-tumor effects can be augmented by genetically modified cells, such as T cells, hematopoietic progenitor cells, peripheral blood (PB) derived T cells (including T celsl from G-CSF-mobilized peripheral blood), and umbilical cord blood (UCB) derived T cells rendered specific for CD 19, a molecule constitutively expressed on B-cell malignancies. Typically T cell specificity is achieved by electrotransfer of an expression cassette encoding hCAR.

[0014] The hCAR may be a chimeric receptor comprising one or more activation endodomain(s), such as a CD3-ζ-derived activation domain. Additional T-cell activation motifs include, but are not limited to CD28, OX-40, 4-1BB. In certain aspects the activation domain can also include a CD28 transmembrane and/or activation domain. In a further aspect the hCAR encoding region and/or expression cassette codon optimized for expression in human cells and subjects, e.g., in one embodiment the scFv region obtained from V _H and V _L sequences of a CD19-specifϊc human antibodies are incorporated into the CD 19 binding segment of the hCAR (for example see U.S. Patent 7,109,304, which is incorporated herein by reference in its entirety). In another embodiment, the hCAR expression cassette is episomally maintained or integrated into the genome of the recombinant cell or . In certain aspects the expression cassette is comprised in a nucleic acid capable of integration by using an integrase mechanism, a viral vector such as a retroviral or a nonviral vector such as transposon mechanism. In a further embodiment the expression cassette is included in a transposon based nucleic acid. In a particular embodiment, the expression cassette is part of a two component Sleeping Beauty (SB) or piggyBac system that utilizes a transposon and transposase for enhanced non-viral gene transfer.

[0015] Recombinant hCAR expressing cells can be numerically expanded to clinically- meaningful numbers. One example of such expansion uses artificial antigen presenting cells (aAPC). Recombinant h9CAR expressing cells can be verified and identified by flow cytometry and western blot analyses. Recombinant hCAR expressing T cells, expressing a CD19-specifϊc CAR can recognize and kill CD 19 expressing target cells. In a further aspect, hCAR can be expressed into Universal cells, that can be infused across transplantation barriers, to help prevent immunogenicity. The hCAR can be used along with human genes for imaging (such as by positron emission tomography, PET) and conditional ablation of T cells, in the event of cytotoxicity. The recombinant cells of the invention can be used in CD19-specifϊc cellular therapies.

[0016] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. The embodiments in the Example section are understood to be embodiments of the invention that are applicable to all aspects of the invention.

[0017] The terms "inhibiting," "reducing," or "prevention," or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.

[0018] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0019] It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0020] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0021] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

[0022] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0023] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications

within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0025] FIG. 1 Schematic representation of the construction of the CD 19 specific hCAR. (A) Diagram showing the various components of the codon optimized hCAR. (B) Pictorial flow chart of the generation of the transposon vector hCD 19RCD28mZ (CoOp)/PT-MNDU3. (C) Integrity of the final transposase vector hCD19RCD28mZ (CoOp)/PTMNDU3 as determined by the restriction enzyme digestion analysis; Lane 1, 1 Kb ladder; Lane 2, PvuII digestion generates fragments of 674 bp, 954 bp, 1293 bp, 1514 bp, and 2364 bp.

[0026] FIG. 2 Characterization of hCAR expression after electro-transfer of SB plasmid system. (A) Expression of hCAR on CD4 ⁺ and CD8 ⁺ T cells after electro-transfer of SB transposon and transposase at 24 h and 28 days (4 weeks) of co-culture on gamma-irradiated K562-derived aAPC expressing tCD19, IL-15, 4-1BBL. (B) Western blot analysis of hCAR expression detected by mAb specific for CD3-zeta. Whole-cell protein (20 microgram) lysates from primary T cells genetically modified with hCoOpCD19RCD28, Transposase alone (lane 1), Transposon and Trasposase showing ~75 KDa chimeric protein), Transposon alone (lane 3), CD19R CD28 ⁺ Jurkat cells (lane 4 showing ~75 KDa chimeric protein) or no DNA control parental Jurkat (lane 5) were resolved by SDS-PAGE under reducing conditions.

[0027] FIG. 3 Redirected specificity of T cells genetically modified with hCAR. Killing of CD19 ⁺ target cells (HLA class I/II neg K562 parental cells transfected to express firefly luciferase and those transfected to express truncated CD 19 and firefly luciferase) in a 4 hour bioluminescence assay. Background lysis of CD19neg (parental K562 expressing luciferase) cells is shown at each E:T ratio is shown.

[0028] FIG. 4 Shows an optimized human CD 19 sequence.

[0029] FIG. 5 Expression of hCD19RCD28 (a CD19-specifϊc CAR expressing an all- human sequence. (A) Expansion of hCAR ⁺ T cells as assessed 1 and 21 days after electroporation and co-culture on γ - irradiated K562-aAPC (expressing CD19, CD64, CD86, 4- IBBL, mIL-15. (B) Specific lysis of CD 19+ tumor targets by hCAR ⁺ compared with hCARneg (no DNA) T cells. (C) Release of perform by hCAR ⁺ when compared to hCARneg (no DNA) control T cells.

[0030] FIG. 6 Sleeping Beauty expression vectors. (A) CoOphCD19RCD28/pT-pSBSO (Transposon). EFl-α promoter; CoOphCD19RCD28, codon-optimized hCD19RCD28 CAR; IR/DR, SB-inverted/direct repeats; bGh pAn, polyadenylation signal from bovine growth hormone; KanR, Kanamycin resistant gene. (B) pCMV-SBl l (Transposase). SBI l, SBI l- transposase; CMV promoter, cytomegalovirus (CMV) enhancer/promoter; SV40 pAn, polyadenylation signal from SV40.

[0031] FIG. 7 Schematic of electroporation of SB plasmids and propagation of hCAR ⁺ T cells on K562-aAPC.

[0032] FIG. 8 Schematic of one example of a hCAR depicting various amino acid segments of the chimeric protein.

DETAILED DESCRIPTION OF THE INVENTION

[0033] Hematopoietic stem-cell transplantation with ex vzvo-expanded donor-derived tumor-specific T cells can be used to augment the graft-versus-leukemia (GVL) and to reduce the incidence of leukemic relapse without exacerbating graft- versus-host disease. Genetically modified peripheral- and umbilical cord blood-derived T cells rendered specific for CD 19, a molecule constitutively expressed on B-cell malignancies can augment GVL-effect. A redirected specificity has been achieved by expressing a chimeric antigen receptor (CAR) that recognizes CD 19 via the scFv of a murine CD19-specific monoclonal antibody (mAb) fused to T-cell activation endodomains (US publication 2004/0126363, which is incorporated herein by reference in its entirety). However, T cells bearing immunogenic transgenes, such as those coding for mouse/rodent proteins, bacterial selection genes, or viral suicide genes, are targeted by the recipient's immune system and deleted resulting in a limited persistence of the recombinant cells in a target subject.

[0034] The present invention provides additional methods and compositions for production of recombinant T cell expressing a human CD19-specific CAR encoded by all-human transgenes to reduce the risk of clearance of adoptively transferred genetically modified T cells due to a host-versws-graft immune-mediated reaction. Primary human T cells or progenitor cells can be modified with a hCD19CAR transgene. In certain aspects, the expression cassette encoding hCD19CAR can be comprised in a transposon and integrated in the genome of a target cell by transposition, for example transposition can be accomplished using Sleeping Beauty (SB) or similar transposition. Recombinant T cells can be produced, for example, by co-culture with artificial antigen presenting cells expressing CD 19 antigen and co-stimulatory molecules, resulting in expansion and stable expression of hCD19CAR without the need for concomitant drug selection. The hCD19CAR positive T cells can be detected by flow cytometry and Western blot analysis and demonstrated specific lysis of

+ CD 19 tumor targets, as well as other method known in the art.

I. HUMAN CD19-SPECIFIC CHIMERIC ANTIGEN RECEPTOR (hCAR) [0035] The application of T-cell therapy as a treatment modality for B cell malignancy has been limited by the paucity of molecularly-defmed tumor antigens capable of eliciting a robust T-cell response and the difficulty of isolating these T cells from a tumor-bearing host. The most robust and successful example of T-cell therapy occurs after allogeneic hematopoietic stem-cell transplantation (HSCT) where the engrafted donor-derived T cells recognize recipient tumor-associated alloantigens in the context of major histocompatibility complex (MHC). However, the graft- versus-leukemia (GVL)-effect for acute lymphoblastic leukemia (ALL) after allogeneic-HSCT is incomplete resulting in relapse as the major cause of mortality. To augment the GVL-effect and potency of T cell immunotherapy, the inventors developed a human chimeric antigen receptor (CAR) to redirect the specificity of T cells for CD 19. Typically, CD 19 is expressed on B-lineage ALL (B-ALL) and other malignant B cells. T cells can be genetically modified to recognize (CD 19 ) B-ALL blasts and other malignant B cells independent of MHC and despite the absence of T-cell costimulatory molecules on leukemic cells.

[0036] Chimeric antigen receptors (CARs) are designed for adoptive immunotherapy by connecting an extracellular antigen-binding domain to a transmembrane domain and an intracellular signaling domain (endodomain). Chimeric antigen receptors have been described in U.S. Patent 5,359,046, including functional antigen-specific receptors generated

in B cells (Sanchez et al, J. Exp. Med., 178:1049-1055 (1993)) and T cells (Burkhardt et al, MoI. Cell. Biol, 14:1095-1103 (1994)) by fusing the Igα and Igβ signal transduction chains to IgM.

[0037] It is a promising anti-tumor approach to eradicate tumor cells by adoptive transfer of T cells expressing chimeric antigen receptors to recognize specific antigens presented on tumor cells and activate T cells to specifically lyse these tumor cells. A critical aspect of this chimeric antigen receptor strategy is the selection of target epitopes that are specifically or selectively expressed on tumor, are present on all tumor cells, and are membrane epitopes not prone to shed or modulate from the cell surface.

[0038] CD 19, a cell surface glycoprotein of the immunoglobulin superfamily, is a potentially attractive target for antibody therapy of B cell-associated malignancies. This antigen is absent from hematopoietic stem cells, and in healthy individuals its presence is exclusively restricted to the B-lineage and possibly some follicular dendritic cells (Scheuermann, et al (1995) Leuk Lymphoma 18, 385-397). In fact, it is present on B cells from earliest recognizable B-lineage cells during development to B-cell blasts but is lost on maturation to plasma cells. Furthermore, CD 19 is not shed from the cell surface and rarely lost during neoplastic transformation (Scheuermann, 1995). The protein is expressed on most malignant B-lineage cells, including cells from patients with chronic lymphocytic leukemia (CLL), Non-Hodgkin lymphoma (NHL), and acute lymphoblastic leukemia (ALL) (Uckun, et al (1988) Blood 71, 13-29). CD19 primarily acts as a B cell co-receptor in conjunction with CD21 and CD81. Upon activation, the cytoplasmic tail of CD 19 becomes phosphorylated which leads to binding by Src-family kinases and recruitment of PI-3 kinase.

[0039] Endowing T cells or NK cells with tumor specificity by gene transfer of cDNA constructs encoding engineered antigen receptors is one strategy for generating tumor- reactive cytolytic T cells (CTL) for therapy. (Weiss et al, Semin. Immunol. 3:313, 1991; Gross et al, supra; Hedrick et al, Int. Rev. Immunol. 10:279, 1993). Cytolytic T cells (CTL) are immunologic effector cells that have the capacity to specifically recognize and directly lyse target cells (Henckart, Semin. Immunol. 9:85, 1997). Re-infusion oϊ ex vivo expanded tumor-specific CD8 ⁺ CTL clones can mediate tumor eradication as demonstrated in animal model systems (Greenberg, Adv. Immunol. 49:281,1991). A growing number of genes encoding proteins expressed by human tumors that elicit T cell responses have been identified by expression cloning technologies (Robbins et al, Current Opin. Immunol. 8:628,

1996; De Plaen et al, Methods 12:125, 1997). The feasibility of isolating T cells from cancer patients with specificity for these molecularly defined tumor antigens is currently being evaluated but remains a significant challenge to the clinical application of adoptive T cell therapy for malignant disease (Yee et al, J. Immunol. 157:4079, 1996).

[0040] Chimeric antigen receptor molecules are distinguished by their ability to both bind antigen and transduce activation signals via immunoreceptor activation motifs (IAM's) present in their cytoplasmic tails. Receptor constructs utilizing an antigen-binding moiety generated from single chain antibodies (scFv) afford the additional advantage of being "universal" in that they bind native antigen on the target cell surface in an HLA-independent fashion. Several laboratories have reported on scFv constructs fused to sequences coding for the intracellular portion of the CD3 complex's zeta chain (ζ), the Fc receptor gamma chain, and sky tyrosine kinase (Eshhar et al, supra; Fitzer-Attas et al, J. Immunol. 160:145, 1998). Re-directed T cell effector mechanisms including tumor recognition and lysis by CTL have been documented in several murine and human antigen-scFv:ζ systems (Eshhar, Cancer Immunol. Immunother. 45:131, 1997; Altenschmidt et al, J. MoI. Med. 75:259, 1997; Brocker et al, Adv. Immunol. 68:257, 1998.

[0041] To date non-human antigen binding regions are typically used in constructing a chimeric antigen receptor. A potential problem with using non-human antigen binding regions, such as murine monoclonal antibodies, is the lack of human effector functionality and inability to penetrate into tumor masses. In other words, such antibodies may be unable to mediate complement-dependent lysis or lyse human target cells through antibody- dependent cellular toxicity or Fc-receptor mediated phagocytosis to destroy cells expressing CAR.. Furthermore, non-human monoclonal antibodies can be recognized by the human host as a foreign protein and, therefore, repeated injections of such foreign antibodies can lead to the induction of immune responses leading to harmful hypersensitivity reactions. For murine -based monoclonal antibodies, this is often referred to as a Human Anti-Mouse Antibody (HAMA) response. Therefore, the use of human antibodies is more preferred because they do not elicit as strong a HAMA response as murine antibodies. Similarly, the use of human sequences in the CAR can avoid immune-mediated recognition and therefore elimination by endogenous T cells that reside in the recipient and recognized processed antigen in the context of HLA.

[0042] To minimize immunogenicity to xenogenic parts of previously developed CD 19- specific chimeric antigen receptors, certain aspects of the present invention provide a chimeric antigen receptor to redirect the specificity of T cells for CD 19 expressed on malignant B cells encoded by all human genes for the treatment of CD 19 associated diseases such as B cell lymphomas and leukemias and autoimmune disorders in humans and other mammals without the adverse responses associated with using murine-derived chimeric antigen receptors. The polypeptides thereof of the present invention can be used alone, conjugated to at least one therapeutic agent or in combination with other treatment modalities, for example, chemotherapy, radiotherapy and hematopoietic stem-cell transplantation. As used herein, the term "antigen" is a molecule capable of being bound by an antibody or T-cell receptor. An antigen is additionally capable of inducing a humoral immune response and/or cellular immune response leading to the production of B- and/or T- lymphocytes. The structural aspect of an antigen that gives rise to a biological response is referred to herein as an "antigenic determinant." B-lymphocytes respond to foreign antigenic determinants via antibody production, whereas T-lymphocytes are the mediator of cellular immunity. Thus, antigenic determinants or epitopes are those parts of an antigen that are recognized by antibodies, or in the context of an MHC, by T-cell receptors. An antigenic determinant need not be a contiguous sequence or segment of protein and may include various sequences that are not immediately adjacent to one another.

II. hCAR NUCLEIC ACIDS AND ENCODED POLYPEPTIDES

[0043] The present invention involves nucleic acids, including nucleic acids encoding a human CD19-specific chimeric T cell receptor (hCAR) polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human CD 19 binding region or domain. In certain embodiments the binding region can comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and/or antigen binding fragment thereof. A complementarity determining region (CDR) is a short amino acid sequence found in the variable domains of antigen receptor (e.g., immunoglobulin and T cell receptor) proteins that complements an antigen and therefore provides the receptor with its specificity for that particular antigen. Each polypeptide chain of an antigen receptor contains three CDRs (CDRl, CDR2 and CDR3). Since the antigen receptors are typically composed of two polypeptide chains, there are six CDRs for each antigen receptor that can come into contact with the antigen — each heavy and light chain contains three CDRs. Since most

sequence variation associated with immunoglobulins and T cell receptors are found in the CDRs, these regions are sometimes referred to as hypervariable domains. Among these, CDR3 shows the greatest variability as it is encoded by a recombination of the VJ (VDJ in the case of heavy chain) regions.

[0044] It is contemplated that the CD 19 human CAR nucleic acids are human genes to enhance the persistence of recombinant cells and enhance cellular immunotherapy for human patients.

[0045] In a specific embodiment, the invention includes a full length hCD19CAR cDNA or coding region, designated SEQ ID NO:3. The CD 19 binding regions or domain can comprise a fragment of the V _H and V _L chains of a single-chain variable fragment (scFv) derived from a human monoclonal antibody to CD19, such as those described in U.S. Patent 7,109,304. The fragment can also be any number of different CD 19 binding domains of a human CD 19- specifϊc antibody. In a more specific embodiment, the fragment is a CD19-specifϊc scFv encoded by a sequence (SEQ ID NO:3) that is optimized for human codon usage for expression in human cells.

[0046] The arrangement could be multimeric such as a diabody or multimers. The multimers are most likely formed by cross pairing of the variable portion of the light and heavy chains into what has been referred to by Winters as a diabody. The hinge portion of the construct can have multiple alternatives from being totally deleted, to having the first cysteine maintained, to a proline rather than a serine substitution, to being truncated up to the first cysteine. The Fc portion can be deleted, although there is data to suggest that the receptor preferably extends from the membrane. Any protein which is stable and dimerizes can serve this purpose. One could use just one of the Fc domains, e.g., either the CH2 or CH3 domain from human immunoglobulin. One could also use the hinge, CH2 and CH3 region of a human immunoglobulin that has been modified to improve dimerization.

[0047] The intracellular signaling domain of the chimeric receptor of the invention is responsible for activation of at least one of the normal effector functions of the immune cell in which the chimeric receptor has been placed. The term "effector function" refers to a specialized function of a differentiated cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Effector function in a memory or memory-type T cell includes antigen-dependent proliferation. Thus the term

"intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain will be employed, in many cases it will not be necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain may find use, such truncated portion may be used in place of the intact chain as long as it still transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal. Examples include the zeta chain of the T cell receptor or any of its homologs (e.g., eta, delta, gamma or epsilon), MBl chain, B29, Fc RIII, Fc RI and combinations of signaling molecules such as CD3ζ and CD28, 4-1BB, OX40 and combination thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the families of activating proteins can be used, such as FcγRIII and FcεRI. See Gross et al, Stancovski et al, Moritz et al, Hwu et al, Weijtens et al, and Hekele et al, for disclosures of cTCR's using these alternative transmembrane and intracellular domains. In a preferred embodiment, the human CD3ζ intracellular domain was taken for activation.

[0048] The CD19-specific extracellular domain and the intracellular signaling-domain are linked by a transmembrane domain such as the human IgG ₄ Fc hinge and Fc regions. Alternatives include the human CD4 transmembrane domain, the human CD28 transmembrane domain, the transmembrane human CD3ζ domain, or a cysteine mutated human CD3ζ domain, or other transmembrane domains from other human transmembrane signaling proteins such as CD 16 and CD8 and erythropoietin receptor.

[0049] More preferably, the CD 19 human CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and a modified CD28 intracellular signaling domain. Other costimulatory receptors include, but not limited to one or more of CD28, OX-40, DAPlO, and 4-1BB. In addition to a primary signal initiated by CD3ζ, an additional signal provided by a human costimulatory receptor inserted in a human CAR is important for fully activation of T cells and could help improve in vivo persistence and the therapeutic success of the adoptive immunotherapy.

[0050] The invention is described herein primarily with reference to the specific human CD19RCD28 and receptor of SEQ ID NOs :1 and 2, but the invention is not limited to that

specific construct and receptor. The present invention also encompasses natural variants of strains that have slightly different nucleic acid sequences but encode a CAR comprising a human-derived CD 19 monoclonal antibody or fragment thereof. The invention also includes nucleic acids of CD 19 human CAR comprising a sequence encoding a derivative of a human CD 19 monoclonal antibody or fragment thereof with minimal amino acid changes, but that possess the same activity. The invention also includes nucleic acids of a CD19-specific CAR derived from a rodent CD19-specific antibody in which the antigen regions were swapped for human motifs. The invention also includes nucleic acids of a CD19-specific CAR derived from a rodent CD19-specific antibody which was de -immunized using empiric approaches and in silica algorithms to reduce immunogenicity in primates.

[0051] In particular embodiments, the invention concerns isolated nucleic acid segments and expression cassettes incorporating DNA sequences that encode hCD19CAR. Vectors of the present invention are designed, primarily, to deliver desired genes to immune cells, preferably T cells under the control of regulated eukaryotic promoters, for example, MNDU3 promoter or ELFlapha promoter, or Ubiquitin promoter.. Also, the vectors may contain a selectable marker if, for no other reason, to facilitate their manipulation in vitro.

III. METHODS AND COMPOSITIONS RELATED TO hCD19CAR

[0052] In certain aspects, the invention includes a method of making and/or expanding the CD19-specific redirected T cells which comprises transfecting T cells or NK cells, or cells derived from hematopoietic stem/progenitor cells, with an expression vector containing a DNA construct encoding the hCAR, then stimulating the cells with CD19 ⁺ cells, recombinant CD 19, or an antibody to the receptor to cause the cells to proliferate.

[0053] In another aspect, this invention is a method of stably transfecting and re-directing T cells by electroporation, or other non-viral gene transfer (such as, but not limited to sonoporation) using naked DNA. Most investigators have used viral vectors to carry heterologous genes into T cells. By using naked DNA, the time required to produce redirected T cells can be reduced. "Naked DNA" means DNA encoding a chimeric T cell receptor (TCR) contained in an expression cassette or vector in proper orientation for expression. The electroporation method of this invention produces stable transfectants which express and carry on their surfaces the chimeric TCR (cTCR).

[0054] "Chimeric TCR" means a receptor which is expressed by T cells and which comprises intracellular signaling, transmembrane and extracellular domains, where the extracellular domain is capable of specifically binding in an MHC unrestricted manner an antigen which is not normally bound by a T cell receptor in that manner. Stimulation of the T cells by the antigen under proper conditions results in proliferation (expansion) of the cells and/or production of IL-2. The CD19-specific chimeric receptor of this invention is an example of a chimeric TCR. However, the method is applicable to transfection with chimeric TCRs which are specific for other target antigens, such as chimeric TCRs that are specific for HER2/Neu (Stancovski et al., supra), ERBB2 (Moritz et al., supra), folate binding protein (Hwu et al., supra), renal cell carcinoma (Weitjens et al., supra), and HIV-I envelope glycoproteins gpl20 and gp41 (Roberts et al, Blood 84:2878, 1994).

[0055] In certain aspects, the T cells or NK cells are primary human T cells, such as T cells derived human peripheral blood mononuclear cells (PBMC), PBMC collected after stimulation with G-CSF, bone marrow or umbilical cord blood. Conditions include the use of mRNA and DNA and electroporation. Following transfection the cells may be immediately infused. In certain aspects, following transfection, the cells may be propagated for days, weeks, or months ex vivo as a bulk population within about 1, 2, 3, 4, 5 days or more following gene transfer into cells. In a further aspect, following transfection, the transfectants are cloned and a clone demonstrating presence of a single integrated or episomally maintained expression cassette or plasmid, and expression of the chimeric receptor is expanded ex vivo. The clone selected for expansion demonstrates the capacity to specifically recognize and lyse CD 19 expressing target cells. The recombinant T cells or T cell precursor may be expanded by stimulation with IL-2, or other cytokines that bind the common gamma- chain {e.g., IL-7, IL-15, IL-21 and others). The recombinant T cells or T cell precursor may be expanded by stimulation with artificial antigen presenting cells. The recombinant T cells may be expanded on artificial antigen presenting cell or with an antibody, such as OKT3, which cross links CD3 on the T cell surface. Subsets of the recombinant T cells may be deleted on artificial antigen presenting cell or with an antibody, such as Campath, which binds CD52 on the T cell surface. In a further aspect, the genetically modified cells may be cryopreserved.

[0056] This invention also represents the targeting of a B cell malignancy or disorder including B cells, with the cell-surface epitope with CD19-specific using a redirected

immune cell, preferably T cells. Malignant B cells are an excellent target for redirected T cells, as B cells can serve as immunostimulatory antigen-presenting cells for T cells. Preclinical studies that support the anti-tumor activity of adoptive therapy with donor- derived CD19-specific T cells bearing a human or humanized CAR include, (i) redirected killing of CD19 ⁺ targets, (ii) redirected secretion/expression of cytokines after incubation with CD19 ⁺ targets/stimulator cells, (iii) sustained proliferation after incubation with CD19 ⁺ targets/stimulator cells.

[0057] Cytokine production by the CD19-specifϊc scFvFc:ζ expressing Jurkat clones when co-cultured with CD19 ⁺ B-cell malignancy does not require the addition of professional antigen presenting cells to culture or pharmacologic delivery of a co-stimulatory signal by the phorbal ester PMA. The function of the CD19R:ζ chimeric immunoreceptor in T cells was first assessed by expressing this scFvFc:ζ construct in primary human T cell clones. Clones secrete cytokines (IFN-γ, TNF-α, and gm-CSF) specifically upon co-culture with human CD19 ⁺ leukemia and lymphoma cells. Cytokine production by CD19-specific clones can be blocked in part by the addition to culture of the anti-CD 19 specific antibody HIB 19. Anti- CD20 antibody does not block cytokine production thereby demonstrating the specificity of the CD19R:ζ chimeric immunoreceptor for CD 19 on the tumor cell surface. Typically, genetically modified T cells display high levels of cytolytic activity in standard 4-hr chromium release assays against human CD19 ⁺ leukemia and lymphoma cell lines cell lines and do not kill other tumor lines that are devoid of CD 19. These preclinical studies support the anti-tumor activity of adoptive therapy with donor-derived hCAR-expressing T cell clones in patients that relapse following HLA-matched allogeneic bone marrow transplantation.

[0058] CD 19 is not tumor-specific and adoptive transfer of cells with this specificity is expected to kill the subset of non-transformed B cells which express CD 19. Although CD 19 is not expressed by hematopoietic stem cells or mature plasma cells, this cross-reactivity may exacerbate the humoral immunodeficiency of patients receiving chemotherapy and/or radiotherapy. Another aspect of the invention includes equipping recombinant T cells with a suicide gene such as the human thymidine kinase gene and herpes virus thymidine kinase gene and derivatives allows for in vivo ablation of transferred cells following adoptive transfer with pharmacologic doses of gancyclovir and is a strategy for limiting the duration or in vivo persistence of transferred cells.

[0059] hCAR-expressing T cells of this invention can be used to treat patients with CD19 ⁺ B-cell malignancies and B-cell mediated autoimmune diseases, including for example, acute lymphoblastic leukemia. High relapse rates observed following autologous transplantation for leukemia can be reduced with post-transplant in vivo treatment with adoptively transferred CD19-specifϊc redirected T cells to purge CD19 ⁺ leukemic stem cells. CD19-specifϊc redirected T cells can be used to treat lymphoma patients with refractory or recurrent disease. The CD19 ⁺ redirected T cells can be administered following lymphodepleting therapy and myeloablative chemotherapy and stem cell rescue, when tumor burden and normal CD19 ⁺ cell burden are at a nadir.

[0060] Patients can be treated by infusing therapeutically effective doses of CD8 CD19- specific redirected T cells in the range of about 10 ⁴ to 10 ¹² or more cells per square meter of body surface (cells/m ). The infusion will be repeated as often and as many times as the patient can tolerate until the desired response is achieved. The appropriate infusion dose and schedule will vary from patient to patient, but can be determined by the treating physician for a particular patient. IL-2 and IL-7 can be co-administered to expand infused cells post- infusion.

[0061] The dosing schedule may be based on an alternate continuous infusion strategy. CD19-specific redirected T cells can be administered as a strategy to support CD8 ⁺ cells as well as initiate/augment a Delayed Type Hypersensitivity response against CD 19+ target cells. The dosing may include giving chemotherapy or antibody therapy with the T cells.

[0062] It is known that chimeric immune receptors are capable of activating target-specific lysis by phagocytes, such as neutrophils and NK cells. Thus the present invention also contemplates the use of chimeric T-cell receptor DNA to transfect into non-specific immune cells including neutrophils, macrophages and NK cells. Furthermore, the present invention contemplates the use of chimeric T-cell receptor DNA to transfect stem cells prior to stem cell transplantation procedures.

[0063] The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art.

IV. IMMUNE SYSTEM AND IMMUNOTHERAPY

[0064] In one embodiment of the present invention, B cell lineage malignancy or disorder is treated by transfer of a redirected T cell of eliciting a specific immune response. Thus, a basic understanding of the immunologic responses is necessary.

[0065] The cells of the adaptive immune system are a type of leukocyte, called a lymphocyte. B cells and T cells are the major types of lymphocytes. B cells and T cells are derived from the same pluripotential hematopoietic stem cells, and are indistinguishable from one another until after they are activated. B cells play a large role in the humoral immune response, whereas T-cells are intimately involved in cell-mediated immune responses. They can be distinguished from other lymphocyte types, such as B cells and NK cells by the presence of a special receptor on their cell surface called the T cell receptor (TCR). In nearly all other vertebrates, B cells and T-cells are produced by stem cells in the bone marrow. T- cells travel to and develop in the thymus, from which they derive their name. In humans, approximately 1-2% of the lymphocyte pool recirculates each hour to optimize the opportunities for antigen-specific lymphocytes to find their specific antigen within the secondary lymphoid tissues.

[0066] T lymphocytes arise from hematopoietic stem cells in the bone marrow, and migrate to the thymus gland to mature. T cells express a unique antigen binding receptor on their membrane (T-cell receptor), which can only recognize antigen in association with major histocompatibility complex (MHC) molecules on the surface of other cells. There are at least two populations of T cells, known as T helper cells and T cytotoxic cells. T helper cells and T cytotoxic cells are primarily distinguished by their display of the membrane bound glycoproteins CD4 and CD8, respectively. T helper cells secret various lymphokines that are crucial for the activation of B cells, T cytotoxic cells, macrophages and other cells of the immune system. In contrast, a T cytotoxic cells that recognizes an antigen-MHC complex proliferates and differentiates into an effector cell called a cytotoxic T lymphocyte (CTL). CTLs eliminate cells of the body displaying antigen, such as virus infected cells and tumor cells, by producing substances that result in cell lysis. Natural killer cells (or NK cells) are a type of cytotoxic lymphocyte that constitutes a major component of the innate immune system. NK cells play a major role in the rejection of tumors and cells infected by viruses. The cells kill by releasing small cytoplasmic granules of proteins called perform and granzyme that cause the target cell to die by apoptosis.

[0067] B cell identifies pathogens when antibodies on its surface bind to a specific foreign antigen. This antigen/antibody complex is taken up by the B cell and processed by proteolysis into peptides. The B cell then displays these antigenic peptides on its surface MHC class II molecules. This combination of MHC and antigen attracts a matching helper T cell, which releases lymphokines and activates the B cell. As the activated B cell then begins to divide, its offspring (plasma cells) secrete millions of copies of the antibody that recognizes this antigen. These antibodies circulate in blood plasma and lymph, bind to pathogens expressing the antigen and mark them for destruction by complement activation or for uptake and destruction by phagocytes. Antibodies can also neutralize challenges directly, by binding to bacterial toxins or by interfering with the receptors used by viruses and bacteria to infect cells.

[0068] NK-cells or natural killer cells are defined as large granular lymphocytes that do not express T-cell antigen receptors (TCR) or Pan T marker CD3 or surface immunoglobulins (Ig) B cell receptor but that usually express the surface markers CD 16 (FcγRIII) and CD56 in humans, and NKl .1/NKl .2 in certain strains of mice.

[0069] Antigen-presenting cells, which include macrophages, B lymphocytes, and dendritic cells, are distinguished by their expression of a particular MHC molecule. APCs internalize antigen and re-express a part of that antigen, together with the MHC molecule on their outer cell membrane. The major histocompatibility complex (MHC) is a large genetic complex with multiple loci. The MHC loci encode two major classes of MHC membrane molecules, referred to as class I and class II MHCs. T helper lymphocytes generally recognize antigen associated with MHC class II molecules, and T cytotoxic lymphocytes recognize antigen associated with MHC class I molecules. In humans the MHC is refereed to as the HLA complex and in mice the H-2 complex.

[0070] The T cell receptor or TCR is a molecule found on the surface of T lymphocytes (or T cells) that is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. It is a heterodimer consisting of an alpha and beta chain in 95% of T cells, while 5% of T cells have TCRs consisting of gamma and delta chains. Engagement of the TCR with antigen and MHC results in activation of its T lymphocyte through a series of biochemical events mediated by associated enzymes, co- receptors and specialized accessory molecules. In immunology, the CD3 antigen (CD stands for cluster of differentiation) is a protein complex composed of four distinct chains (CD3γ,

CD3δ and two times CD3ε) in mammals, that associate with molecules known as the T cell receptor (TCR) and the ζ-chain to generate an activation signal in T lymphocytes. The TCR, ζ-chain and CD3 molecules together comprise the TCR complex. The CD3γ, CD3δ and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. The transmembrane region of the CD3 chains is negatively charged, a characteristic that allows these chains to associate with the positively charged TCR chains (TCRα and TCRβ). The intracellular tails of the CD3 molecules contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM for short, which is essential for the signaling capacity of the TCR.

[0071] CD28 is one of the molecules expressed on T cells that provide co-stimulatory signals, which are required for T cell activation. CD28 is the receptor for B7.1 (CD80) and B7.2 (CD86). When activated by Toll-like receptor ligands, the B7.1 expression is upregulated in antigen presenting cells (APCs). The B7.2 expression on antigen presenting cells is constitutive. CD28 is the only B7 receptor constitutively expressed on naive T cells. Stimulation through CD28 in addition to the TCR can provide a potent co-stimulatory signal to T cells for the production of various interleukins (IL-2 and IL-6 in particular).

[0072] The strategy of isolating and expanding antigen-specific T cells as a therapeutic intervention for human disease has been validated in clinical trials (Riddell et al, Science 257:238, 1992; Walter et al, N. Engl. J. Med. 333:1038, 1995; Heslop et al, Nat. Med. 2:551, 1996).

[0073] Malignant B cells appear to be an excellent target for redirected T cells, as B cells can serve as immunostimulatory antigen-presenting cells for T cells (Glimcher et al, 1982). Lymphoma, by virtue of its lymph node tropism, is anatomically ideally situated for T cell- mediated recognition and elimination. The localization of infused T cells to lymph node in large numbers has been documented in HIV patients receiving infusions of HlV-specific CD8 ⁺ CTL clones. In these patients, evaluation of lymph node biopsy material revealed that infused clones constituted approximately 2-8% of CD8 ⁺ cells of lymph nodes. Lymph node homing might be further improved by co-transfecting T cells with a cDNA construct encoding the L-selection molecule under a constitutive promoter since this adhesion molecule directs circulating T cells back to lymph nodes and is down-regulated by in vitro expansion (Chao et al, J. Immunol. 159:1686, 1997). The present invention may provide a method of treating a human disease condition associated with a cell expressing endogenous CD 19

comprising infusing a patient with a therapeutically effective dose of the recombinant human CD19-specifϊc CAR expressing cell as described above. The human disease condition associated with a cell expressing endogenous CD 19 may be selected from the group consisting of lymphoma, leukemia, Non-Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, and B cell associated autoimmune diseases.

[0074] Leukemia - Leukemia or leukemia is a cancer of the blood or bone marrow and is characterized by an abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes). It is part of the broad group of diseases called hematological neoplasms. Leukemia is a broad term covering a spectrum of diseases. Leukemia is clinically and pathologically split into its acute and chronic forms.

[0075] Acute leukemia is characterized by the rapid proliferation of immature blood cells. This crowding makes the bone marrow unable to produce healthy blood cells. Acute forms of leukemia can occur in children and young adults. (In fact, it is a more common cause of death for children in the U.S. than any other type of malignant disease). Immediate treatment is required in acute leukemias due to the rapid progression and accumulation of the malignant cells, which then spill over into the bloodstream and spread to other organs of the body. Central nervous system (CNS) involvement is uncommon, although the disease can occasionally cause cranial nerve palsies. Chronic leukemia is distinguished by the excessive build up of relatively mature, but still abnormal, blood cells. Typically taking months to years to progress, the cells are produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood. Chronic leukemia mostly occurs in older people, but can theoretically occur in any age group. Whereas acute leukemia must be treated immediately, chronic forms are sometimes monitored for some time before treatment to ensure maximum effectiveness of therapy.

[0076] Furthermore, the diseases are classified into lymphocytic or lymphoblastic which indicate that the cancerous change took place in a type of marrow cell that normally goes on to form lymphocytes, and myelogenous or myeloid which indicate that the cancerous change took place in a type of marrow cell that normally goes on to form red cells, some types of white cells, and platelets (see lymphoid cells vs. myeloid cells).

[0077] Acute lymphocytic leukemia (also known as Acute Lymphoblastic Leukemia, or ALL) is the most common type of leukemia in young children. This disease also affects adults, especially those aged 65 and older. Chronic lymphocytic leukemia (CLL) most often affects adults over the age of 55. It sometimes occurs in younger adults, but it almost never affects children. Acute myelogenous leukemia (also known as Acute Myeloid Leukemia or AML) occurs more commonly in adults than in children. This type of leukemia was previously called "acute nonlymphocytic leukemia". Chronic myelogenous leukemia (CML) occurs mainly in adults. A very small number of children also develop this disease.

[0078] Lymphoma - Lymphoma is a type of cancer that originates in lymphocytes (a type of white blood cell in the vertebrate immune system). There are many types of lymphoma.

According to the U.S. National Institutes of Health, lymphomas account for about five percent of all cases of cancer in the United States, and Hodgkin's lymphoma in particular accounts for less than one percent of all cases of cancer in the United States. Because the lymphatic system is part of the body's immune system, patients with a weakened immune system, such as from HIV infection or from certain drugs or medication, also have a higher incidence of lymphoma.

[0079] In the 19th and 20th centuries the affliction was called Hodgkin's Disease, as it was discovered by Thomas Hodgkin in 1832. Colloquially, lymphoma is broadly categorized as Hodgkin's lymphoma and non-Hodgkin lymphoma (all other types of lymphoma). Scientific classification of the types of lymphoma is more detailed. Although older classifications referred to histiocytic lymphomas, these are recognized in newer classifications as of B, T or NK cell lineage.

[0080] Autoimmune disease - Autoimmunity is the failure of an organism to recognize its own constituent parts (down to the sub-molecular levels) as "self," which results in an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease. Prominent examples include Coeliac disease, diabetes mellitus type 1 (IDDM), systemic lupus erythematosus (SLE), Sjogren's syndrome, multiple sclerosis (MS), Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, and rheumatoid arthritis (RA).

[0081] Inflammatory diseases, including autoimmune diseases are also a class of diseases associated with B-cell disorders. Examples of autoimmune diseases include, but are not

limited to acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, post-streptococcalnephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitisubiterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis, and fibrosing alveolitis. The most common treatments are corticosteroids and cytotoxic drugs, which can be very toxic. These drugs also suppress the entire immune system, can result in serious infection, and have adverse affects on the bone marrow, liver and kidneys. Other therapeutics that has been used to treat Class III autoimmune diseases to date have been directed against T cells and macrophages. There is a need for more effective methods of treating autoimmune diseases, particularly Class III autoimmune diseases.

V. EXAMPLES [0082] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

EXAMPLE 1 - SLEEPING BEAUTY-MEDIATED GENE TRANSFER OF HCAR TRANSPOSON IN PRIMARY T CELLS AND ITS EXPRESSION

[0083] After using the Nucleofector to import plasmid DNA into quiescent T cells, the inventors observed that PB- derived electroporated T cells readily expressed the CAR

transposon (FIG. 2A). Consistent with earlier observations T cells that were mock electroporated (No DNA) did not show any expression of CAR, 24 hours after electroporation (0.25% on CD4 ⁺ and 0.79 % on CD8 ⁺ ) while cells electroporated with the transposon and transposase expressed CAR (5.97% on CD4 ⁺ and 3.53% on CD8 ⁺ ) (FIG. 2A). These data demonstrate that the Nucleofector technology can introduce SB DNA plasmids into quiescent primary T cells. To select for PB- derived T cells with integrated transposon, the genetically modified cells were co-cultured with γ-radiated aAPC (K562 genetically modified to express tCD19, 4-1BBL, IL-15-Fc) at a ratio of 1 :2 (T cell to aAPC). After 4 weeks of continuous co-culture (γ-radiated aAPC re-added every 7 days) CAR expression in T cells electroporated with transposon and transposase was maintained (3.57 % on CD4 ⁺ and 1.22% on CD8 ⁺ cells) (FIG. 2A). In addition, as shown in FIG. 2B, a 75 kDa band corresponding to the size of the h-CAR was observed when probed with anti-CD3-ζ while no band of similar size was observed in T cells electroporated with no DNA, and either transposon or transposase alone.

EXAMPLE 2 - REDIRECTED FUNCTION OF HCAR+ T CELLS AFTER

ELECTRO-TRANSFER OF SB PLASMIDS

[0084] The T cells stably expressing hCAR were evaluated for redirected killing. The genetically modified T cells were able to lyse CD19 ⁺ targets, and specificity of killing was demonstrated by reduced lysis of CD19neg K562 cells (FIG. 3). The inventors demonstrated a 2-fold increase in specific lysis of CD19 ⁺ K562 at E:T ratio of 25:1 and the specific lysis was several fold higher at E:T ratio 5:1 with lack of killing of CD19neg K562. This is consistent with absence of resident NK-cell function in the culture, as these target cells are sensitive to NK-cell mediated lysis.

EXAMPLE 3 - CONSTRUCTION OF HUMAN CD1RCD28MZ, HUMAN CODON OPTIMIZED, CHIMERIC ANTI-CD19 RECEPTOR.

[0085] Based on the VH and VL sequences of the CD19-specific human antibodies REI, and EU respectively described in patent 7,109,304, a scFv was designed and constructed. A full length scFvFcCD28m:ζ cDNA, designated CD19RCD28mZ, consists of a Kozak consensus ribosome binding sequence, the human GM-CSF receptor alpha-chain leader peptide, REI VL, (GSTSGSGKPGSGEGSTKG) whitlow linker peptide, EU VH, human IgG4 hinge and Fc regions, human CD28 mutated transmembrane, and cytoplasmic domains, and human cytoplasmid CD3ζ (FIG. IA). The cDNA sequence was further human codon optimized and synthesized by Geneart (geneart.com). Correct assembly of the CAR gene

was validated from DNA sequence analyses. Sources of DNA preliminary sequences used to construct 2nd-generation CD19-specifϊc codon optimized CAR sub-components, include Human VH and VL sequences of a antibody specific for human CD 19 Antibodies from REI, and EU respectively; human GM-CSF receptor α chain leader peptide CE7 neuroblastoma line (FHCRC); Human IgG4 Fc (residues 161 - 389 of the mature protein) Andrew Raubitschek; Human CD28 Transmembrane and cytoplasmic domains Cloning (Laurence Cooper lab); Human cytoplasmic CD3-ζ chain (residues 31 - 142 of the mature protein) Jurkat cells; human CD19RCD28mZ CAR was human codon optimized and synthesized entirely by Geneart (Germany).

EXAMPLE 3-GENERATION OF THE TRANSPOSON VECTOR HCD19RCD28MZ

(COOPVPT-MNDU3

Inserting h-CAR

[0086] The Sleeping Beauty transposon DNA plasmid pT-MNDU3-EGFP obtained from Dr. Don Kohn's (CHLA) laboratory was digested with Spel and Nrul resulting in the removal of the EGFP gene from the vector. Spel digestion of the plasmid vector leaves a cohesive 5' end which is complementary to Nhel while the digestion with Nrul results in generation of blunt end at the 3 ' .

[0087] The hCD19RCD28mZ (CoOp)/pEK vector containing a codon optimized human chimeric antigen receptor (CAR) hCD19RCD28mZ (CoOp), synthesized by Genart (Germany) was digested with Spel. The cohesive 5' overhang ends left by Spel digestion were then blunted and the vector was digested further with Nhel thus releasing the CAR fragment having a cohesive termini at the 5' end and blunt end at the 3'.

[0088] The CAR fragment obtained from hCD19RCD28mZ (CoOp)/pEK was ligated into the EGFP-deleted PT-MNDU3 vector to generate hCD19RCD28mZ (CoOp)/PT-MNDU3 vector. The final vector thus expresses the CAR under the control of the MNDU3 promoter. Efficient polyadenilation is facilitated by the BGH polyA signal resulting in high levels of steady-state mRNA. The promoter-CAR-polyA cassette is flanked by the transposon inverted repeats for transposition when using the sleeping beauty system. The plasmid can be propagated in bacteria grown in Ampicillin. A pictorial flow chart for the generation of the final transposon vector hCD19RCD28mZ(CoOp)/PT-MNDU3 is presented on FIG. IB. The

integrity of the final vector was determined by restriction enzymes digestion followed by agarose gel electrophoresis (FIG. 1C).

Cell lines and primary human T cells

[0089] HLAnuii K562 (erythroleukemia) cells were obtained from American Type Culture Collection (Manassas, VA). They were cultured in HyQ RPMI 1640 (Hyclone, Logan, UT) supplemented with 2mM Glutamax-1 (Gibco-Invitrogen, Carlsbad, CA), and 10% heat- inactivated defined fetal calf serum (FCS) (Hyclone), referred to as culture media. Human T cells were isolated by density gradient centrifugation over Ficoll-Paque-Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), from UCB or PB mononuclear cells after consent and were cultured in CM.

Generation of aAPC

[0090] The parental K562 cell line transduced with lentiviral vectors expressing CD64, tCD19, IL- 15 GFP, 4- IBBL, CD86 were a kind gift from Carl June and associates, University of Pennsylvania, Philadelphia. These cells were cultured in HyQ RPMI 1640 (Hyclone, Logan, UT) supplemented with 2mM Glutamax-1 (Gibco-Invitrogen, Carlsbad, CA), and 10% heat inactivated defined fetal calf serum (FCS).

Electroporation and aAPC-T cell co-culture

[0091] On day 0, PB (10 ⁷ ) were suspended in 100 μL of Amaxa Nucleofector solution (CD34 kit, Cat# VPA-1003) and mixed with 5 μg of supercoiled Coθ _P hCD19RCD28/pT- MNDU3 and 5 μg pCMV-SBl l plasmids, transferred to a cuvette, and electroporated (Program U- 14). After a 10 minute room temperature incubation the cells were transferred to a 6-well plate containing 3 to 4 mL incomplete phenol-free RPMI and rested for 2 to 3 hours. The cells were cultured overnight in 6 to 7 mL 10% phenol-free RPMI and stimulated the next day (day 1) with γ-irradiated (100 Gy) aAPC at a 1 :2 T cell/aAPC ratio. The γ- irradiated aAPC were re-added every 7 days. Recombinant human IL-2 (rhIL-2; Chiron, Emeryville, CA) was added to the cultures at 50 U/mL every on a Monday- Wednesday- Friday schedule beginning day 1 of each 7-day expansion cycle. T cells were enumerated every 7 days and viable cells counted based on trypan blue exclusion.

Western Blot

[0092] Expression of the chimeric 75-kDa (CD19RCD28) CD3-ζ was accomplished using a primary mouse anti-human CD3-ζ mAb (1 μg/mL) (BD Biosciences, San Jose, CA) and secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1 :75,000; Pierce, Rockford, IL) under reducing conditions, based on methods previously described (1). Protein lysates were transferred onto nitrocellulose membrane using iBlot Dry Blotting System (Invitrogen) and developed with SuperSignal West Femto Maximum Sensitivity substrate (Pierce) per the manufacturer's instructions and chemiluminiscence was captured after 1- minute exposure using VersaDoc MP 4000 Imaging System (BioRad, Hercules, CA).

Flow cytometry

[0093] Fluorochrome-conjugated reagents were obtained from BD Biosciences (San Jose, CA): anti-CD4, anti-CD8. Affinity purified F(ab')2 fragment of PE-conjugated goat antihuman Fcγ (Caltag Cat # Hl 0104) was used at 1/20 dilution to detect cell surface expression of CD19-specific CAR. Blocking of nonspecific antibody binding was achieved using FACS wash buffer (2% FBS in PBS). Data acquisition was on a FACSCalibur (BD Biosciences) using CellQuest version 3.3 (BD Biosciences). Analyses and calculation of percentage of Fc positive T cells was undertaken using Flojo software version 3.00.007 (Thornhill, Ontario, Canada).

Non-radioactive cytotoxic assay.

[0094] The cytolytic activity of T cells was determined by 4 hour bioluminescence luciferase assay. hCD19-specific T cells were incubated with 5x103 firefly luciferase expressing cells target in a 96-well plate (Costar, Cambridge, MA). After 4 h of incubation at 37o C these cells were added to 20 μl of luciferin substrate aliquoted into each well of a 96- well Opti-plate (the final concentration of luciferin in each well is 0.14 mg/ml). The plate is incubated at 37o C for 10 min. The percentage of specific cytolysis was calculated from the release of luminescence recorded as counts per minute, using a TopCount NXT(Perkin-Elmer Life and Analytical Sciences, Inc., Boston, MA).

EXAMPLE 4-GENERATION AND EXPANSION OF HCAR-EXPRE S SING CELLS

[0095] The hCAR which was cloned into the Sleeping Beauty (SB) transposon is as described above. Primary T cells were modified by non-viral gene transfer using this SB

transposon-mediated system to express hCAR. The inventors observed that hCAR ⁺ T cells expanded on CD19-specifϊc K562 derived artificial antigen presenting cells (aAPC). A 10- fold expansion of CD19-specific T cells was observed in 21 days as opposed to no expansion of cells electroporated without DNA plasmid (FIG. 5A). The cytolytic ability of expanded hCAR modified T cells was evaluated by specific killing of CD19 ⁺ K562 target cells. T cells mock-transfected without DNA had background cytolytic activity compared to T cells expressing the hCAR (FIG. 5B). Further, the ability of these hCAR ⁺ cytotoxic T cells to release perform, a pore-forming protein, was evaluated by intracellular flow cytometry assay. It was observed that about 35% of the hCAR ⁺ cells released perforin when compared to cells transfected without DNA, confirming their ability to mediate lysis of tumor targets (FIG. 5C).

[0096] The inventors subcloned the hCD19RCD28 gene into a SB transposon which has been proposed as a clinical vector as described in FIG. 6. Currently this plasmid is being sequenced to ensure the accuracy of the hCAR transgene. After which, primary T cells will be electroporated with this vector in the presence of transposase by non-viral gene transfer method with this vector and hCAR+ T cells will be propagated and expanded as shown in FIG. 7.

[0097] The hCAR ⁺ T cells will be evaluated for their redirected specificity by a (i) chromium release assay, (ii) release of ThI cytokines like IFN-γ and TNF-α, (iii) Their effector and memory phenotype will be assessed by multi-parameter flow cytometry analysis, (iv) Their TCR-Vβ repertoire will be analyzed by flow cytometry and (v) safety issues will be addressed by analyzing for the presence or absence of SBl 1 transposase by PCR, number of copies of hCAR transgene integrated by fluorescence in-situ hybridization.