Chimeric antigen receptor targeting CD19

Abstract

To provide compositions that can be used in methods for treating B-cell malignancies and that have reduced toxicity and immunogenicity in humans.SOLUTION: A pharmaceutical composition comprises as an active ingredient a host cell expressing a chimeric antigen receptor (CAR) directed against CD19, where the CAR comprises specific 13 types of amino acid sequences.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application is a continuation of U.S. Provisional Patent Application No. 62 / 006,313, filed June 2, 2014, which is incorporated by reference. Claim the benefits of STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with federal support by the National Cancer Institute, National Institutes of Health under Project No. Z01 BC001415. The federal government has certain rights in this invention.

[0002] Incorporation by reference of electronically submitted property The computer readable nucleotide / amino acid sequence listing, filed herewith and identified as follows, is incorporated herein by reference in its entirety: One 58,356 byte ASCII (text) file, named "720755_ST25.TXT," created June 1, 2015. . [Background technology]

[0003] Background of the Invention B-cell malignancies, such as lymphomas and leukemias, arise when the regulation of B-cell differentiation and activation is disrupted. Malignancies of mature B cells include follicular lymphoma, mantle cell lymphoma, and leukemia. lymphoma, Burkitt's lymphoma, multiple myeloma, diffuse large B-cell lymphoma, Hodgkin's lymphoma These include lymphoma, lymphoplasmacytic lymphoma, marginal zone lymphoma, and chronic lymphocytic leukemia (Shaffer et al., Nature Reviews Immunology, 2:920-933 (2002)). , e.g., chemotherapy, therapeutic monoclonal antibodies (e.g., Rituximab (RITUXAN) TM ) and allogeneic stem cell transplantation (alloHSCT) are It does not cure malignancies (e.g., Dreger et al., Leukemia, 21(1):12-17(2007) ;Gribben, JG, Blood, 109(11):4617-4626(2007); and Armitage, JO, Blood, 110(1):29-36 (2007)). In particular, monoclonal antibodies are not therapeutic as single agents, and alloHSCT is associated with high levels of mortality and morbidity (see, e.g., Dreger et al., See, supra, Armitage et al., supra, and McLaughlin et al., Journal of Clinical Oncology, 16(8):2825-2833 (1998).

[0004] T cells can be genetically engineered to express chimeric antigen receptors (CARs), which are fusion proteins composed of an antigen recognition portion and a T cell activation domain (see, e.g., Kershaw et al., supra, Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724(1993), and Sadelain et al., Curr. Opin. Immunol., 21(2):215-223(2009)). B cell lineage Adoptive T cell approaches utilizing CD19-targeting CARs have been developed for various malignancies. (e.g., Jensen et al., Biology of Blood and Marrow Transplantation, 16:1245-1256(2010);Kochenderfer et al., Blood, 116(20):4099-4102(2010);Porter et al., The New England Journal of Medicine, 365(8):725-733(2011);Savoldo et al. al., Journal of Clinical Investigation, 121(5):1822-1826(2011), Cooper et al., Blood, 101(4):1637-1644(2003);Brentjens et al., Nature Medicine, 9(3):279-286(2003);Kalos et al., Science Translational Medicine, 3(95):95ra73(2011);Cheadle et al., Journal of Immunology, 184(4):1885-1896(2010);Brentjens et al., Clinical Cancer Research, 13(18 Pt 1):5426-5435(2007);Kochenderfer et al., Blood, 116(19):3875-3886(2010);Brentjens et al., Blood, 118(18):4817-4828(2011); and Kochenderfer et al., Blood, December 8, 2011 (see publication ahead of print (2012)). The B cell antigen CD19 is expressed is restricted to normal and malignant B cells and has therefore been selected as a target for CARs ( See, e.g., Nadler et al., Journal of Immunology, 131(1):244-250 (1983). .

[0005] One of the drawbacks associated with anti-CD19 CAR therapies reported to date is that they can induce significant toxicity associated with elevated serum cytokine levels. The development of a human anti-mouse immune response is also a potential risk associated with current anti-CD19 CARs containing murine sequences. (See, e.g., Jensen et al., supra; Lamers et al., Blood, 117(1):72-82(2011); and Maus et al., Cancer Immunol Res, 2:112-120(2014)). Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, compositions that can be used in methods for treating B-cell malignancies include those comprising: There is a need for compositions with low toxicity and immunogenicity in humans. The present invention provides such compositions and methods. [Means for solving the problem]

[0007] Brief summary of the invention The present invention provides an isolated or purified chimeric antigen receptor (CAR) against CD19, comprising the sequence No. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. The present invention provides a CAR comprising the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

[0008] Furthermore, the present invention relates to isolated or purified nucleic acid sequences encoding said CARs, vectors comprising such nucleic acid sequences, isolated T cells comprising such vectors, and isolated T cells comprising such single A method for destroying malignant B cells by contacting isolated T cells with a population of malignant CD19-expressing B cells in vivo or ex vivo is provided.

[0009] The present invention also relates to the use of the following elements present in SEQ ID NO: 4 or SEQ ID NO: 9: (i) extracellular spacer an isolated or isolated antibody comprising (i) a transmembrane domain derived from a human CD8α molecule, and (iii) an intracellular T cell signaling domain derived from a human CD28 molecule, a human CD27 molecule, and a human CD3ζ molecule; also provides purified CAR.

[0010] The present invention relates to the following elements present in SEQ ID NO: 10 or SEQ ID NO: 11: (i) extracellular spacer The present invention provides an isolated or purified CAR comprising (i) a transmembrane domain derived from a human CD8α molecule, and (iii) an intracellular T cell signaling domain derived from a human CD28 molecule, a human CD27 molecule, and an FcεRI gamma chain. [Brief explanation of the drawings]

[0011] Brief description of each figure in the drawing [Figure 1] Figure 1 is a graph depicting the results of an experiment illustrating the in vitro survival of T cells expressing the indicated CARs as described in Example 2. At day 7 of culture, the percentages of T cells expressing the indicated CARs were as follows: FMC63-28Z, 71%; FMC63-CD828Z, 88%; and FMC63-CD8BBZ, 87%. [Figure 2] 2A-2D are FACs plot images illustrating the expression of the indicated fully human CARs containing the CD27 intracellular signaling domain on the surface of T cells. The plots are gated on viable CD3+ lymphocytes. [Figure 3] Figures 3A and 3B are FACs plot images illustrating the expression of 47G4-CD828Z CAR on the surface of T cells (Figure 3A) compared to non-transduced controls (Figure 3B). The plots are gated on viable CD3+ lymphocytes. [Figure 4]Figures 4A and 4B are graphs depicting experimental results illustrating TNF production by T cells expressing FMC63-28Z, FMC63-CD828Z, or FMC63-CD8BBZ CARs in the CD19+ T cell lines CD19-K562 (Figure 3A) and NALM6 (Figure 3B). A standard TNF ELISA was performed to measure the amount of TNF (pg / mL) in the culture supernatant. TNF levels were normalized to the T cell fraction in each culture expressing each CAR. Results show the mean and standard error of the mean of normalized TNF levels from two different donors. [Figure 5] Figure 5 is a graph depicting experimental results illustrating IFNγ production by T cells expressing the 47G4-CD828Z CAR in the CD19+ T cell lines CD19-K562 and NALM6. A549, TC71, and CCRF-CEM are CD19-negative cell lines. [Figure 6-1] Figures 6A and 6B are FACs plots illustrating that T cells transduced with the indicated CARs degranulated in a CD19-specific manner, as measured by upregulation of CD107a. [Figure 6-2] Figures 6C and 6D are FACs plots illustrating that T cells transduced with the indicated CARs degranulated in a CD19-specific manner, as measured by upregulation of CD107a. [Figure 7] Figures 7A-7C are FACs plots illustrating that T cells expressing the indicated CARs can proliferate in response to CD19, as measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence. T cells expressing the indicated CARs were cultured for 4 days with either the CD19+ cell line CD19-K562 (closed curve) or the CD19-negative cell line NGFR-K562 (open curve) in IL-2-free medium. All plots are gated on viable CD3+CAR+ lymphocytes. [Figure 8]Figure 8 is a graph depicting the results of an experiment demonstrating that T cells transduced with the MSGV-FMC63-CD828Z plasmid encoding the FMC63-CD828Z CAR are cytotoxic to primary chronic lymphocytic leukemia (CLL) cells. [Figure 9] Figure 9 is a graph depicting the results of an experiment demonstrating that T cells expressing either the FMC63-28Z CAR or the 47G4-CD8CD28Z CAR reduce NALM6 tumor size in NSG immunodeficient mice. DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description of the Invention The present invention provides an isolated or purified chimeric antigen receptor (CAR), which comprises an antigen recognition portion and a T cell activation portion. The chimeric antigen receptor (CAR) is a CAR that inhibits T cell signaling. or the antigen-binding domain of an antibody (e.g., a single-chain variable CARs are artificially constructed hybrid proteins or polypeptides containing monoclonal antibody fragments (scFv). CARs utilize the antigen-binding properties of monoclonal antibodies to target non-MHC-restricted antigens. Redirect T cell reactivity and specificity to selected targets in a manner MHC-unrestricted antigen recognition confers on T cells expressing CARs the ability to recognize antigens independently of antigen processing, thus bypassing a major tumor escape mechanism. Furthermore, when expressed in T cells, CARs advantageously bind to endogenous T cell receptors (TCRs). ) does not dimerize with the alpha and beta chains of

[0013] "Isolated" means that a substance (e.g., a protein or nucleic acid) is removed from its natural environment. "Purified" means that a given substance (e.g., a protein or nucleic acid) is either removed from nature (e.g., genomic DNA and mRNA) or synthesized. "Purity" refers to increased purity, whether derived from a nucleic acid (e.g., cDNA) and / or amplified under laboratory conditions, where "purity" is a relative term and not "absolute purity." However, it should be understood that nucleic acids and proteins may be formulated with diluents or adjuvants and still be isolated for practical purposes. For example, proteins may be prepared using a diluent or adjuvant that is used to introduce them into cells. When used, it is typically mixed with an acceptable carrier or diluent.

[0014] The CAR of the present invention targets CD19 (B lymphocyte antigen CD19, also known as B4 and CVID3). CD19 contains the antigen recognition moiety. It is expressed exclusively by hematopoietic B lymphocytes and follicular dendritic cells. It is a cell surface molecule expressed by B lineage-restricted antigens. Most pre-B cells, as well as most non-T cell acute lymphoblastic leukemia cells and B cells CD19 is present on type 2 chronic lymphocytic leukemia cells (Tedder and Isaacs, J. Immun., 143:712-717(1989)). CD19, along with CD21 and CD81, primarily functions as a B cell coreceptor (Bradbury et al., J. Immunol., 149(9):2841-2850(1992); Horvath et al., J. Biol. Chem., 273(46):30537-30543(1998); and Imai et al., J. Immunol., 155(3):1229-1239(1995)). Upon activation, the cytoplasmic tail of CD19 is phosphorylated, leading to binding by Src family kinases and recruitment of PI-3 kinase. CD19 has also been shown to interact with other cell signaling proteins, e.g., B These include Lyn tyrosine protein kinase, the major Src kinase in cells (Fujimoto et al., Immunity, 13:47-57(2000)), CD82 (Imai et al., supra), complement receptor 2 (Bradbury et al., supra; and Horvath et al., supra), and VAV2 (Doody et al., EMBO J., 19(22):6173-6184(2000)).

[0015] The CAR of the present invention is an antigen-binding fragment comprising a monoclonal antibody against CD19 or an antigen-binding portion thereof. As used herein, the term "monoclonal antibody" refers to a group of antibodies produced by a single clone of B cells that bind to the same epitope. In contrast, "polyclonal antibodies" refer to antibodies produced by a variety of B cells, It refers to a population of antibodies that bind to various epitopes of the same antigen. The antibody may be a whole antibody or an antibody fragment. A whole antibody typically consists of four polypeptides. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. The regions have the same general structure, and each region is divided into four frames with relatively conserved sequences. The framework regions are bounded by three complementarity-determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody. It is involved in antibody binding.

[0016] The terms "fragment of an antibody," "antibody fragment," "functional fragment of an antibody," and "antigen-binding portion" are used interchangeably herein. one or more fragments or portions of an antibody that are modified and retain the ability to specifically bind to an antigen (See generally, Holliger et al., Nat. Biotech., 23(9):1126-1129(2005)). The antigen recognition portion of the CAR of the present invention can comprise any CD19-binding antibody fragment. Antibody fragments desirably contain, for example, one or more CDRs, variable regions (or portions thereof), Examples of antibody fragments include (i) a Fab flag, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a single antibody fragment. (iv) an Fv fragment consisting of the VL and VH domains of the Fv fragment; single-chain Fvs (scFvs), which are monovalent molecules consisting of two domains (i.e., VL and VH) joined by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16:778 (1998)), and (v) diabodies (dimers of polypeptide chains Thus, each polypeptide chain comprises a VH connected to a VL by a peptide linker, Examples of suitable linkers include, but are not limited to, a linker that is too short to allow pairing between VH and VL on the same polypeptide chain, thereby promoting pairing between complementary domains on different VH-VL polypeptide chains to form a dimeric molecule having two functional antigen-binding sites. Antibody fragments are known in the art and are described in more detail in, for example, U.S. Patent Application Publication No. 2009 / 0093024 A1. In a preferred embodiment, the antigen-recognition portion of the CAR of the present invention comprises an anti-CD19 single-chain Fv (scFv).

[0017] Antigen-binding portions or fragments of monoclonal antibodies can be of any size, so long as the portion binds to CD 19. In this regard, antigen-binding portions or fragments of monoclonal antibodies to CD 19 (also referred to herein as "anti-CD19 monoclonal antibodies") desirably contain between about 5 and 18 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of the foregoing values). It contains one or more CDRs including

[0018] In one embodiment, the CAR of the present invention comprises an anti-CD19 antibody comprising the variable region of an anti-CD19 monoclonal antibody. The antigen recognition portion comprises a variable region of a mouse or human anti-CD19 monoclonal antibody. The anti-CD19 monoclonal antibody may be obtained or derived from a mammal (including, but not limited to, a mouse, a rat, or a human). Preferably, the antigen recognition portion comprises a variable region of a mouse or human anti-CD19 monoclonal antibody. In this regard, the antigen recognition portion comprises a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region of a mouse or human anti-CD19 monoclonal antibody. Preferably, the antigen recognition portion of the CAR of the present invention comprises a variable region of a light chain, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region of a mouse or human anti-CD19 monoclonal antibody. The recognition portion comprises the light chain variable region and the heavy chain variable region of a murine or human anti-CD19 monoclonal antibody. The FMC63 antibody (described in Nicholson et al., Molecular Immunology, 34(16-17):1157-1165 (1997)) is an example of a murine anti-CD19 monoclonal antibody that can be used in the present invention. The variable region of the FMC63 monoclonal antibody is used in CARs being tested in clinical trials. (e.g., Kochenderfer et al., Nature Review Clinical Oncol., 10(5);267-276(2013); Porter et al., New Eng. J. Med., 365(8):725-733(2011); Kalos et al., Science Translational Medicine, 3(95):95ra73(2011); Kochenderfer et al., Blood, 116(20):4099-4102(2010); and Kochenderfer et al., Blood, 119(12):2709-2720(2012)). 47G4 antibody (described in U.S. Patent Application Publication No. 2010 / 0104509) ) is an example of a human anti-CD19 monoclonal antibody that can be used in the present invention.

[0019] In another embodiment, the CAR of the present invention comprises a signal sequence. The signal sequence may be positioned at the amino terminus of the recognition portion (e.g., the variable region of an anti-CD19 antibody). The signal sequence may include any suitable signal sequence. In one embodiment, the signal sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal sequence or a CD8α signal sequence. For example, a CAR of the present invention comprising a mouse anti-CD19 scFv may include a GM-CSF signal sequence, while a CAR of the present invention comprising a human anti-CD19 scFv may include a CD8α signal sequence. .

[0020] In another embodiment, the CAR of the invention comprises an extracellular spacer sequence. The -sequence is a short amino acid sequence that enhances antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2):89-99(2004)), and is the antigen-recognition moiety (e.g., anti-CD19 scFv). and the T cell activation moiety. An extracellular spacer sequence may be placed between any transmembrane tag. In one embodiment, for example, the extracellular spacer sequence is derived from a human CD8α molecule or a human CD28 molecule.

[0021] The CAR of the present invention also comprises a transmembrane domain. The transmembrane domain can be any of the domains known in the art. For example, the transmembrane domain can be obtained or derived from a CD8α molecule or a CD28 molecule. CD8 is a molecule that binds to T cells. It is a transmembrane glycoprotein that acts as a co-receptor for the TCR and is primarily involved in the repair of damaged cells. CD8 is expressed on the surface of cytotoxic T cells. The most common form of CD8 exists as a dimer composed of CD8α and CD8β chains. CD28 is expressed on T cells and provides costimulatory signals necessary for T cell activation. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2). In a preferred embodiment, CD8α and CD28 are of human origin.

[0022] The CARs of the present invention comprise a T cell activation moiety. The T cell activation moiety comprises at least one intracellular (i.e., cytoplasmic) T cell signaling domain (also referred to as a "costimulatory domain"). The most common intracellular T cell signaling domain used in CARs is CD3 zeta (CD3ζ), which associates with TCRs to generate signals and contains immunoreceptor tyrosine-based activation motifs (ITAMs). Preferably, the T cell activation moiety activates multiple (i.e., two or more) intracellular T cells. The intracellular T cell signaling domain includes a signaling domain derived from a CD28 molecule, a CD3 zeta (ζ) molecule or a modified version thereof, or a human high-affinity IgE receptor (FcεRI) gamma chain. , CD27 molecules, OX40 molecules, 4-1BB molecules, or other intracellular signaling molecules known in the art. As mentioned above, CD28 is a T cell marker important for T cell costimulation. 4-1BB, also known as CD137, provides a potent costimulatory signal to T cells. CD27 signals, promotes differentiation, and enhances long-term survival of T lymphocytes. CD27 is a member of the TNF receptor superfamily and is required for the generation and long-term maintenance of T cell immunity. The human high-affinity IgE receptor (FcεRI) is a tetrameric receptor complex consisting of one alpha chain, one beta chain, and two gamma chains linked by disulfide bridges. FcεRI is constitutively expressed on mast cells and basophils and is inducible on eosinophils. In a preferred embodiment, the intracellular T cell signaling domain is of human origin.

[0023] The CAR of the present invention comprises any one of the above transmembrane domains and the above intracellular T cell signaling domain. Any combination of one or more (e.g., 1, 2, 3, or 4) of the following binding domains: For example, the CAR of the present invention may comprise a CD28 transmembrane domain and an intracellular T cell signaling domain of CD28 and CD3ζ. Alternatively, for example, a CAR of the invention may comprise a CD8α transmembrane domain and the intracellular T cell signaling domains of CD28, CD3ζ, FcεRI gamma chain, and / or 4-1BB. In another embodiment, a CAR of the invention may comprise a CD8α transmembrane domain and the intracellular T cell signaling domains of CD28, CD3ζ, and CD27. In yet another embodiment, a CAR of the invention may comprise a CD28 transmembrane domain and the intracellular T cell signaling domains of CD27, 4-1BB, and FcεRI gamma chain.

[0024] The present invention further provides an isolated or purified antibody encoding the chimeric antigen receptor (CAR) of the present invention. Nucleic acid sequences are provided. "Nucleic acid sequence" is intended to encompass polymers of DNA or RNA, i.e., polynucleotides, which can be single- or double-stranded and can contain non-natural or modified nucleotides. As used herein, the terms "nucleic acid" and "polynucleotide" refer to polymeric forms of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and, thus, include double- and single-stranded DNA, as well as double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides, such as, but not limited to, methylated and / or capped polynucleotides.

[0025] The CARs of the invention can contain any number of amino acids, provided that the CAR retains its biological activity (e.g., the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent disease in a mammal). For example, a CAR can contain 50 or more amino acids (e.g., , 60 or more, 100 or more, or 500 or more) amino acids, but may contain fewer than 1,000 amino acids (e.g., 900 or less, 800 or less, 700 or less, or 600 or less). Preferably, the CAR contains from about 50 to about 700 amino acids (e.g., about 70, about 80, about 90, about 150, about 200, about 300, about 400, about 550, or about 650 amino acids), about 100 to about 500 amino acids (e.g., about 125, about 175, about 225, about 250, about 275, about 325, about 350, about 375, about 425, about 450, or about 475 amino acids), or a range defined by any two of the foregoing values.

[0026] The scope of the present invention includes functional portions of the CARs of the invention as described herein. When used with respect to a CAR, the term "functional portion" refers to any of the CARs of the invention. any part or fragment thereof, and the part or fragment is a CAR (the part or fragment thereof) A fragment is a portion of a CAR that retains the biological activity of the parent CAR. The molecule may recognize the target cell to a similar extent, to the same extent, or to a greater extent than the parent CAR, for example. or to detect, treat, or prevent disease. In relation to the nucleic acid sequence encoding the parent CAR, the nucleic acid sequence encoding a functional portion of the CAR comprises, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR. It may encode a protein.

[0027] A functional portion of a CAR may contain additional amino or carboxy termini at the amino or carboxy termini or at both ends of the portion. Preferably, the amino acid sequence of the parent CAR may include amino acids that are not found in the amino acid sequence of the parent CAR. The additional amino acids do not interfere with the biological function of the functional moiety (e.g., recognizing a target cell, detecting cancer, treating or preventing cancer, etc.) More desirably, the additional amino acids enhance the biological activity of the CAR compared to the biological activity of the parent CAR.

[0028] The present invention also provides functional variants of the CAR of the present invention. The term "functional variant" refers to a variant of a CAR of the present invention that has a substantial or significant sequence difference. A CAR, polypeptide or protein having identity or similarity, and the functional variant A functional variant refers to a mutant of CAR that retains the biological activity of CAR (of which the functional variant is a variant). A functional variant may, for example, bind to a target to a similar extent, to the same extent, or to a greater extent than the parent CAR. These include variants of the CARs described herein (parent CARs) that retain the ability to recognize cells. In relation to the nucleic acid sequence encoding the parent CAR, a nucleic acid sequence encoding a functional variant of a CAR may be, for example, about 10% identical, about 25% identical, about 30% identical, or about 50% identical to the nucleic acid sequence encoding the parent CAR. , about 65% identical, about 80% identical, about 90% identical, about 95% identical, or about 99% identical.

[0029] A functional variant can, for example, comprise the amino acid sequence of a CAR of the invention with at least one conservative amino acid substitution. The phrase "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that shares common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York, 1999). (1979)). Such an analysis allows the definition of groups of amino acids that preferentially exchange with each other and are therefore most similar to each other in their effect on the overall structure of the protein (Schulz, GE and Schirmer, RH, supra). Examples of conservative mutations include , amino acid substitutions of amino acids within the same amino acid subgroup, for example, substitution of arginine with lysine and vice versa (so that a positive charge can be maintained); substitution of aspartic acid with glutamic acid and vice versa (so that a negative charge can be maintained); substitution of threonine with serine (so that a free -OH can be maintained); and substitution of asparagine with glutamine (so that a free -NH2 can be maintained). (so that it can be maintained), etc.

[0030] Alternatively or additionally, functional variants have at least one non-conservative amino acid substitution. The amino acid sequence of the parent CAR may include a "non-conservative mutation" that is a change in amino acid position between different groups. Non-conservative amino acid substitutions involve substitutions such as substitutions of tryptophan with lysine or serine with phenylalanine. In this case, it is preferred that the non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions do not improve the biological activity of the functional variant compared to the parent CAR. The biological activity of the functional variant may be enhanced so as to increase the biological activity.

[0031] The CARs of the present invention (including functional portions and functional variants thereof) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, Homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydrogen hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenyl phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylalanine, Dihexylglycine, Indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, Aminomalic acid, Aminomalic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, Ornithine, α-Aminocyclopentanecarboxylic acid, α-Aminocyclohexanecarboxylic acid, α-Aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-Diaminobutyric acid, α,β- Examples include diaminopropionic acid, homophenylalanine and α-tert-butylglycine.

[0032] The CAR of the present invention (including functional portions and functional variants thereof) can be modified by glycosylation, amidation, or the like. , carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted into an acid addition salt, and / or optionally dimerized or polymerized, or conjugated.

[0033] The present invention also provides a CAR (i.e., any antigen recognition site) directed against any target molecule of interest. the extracellular spacer, the transmembrane domain and the intracellular T cell signaling domain In another embodiment, the present invention provides a CAR comprising any one of the T cell signaling domains in any combination. For example, a CAR of the present invention can comprise (i) an extracellular spacer, (i) a transmembrane domain derived from a human CD8α molecule, and (iii) an intracellular T cell signaling domain derived from a human CD3 zeta (CD3ζ) molecule and a human CD28 molecule (as used in the CAR of SEQ ID NO: 1). In another embodiment, a CAR of the present invention comprises (i) an extracellular spacer, (i) a transmembrane domain derived from a human CD8α molecule, and (iii) an intracellular T cell signaling domain derived from a human CD28 molecule, a human CD27 molecule, and a human CD3ζ molecule. In another embodiment, the CAR of the present invention comprises (i) an extracellular spacer, (ii) a transmembrane domain derived from a human CD8α molecule, and (iii) a signaling domain derived from a human CD28 molecule, a human CD27 molecule, and an FcεRI gamma chain. In yet another embodiment, the CAR of the invention comprises (i) an extracellular spacer, (ii) a transmembrane domain derived from a human CD8α molecule, and (iii) a transmembrane domain derived from a human CD28 molecule and an FcεRI gamma chain. The CAR may comprise an intracellular T cell signaling domain that mediates T cell signaling (as used in the CAR of SEQ ID NO: 12).

[0034] In a preferred embodiment, the CAR of the invention comprises or consists of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0035] The CARs of the present invention can be produced using methods known in the art. For example, nucleic acid sequences, polynucleotides, Peptides and proteins can be produced recombinantly using standard recombinant DNA methodologies. (e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994). Additionally, synthetically produced nucleic acid sequences encoding CAR can be used in plants, The nucleic acid sequences may be isolated and / or purified from sources such as bacteria, insects, or mammals (e.g., rats, humans, etc.). Methods of isolation and purification are well known in the art. Alternatively, the nucleic acid sequences described herein may be commercially synthesized. In this regard, the nucleic acid sequences may be synthetic, recombinant, isolated, and / or purified.

[0036] The present invention also provides a vector comprising a nucleic acid sequence encoding a CAR of the present invention. The vector can be, for example, a plasmid, cosmid, viral vector (e.g., retrovirus or adenovirus), or phage. Suitable vectors and methods for preparing vectors are well known in the art (see, e.g., Sambrook et al., supra, and Ausubel et al., supra). (see).

[0037] In addition to the nucleic acid sequence encoding the CAR of the invention, the vector preferably comprises a promoter Expression control sequences include those that provide for expression of a nucleic acid sequence in a host cell, such as enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), etc. Exemplary expression control sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990). It is described in.

[0038] Numerous promoters, including constitutive, inducible, and repressible promoters, from a variety of different sources are well known in the art. Representative sources of promoters include, for example, viral, mammalian, insect, plant, yeast, and bacterial, and suitable promoters from these sources are readily available or can be synthetically produced based on sequences publicly available, for example, from depositories such as the ATCC and other commercial or private sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include, for example, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone inducible system (No et al., Proc. Natl. Acad. Sci., 93:3346-3351(1996)), and the T-REX system. TM (Invitrogen, Carlsbad, CA), LACSWITCH TM system (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al., Nuc. Acid. Res., 27:4324-4327 (1999); Nuc. Acid. Res., 28:e99 (2000); U.S. Patent No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308:123-144 (2005)).

[0039] As used herein, the term "enhancer" refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. They can be located many kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, DNA methylation patterns, or changes in DNA structure. Numerous enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (e.g., from depositories such as the ATCC and other commercial or private sources). Many polynucleotides containing promoters (e.g., the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, within, or downstream of a coding sequence. The term "Ig enhancer" refers to an enhancer element derived from an enhancer region mapped within the immunoglobulin (Ig) locus (such enhancers include, for example, the heavy chain (mu) 5' enhancer, the light chain (kappa) 5' enhancer, the kappa and mu intro enhancer, and the mu enhancer. Examples of such enhancers include nucleotide enhancers, nucleotide enhancers, and 3' enhancers (see generally Paul WE (ed.), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353-363; and U.S. Patent No. 5,885,827).

[0040] A vector may also contain a "selectable marker gene." As used herein, the term "selectable marker gene" refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected or not selected in the presence of a corresponding selection agent. Suitable selectable marker genes are known in the art and are described, for example, in International Patent Application Publications WO 1992 / 08796 and WO 1994 / 28143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77:3567(1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527(1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072(1981); Colberre-Garapin et al., J. Mol. Biol., 150:1(1981); Santerre et al. al., Gene, 30:147(1984); Kent et al., Science, 237:901-903(1987); Wigler et al., Cell, 11:223(1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026(1962); Lowy et al., Cell, 22:817(1980); and U.S. Patent Nos. 5,122,464 and 5,770,359.

[0041] In some embodiments, the vector is an "episomal expression vector" or "episome," which is replicable in a host cell and, under appropriate selective pressure, persists in the host cell as an extrachromosomal segment of DNA (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available episomal expression vectors: Epstein-Barr virus nuclear antigen 1 (EBVN1) These include, but are not limited to, episomal plasmids that utilize the T antigen and SV40 origin of replication in place of EBNA1 and oriP. The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA), and pBK-CMV from Stratagene (La Jolla, CA) are non-limiting examples of episomal vectors that use the T antigen and SV40 origin of replication in place of EBNA1 and oriP.

[0042] Other suitable vectors include those that can be randomly integrated into the DNA of a host cell or that can be expressed. Integrating expression vectors are included, which may contain recombination sites that allow for specific recombination between the vector and the host cell chromosome. Endogenous expression regulatory sequences of the host cell chromosome can be utilized to effect expression of the protein. Examples of vectors that integrate in a site-specific manner include, for example, the flp-in system (e.g., pcDNA 1.0) from Invitrogen (Carlsbad, CA). TM 5 / FRT), or the cre-lox system (e.g., Stratagene (La Jolla, pExchange-6 Core Vectors (e.g., those found in the pExchange-6 Core Vectors of the CA) Examples of vectors that randomly integrate into host cell chromosomes include pcDNA3.1 from Invitrogen (Carlsbad, CA) (when introduced in the absence of T antigen) and pcDNA3.1 from Promega (Madison, CA). pCI or pFN10A(ACT)FLEXI TM Examples include:

[0043] Viral vectors may also be used. Exemplary viral expression vectors include adenovirus-based vectors (e.g., the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)), lentivirus-based vectors (e.g., Life Technologies (Carlsbad, CA) lentivirus-based pLP1), and retrovirus Vectors include, but are not limited to, pFB-ERV plus pCFB-EGSH from Stratagene (La Jolla, Calif.). In a preferred embodiment, the viral vector is a lentiviral vector.

[0044] A vector containing a nucleic acid encoding a CAR of the invention can be introduced into a host cell (including any suitable prokaryotic or eukaryotic cell) capable of expressing the CAR. Preferred host cells are those that can be grown easily and reliably, have reasonably fast growth rates, have well-characterized expression systems, and can be easily and efficiently transformed or transfected.

[0045] As used herein, the term "host cell" refers to any type of cell that can contain an expression vector. Host cells can be eukaryotic cells (e.g., plant, animal, fungus, or algae) or prokaryotic cells (e.g., bacteria or protists). Host cells can be cultured cells or primary cells (i.e., isolated directly from an organism, such as a human). Host cells can be adherent cells or suspension cells (i.e., cells that grow in suspension). Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, and the like. These include human ovary cells, monkey VERO cells, COS cells, and HEK293 cells. For purposes of amplifying or replicating the vector, the host cell may be a prokaryotic cell (e.g., DH5α cell). For purposes of producing a recombinant CAR, the host cell may be a mammalian cell. The host cells are preferably human cells. The host cells can be of any cell type, can be derived from any tissue type, and can be at any stage of development. In one embodiment, the host cells are peripheral blood lymphocytes (PBLs), peripheral blood mononuclear cells (PMBCs), natural killer (NK) cells, or the like. ) or T cells. Preferably, the host cells are T cells. Methods for selecting suitable mammalian host cells, as well as methods for transforming, culturing, amplifying, screening and purifying the cells, are known in the art.

[0046] The present invention provides isolated T cells that express a nucleic acid sequence encoding a CAR of the present invention as described herein. The T cells of the present invention can be any T cell, such as, for example, a cultured T cell (e.g., a primary T cell), or a T cell from a cultured T cell line, or a T cell obtained from a mammal. When obtained from mammals, T cells can be obtained from multiple sources (blood, bone marrow, lymph nodes, thymus, or T cells can be obtained from any tissue, including, but not limited to, other tissues or body fluids. The T cells may also be enriched or purified. The T cells are preferably human T cells (e.g., isolated from a human). The T cells may be at any stage of development, including CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (e.g., Th1 and Th2 cells), CD8 + T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naïve T cells, etc. In one embodiment, the T cells are CD8 + T cells is CD4 + T cells. T cell lines are available from, for example, the American Type Culture Collection (ATCC, Manassas, VA) and the German Collection of Microorganisms and Cell Cultures (DSMZ), and include, for example, Jurkat cells (ATCC TIB-152), Sup-T1 cells (ATCC CRL-1942), RPMI 8402 cells (DSMZ ACC-290), Karpas 45 cells (DSMZ ACC-545), and their derivatives. Derivatives of

[0047] The nucleic acid sequence encoding the CAR of the present invention can be "transfected," "transformed," or As used herein, the terms "transfection," "transformation," or "transduction" refer to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation. (See, for example, Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; These include electroporation; cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)). Phage or viral vectors can be delivered in suitable packages. The infectious particles can be propagated in genomic DNA cells (many of which are commercially available) and then introduced into host cells.

[0048] Without being bound by any particular theory or mechanism, it is believed that the CARs of the present invention, by eliciting an antigen-specific response to CD19, provide one or more of the following: targeting and destruction of CD19-expressing cancer cells, reduction or elimination of cancer cells, and / or immunolocalization to the tumor site(s). Thus, the present invention provides a method for the treatment of malignant B cells by promoting immune cell infiltration and enhancing / extending anti-cancer responses. a method for destroying one or more of the isolated T cells, the method comprising: The present invention provides a method for treating a malignant B cell tumor, comprising contacting a tumor cell with a CAR-binding fragment thereof, whereby a CAR is produced and binds to CD19 on the malignant B cell, resulting in the destruction of the malignant B cell. As discussed above, treatment of B cell malignancies typically involves chemotherapy, therapeutic monoclonal antibodies, and allogeneic stem cell transplantation; however, high relapse rates are common in patients undergoing such treatment. As discussed above, CD19 is highly expressed by malignant B cells (see, e.g., Nadler et al., supra), and the methods of the present invention can be used to treat any B cell malignancy known in the art. Malignant tumors of mature B cells include follicular lymphoma, mantle cell lymphoma, and Burkitt's lymphoma. Lymphoma, multiple myeloma, diffuse large B-cell lymphoma, Hodgkin's lymphoma, These include, but are not limited to, lymphoplasmacytic lymphoma, marginal zone lymphoma, and chronic lymphocytic leukemia (Shaffer et al., supra).

[0049] One or more monoclonal antibodies expressing a nucleic acid sequence encoding the anti-CD19 CAR of the invention described herein. The isolated T cells can be contacted with a population of malignant B cells expressing CD19 ex vivo, in vivo, or in vitro. "Ex vivo" refers to an artificial environment outside the living body that minimizes alteration of natural conditions. "In vivo" refers to a method performed in or on a cell or tissue in an environment. In contrast, the term "in vivo" refers to a method performed within a living organism in its normal, intact state. "In vitro" methods, on the other hand, are performed using biological components that have been isolated from their normal biological environment. The methods of the present invention preferably involve ex vivo and in vivo components. In this regard, for example, the isolated T cells described above may be transfected with a T cell encoding an anti-CD19 CAR of the present invention. They can be cultured ex vivo under conditions for expression of the nucleic acid sequence and then directly transferred into a mammal, preferably a human, suffering from a B-cell malignancy. The cell transplantation method is referred to in the art as "adoptive cell transfer (ACT)." In this transplant, immune-induced cells are passively transferred into a new recipient host, transferring the functionality of the immune-induced donor cells to the new host. Adoptive cell transfer methods for treating various types of cancer, including hematological cancers, such as tumors, are known in the art and are disclosed, for example, in Gattinoni et al., Nat. Rev. Immunol., 6(5):383-393(2006); June, CH, J. Clin. Invest., 117(6):1466-76(2007); Rapoport et al., Blood, 117(3):788-797(2011); and Barber et al., Gene Therapy, 18:509-516(2011)).

[0050] When T cells are administered to a mammal, the cells may be allogeneic or autologous to the mammal. In "autologous" administration methods, cells (e.g., hematopoietic stem cells or lymphocytes) are removed from a mammal, stored (optionally modified), and returned to the same mammal. In "allogeneic" administration methods, a mammal receives cells (e.g., hematopoietic stem cells or lymphocytes) from a genetically similar, but not identical, donor. Preferably, the cells are autologous to the mammal.

[0051] The T cells are desirably administered to the human in the form of a composition, such as a pharmaceutical composition. The nucleic acid sequence encoding a CAR of the present invention, or a vector comprising a nucleic acid sequence encoding a CAR, can be formulated into a composition, such as a pharmaceutical composition, and administered to a human. The pharmaceutical composition of the present invention can comprise a population of T cells expressing a CAR of the present invention. The nucleic acid sequence encoding a CAR of the present invention, or a vector comprising a nucleic acid sequence encoding a CAR, can be formulated into a composition, such as a pharmaceutical composition, and administered to a human. The pharmaceutical composition of the present invention can comprise a population In addition to the host cells expressing the CAR of the invention, the pharmaceutical composition may also contain other pharmaceutically active agents or drugs. The pharmaceutical composition may comprise an agent, for example, a chemotherapeutic agent, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In a preferred embodiment, the pharmaceutical composition comprises an isolated T cell expressing a CAR of the invention, more preferably a population of T cells expressing a CAR of the invention.

[0052] The T cells of the present invention may be provided in the form of a salt (e.g., a pharmaceutically acceptable salt). Pharmaceutically acceptable acid addition salts include those derived from mineral acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, etc.). , nitric acid, and sulfuric acid), and salts derived from organic acids (e.g., tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acids such as p-toluenesulfonic acid).

[0053] The choice of carrier may depend on the particular CAR of the present invention, the nucleic acid sequence encoding the CAR, the vector, or the carrier that expresses the CAR. The specificity of the CAR, nucleic acid sequence encoding the CAR, vector, or host cell expressing the CAR will be determined in part by the host cell in which the CAR is expressed and by the particular method used to administer the CAR, nucleic acid sequence encoding the CAR, vector, or host cell expressing the CAR of the invention. Accordingly, there are many different suitable formulations of pharmaceutical compositions of the present invention. For example, the pharmaceutical composition may include preservatives. Suitable preservatives include, for example, methylparaben, propanediol, and the like. Optionally, a mixture of two or more preservatives may be used. The preservative or mixture thereof is typically is present in an amount of about 0.0001% to about 2% by weight of the total composition.

[0054] In addition, a buffering agent may be used in the composition.Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts.Optionally, a mixture of two or more buffering agents may be used.Buffering agent or mixture thereof is typically present in an amount of from about 0.001% to about 4% by weight of the total composition.

[0055] Methods for preparing administrable (e.g., parenterally administrable) compositions are known to those skilled in the art and are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (2005).

[0056] The present invention also provides a method for the production of a CAR, a nucleic acid sequence encoding the CAR, a vector, or a host cell expressing the CAR. The composition containing the compound can be formulated as an inclusion complex (e.g., a cyclodextrin inclusion complex) or liposome. Liposomes can target host cells (e.g., T cells or NK cells) or target specific tissues. Liposomes can function to target the nucleic acid sequence of the present invention. Liposomes can also be used to increase the half-life of the nucleic acid sequence of the present invention.Many methods for preparing liposomes are available, such as those described in Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980) and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

[0057] The compositions may utilize time-released, delayed release, and sustained release delivery systems so that delivery of the compositions of the present invention occurs prior to sensitization of the area to be treated and in sufficient time to cause sensitization. A variety of release delivery systems are available and known to those skilled in the art, and such systems may be particularly suitable for certain composition embodiments of the present invention, which may avoid repeated administration of the composition, thereby increasing convenience for the subject and the physician.

[0058] The composition desirably comprises a compound according to the invention in an amount effective to treat or prevent B-cell malignancies. A host cell expressing a nucleic acid sequence encoding a CAR of the present invention, or a vector containing such a nucleic acid sequence. As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures the disease and / or adverse symptoms resulting from the disease. To this end, the methods of the invention involve the use of host cells expressing a CAR of the invention, or a "therapeutically effective amount" of a composition comprising a vector comprising a nucleic acid sequence encoding a CAR. "Therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. The therapeutically effective amount may vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the CAR to elicit a desired response in the individual. For example, For example, a therapeutically effective amount of a CAR of the invention can bind to CD19 on multiple myeloma cells and destroy them. The amount of destruction.

[0059] Alternatively, the pharmacological and / or physiological effect can be prophylactic, i.e., the effect completely or partially prevents a disease or its symptoms. In this regard, the methods of the invention comprise administering to a mammal susceptible to a B-cell malignancy a "prophylactically effective amount" of a composition comprising a host cell expressing a CAR of the invention or a vector comprising a nucleic acid sequence encoding a CAR. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (eg, prevention of disease onset).

[0060] A typical amount of host cells administered to a mammal (e.g., a human) can be, for example, in the range of 1 million to 100 billion cells; however, amounts less than or greater than this exemplary range are also contemplated by the present invention. For example, a daily dose of the host cells of the present invention is about 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 50 100 million cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), preferably about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or any of the foregoing values). a range defined by any two of the above), more preferably from about 100 million cells to about 50 billion cells (e.g., , about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells The cell population may be about 10,000,000 cells, about 800,000,000 cells, about 900,000,000 cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, or a range defined by any two of the foregoing values).

[0061] The effectiveness of the treatment or prophylaxis can be monitored by periodic evaluation of the treated patient. Depending on the condition, treatment is repeated for repeated administrations over several days or longer until a desired suppression of disease symptoms occurs. However, other administration regimens may be useful and are within the scope of the invention. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0062] Compositions comprising host cells expressing a CAR of the present invention or vectors comprising a nucleic acid sequence encoding a CAR can be administered to a mammal using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, intrapulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. The compositions are preferably suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, intravaginal, and intraperitoneal administration. More preferably, the compositions are administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0063] A composition comprising a host cell expressing a CAR of the invention, or a vector comprising a nucleic acid sequence encoding a CAR, can be administered with one or more additional therapeutic agents, which are co-administered to a mammal. "Coadministering" refers to the administration of a CAR of the present invention in combination with one or more additional therapeutic agents. or the one or more additional therapeutic agents may be administered to a host cell of the invention or a CAR of the invention in a sufficiently close proximity in time such that the one or more additional therapeutic agents can enhance the effect of the agent or agents, or the one or more additional therapeutic agents can enhance the effect of the CAR of the invention. In this regard, the host cells of the invention or the composition comprising the vector of the invention can be administered first, and the one or more additional therapeutic agents can be administered second, or the one or more additional therapeutic agents can be administered first, and the host cells of the invention or the composition comprising the vector of the invention can be administered second. A composition comprising a vector of the invention can be administered second. Alternatively, a host cell or a composition comprising a vector of the invention can be administered second. The composition comprising the vector of the invention and one or more additional therapeutic agents may be administered simultaneously. An exemplary therapeutic agent that may be co-administered with a host cell or composition comprising a vector of the invention is IL-2.

[0064] When a composition comprising a host cell expressing a CAR of the invention, or a vector comprising a nucleic acid sequence encoding a CAR, is administered to a mammal (e.g., a human), the biological activity of the CAR is enhanced by a method known in the art. According to the method of the present invention, CAR binds to CD19 on the surface of malignant B cells, and the malignant B cells are destroyed. The binding of CAR to CD19 on the surface of malignant B cells is Any suitable method known in the art (including, for example, ELISA and flow cytometry) may be used. The ability of a CAR to destroy malignant B cells can be assayed using any suitable method known in the art, for example, the method described in Kochenderfer et al., J. Immunotherapy, 32(7):689-702(2009) and Herman et al. J. Immunological Methods, 285(1):25-40(2004), etc. The biological activity of a CAR can also be measured by assaying the expression of certain cytokines, such as CD107a, IFNγ, IL-2, and TNF. do.

[0065] Those skilled in the art will readily appreciate that the CARs of the present invention can be modified in any number of ways to increase the therapeutic or prophylactic efficacy of the CAR through such modifications. For example, the CAR can be directly The compound (e.g., CAR) can be conjugated to a targeting moiety either directly or indirectly via a linker. The practice is known in the art, see, e.g., Wadwa et al., J. Drug Targeting 3:111 (1995) and U.S. Patent No. 5,087,616.

[0066] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. [Example]

[0067] Example 1 This example demonstrates methods for generating anti-CD19 chimeric antigen receptors (CARs) of the invention.

[0068] A series of anti-CD19 CARs were designed and synthesized. All CARs were engineered using either the murine monoclonal antibody FMC63 (Nicholson et al., Molecular Immunology, 34(16-17):1157-1165 (1997)) or the fully human monoclonal antibody 47G4 (U.S. Patent Application Publication No. 2010 / 0104509). CARs contained antigen recognition domains composed of single-chain variable fragments (scFv) derived from either the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor or the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor. The CARs contained a combination of two or more intracellular T cell signaling domains (or "costimulatory domains") derived from the human CD3 zeta (CD3ζ) molecule, the human CD28 molecule, the human 4-1BB molecule, the human CD27 molecule, and / or the FcεRI gamma chain.

[0069] More specifically, a CAR containing FMC63-derived scFv, a GM-CSF receptor signal sequence, CD8 extracellular and transmembrane components, and the intracellular T cell signaling domains of human CD3ζ and CD28 molecules is constructed. The loading plasmid (denoted FMC63-CD828Z) was prepared using the plasmid MSGV-FMC63-28Z (described in Kochenderfer et al., Journal of Immunotherapy, 32(7):689-702 (2009)) as the starting material. First, the MSGV-FMC63-28Z plasmid was digested with the restriction enzymes NotI and BmgBI (New The entire CD28 portion of this plasmid was removed by digestion with 100 kDa (Biolabs, England, Ipswich, MA). Next, we identified a portion of the extracellular domain and the entire transmembrane domain of the human CD8 molecule, and the cytoplasmic domain of the CD28 molecule. DNA fragments (synthesized by Invitrogen, Carlsbad, CA) encoding the CD8, CD28, and cytoplasmic portions of the CD3ζ molecule were ligated into the digested MSGV-FMC63-28Z plasmid. The sequences of human CD8, CD28, and CD3ζ were obtained from the National Center for Biotechnology Information website. Guidance regarding portions of each molecule to include in CARs was obtained from Kochenderfer et al., Journal of Immunotherapy, 32(7):689-702 (2009).

[0070] Fully human anti-CD19 CARs were generated by utilizing the sequence of the fully human 47G4 monoclonal antibody (described in U.S. Patent Application Publication No. 2010 / 0104509). The 47G4 antibody contains a human kappa light chain. The 47G4 scFv was generated by vaccinating KM strain mice carrying the transgene and a human heavy chain transchromosome. The sequences of the light and heavy chain variable regions of the 47G4 antibody were obtained from U.S. Patent Application Publication No. 2010 / 0104509. The 47G4 scFv was designed to contain, from 5' to 3', the following elements: a CD8 signal sequence, the 47G4 antibody light chain variable region, a linker peptide containing the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 14) (see Cooper et al., Blood, 101(4):1637-1644 (2003)), and the 47G4 The antibody heavy chain variable region. Next, a DNA sequence encoding a CAR was designed, containing the following components from 5' to 3': the 47G4 scFv, part of the extracellular domain and all of the transmembrane domain of the human CD8 molecule, and the cytoplasmic portions of the human CD28 molecule and the human CD3ζ molecule. This CAR was designated 47G4-CD828Z, and the sequence was synthesized by Invitrogen (Carlsbad, CA).

[0071] Using standard methods, the pRRLSIN.cPPT.MSCV.coDMF5.oPRE lentiviral plasmid (described in Yang et al., Journal of Immunotherapy, 33(6):648-658 (2010)) was modified to replace the coDMF5 portion of the plasmid with the 47G4-CD828Z CAR sequence described above. The code is designated as LSIN-47G4-CD8CD28Z.

[0072] A plasmid designated MSGV-47G4-CD8BBZ was constructed by modifying the MSGV-FMC63-CD828Z plasmid using standard methods. The MSGV-47G4-CD8BBZ plasmid encodes a CAR (designated 47G4-CD8BBZ) that contains, from 5' to 3', the 47G4 scFv described above, part of the extracellular domain and all of the transmembrane domain of the human CD8 molecule, a portion of the human 4-1BB (CD137) molecule containing the amino acid sequence RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 15), and the cytoplasmic portion of the CD3ζ molecule.

[0073] The CD28 sequence of the plasmid MSGV-FMC63-CD828Z was replaced with the same 4-1BB sequence contained in MSGV-47G4-CD8BBZ, resulting in a plasmid encoding a CAR designated FMC63-CD8BBZ CAR. A plasmid (designated MSGV-FMC63-CD8BBZ) was constructed.

[0074] DNA encoding the SP6 scFv (Ochi et al., Proc. Natl. Acad. Sci. USA, 80(20):6351-6355(1983)) was ligated into the MSGV-FMC63-CD828Z retroviral vector after excision of the DNA encoding the FMC63 scFv to form MSGV-SP6-CD828Z, which recognizes the hapten 2,4,6-trinitrobenzenesulfonic acid and served as a negative control in some experiments.

[0075] All of the anti-CD19 CARs generated using the above methods are listed in Table 1.

[0076] [Table 1]

[0077] The results of this example demonstrate the generation of anti-CD19 CARs based on fully human and murine monoclonal anti-CD19 antibodies.

[0078] Example 2 This example demonstrates how to generate T cells that express nucleic acid sequences encoding the CARs of the invention. do.

[0079] Replication incompetent gamma retroviruses encoding the above CARs Viruses or lentiviruses were produced and used to transduce T cells. For transient production of gammaretrovirus, 293GP packaging cells (Burns et al., Proc. Natl. Acad. Sci., USA, 90(17):8033-8037(1993)) were transfected with plasmids encoding the CARs described in Example 1, along with a plasmid encoding the RD114 envelope protein. (Porter et al., Human Gene Therapy, 7(8):913-919(1996)) and LIPOFE CTAMINE TM 2000 (Life Technologies, Carlsbad, CA) was used for transfection. Transfected cells were incubated in antibiotic-free D10 medium at 37°C for 6-8 hours. The medium used for transfection was then replaced with fresh D10 medium. The cells were then incubated for an additional 36–48 hours. During and after transfection, 293GP cells were cultured on poly-D-lysine-coated dishes (BD Biosciences, San Jose, CA). The retrovirus-containing supernatant was removed from the dish and centrifuged to remove cell debris. The supernatant was stored at −80°C.

[0080] Supernatants containing lentiviruses encoding each of the CARs described in Example 1 were generated using the protocol described in Yang et al., Journal of Immunotherapy, 33(6):648-658(2010)).

[0081] Peripheral blood mononuclear cells (PBMCs) were thawed and washed once with T cell medium. 50 ng / mL of anti-CD3 monoclonal antibody was added. 1 × 10 in T cell medium containing the monoclonal antibody OKT3 (Ortho, Bridgewater, NJ) and 300 IU / mL IL-2. 6 PBMCs were suspended at a concentration of 100 cells / mL. 20 mL of this supernatant was added to a 75 cm 2 The cells were added to a culture flask (Corning, Corning, NY). The flask was cultured upright at 37°C and 5% CO2 (see, e.g., Kochenderfer et al., Journal of Immunotherapy, 32(7):689-702(2009)).

[0082] Gammaretroviral transduction of T cells was first described using retroviral TM (Takara / Clontech Laboratories, Mountain View, CA) was dissolved in PBS at a concentration of 10 μg / mL. , this RetroNectin TMPBS solution (2 mL) was added to each well of a nontissue-culture-coated 6-well plate (BD Biosciences). The plate was incubated at room temperature (RT) for 2 hours. After incubation, the retronectin TM The solution was aspirated, and 2 mL of blocking solution consisting of Hanks' balanced salt solution (HBSS) supplemented with 2% bovine serum albumin (BSA) was added to each retronectin. TM Coe The plate was incubated at room temperature for 30 minutes. The blocking solution was aspirated and rinsed with a solution of HBSS + 2.5% HEPES. The gammaretroviral supernatant was quickly thawed and diluted 1:1 in T cell medium. 2 mL of the diluted supernatant was then added to each of the retroviral TM was added to the coated wells.

[0083] After adding the supernatant, the plate was centrifuged at 2000 × g for 2 hours at 32 °C. The supernatant was then aspirated from the wells and added to 2 × 10 cells cultured with OKT3 and IL-2 for 2 days. 6 T cells were added to each well. When T cells were added to the retrovirus-coated plates, they were diluted at 0.5 × 10 per mL in T cell medium supplemented with 300 IU / mL of IL-2. 6 After adding T cells to each well, the plate was centrifuged at 1000 x g for 10 minutes and incubated at 37°C overnight. After 24–30 h of incubation, T cells were removed from the plates and resuspended at 0.5 × 10 cells per mL in fresh T cell medium supplemented with 300 IU / mL of IL-2. 6 The cells were suspended at a concentration of 1000 0.05% and incubated at 37°C, 5% CO2. Cultivated.

[0084] For lentiviral transduction of T cells, activated PBMCs were suspended in the lentiviral supernatant supplemented with protamine sulfate and 300 IU / mL IL-2. The cells were centrifuged at 1200 × g for 1 hour. The cells were then cultured at 37°C for 3 hours. The supernatant was then resuspended in RPMI (Mediatech, Inc., Manassas, VA). The supernatant was diluted 1:1 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and IL-2. Cells were cultured overnight in the diluted supernatant, and then returned to culture in AIM V medium supplemented with 5% human AB serum and IL-2.

[0085] The expression of FMC63-based CARs on transduced T cells was evaluated. Specifically, transduced T cells were washed and suspended in FACs buffer (phosphate-buffered saline (PBS) supplemented with 0.1% sodium azide and 0.4% BSA). To detect FMC63 scFv, a biotin-labeled polynucleotide was used. Reclonal goat anti-mouse F(ab)2 antibody (anti-Fab, Jackson Immunoresearch, West Grove, PA) was added. The cells were incubated at 4°C for 25 minutes and washed once. The cells were then buffered with FACS. The cells were suspended in PBS and blocked with normal mouse IgG (Invitrogen, Carlsbad, CA). The cells were then stained with phycoerythrin (PE)-conjugated streptavidin (BD Pharmingen, San Diego, CA), anti-CD4, anti-CD8, and anti-CD3. Flow cytometry was performed using an LSR II microscope. The analysis was performed using a flow cytometer (BD Biosciences) and FlowJo software (Treestar, Inc. Ashland, OR). The expression of CARs was assessed using almost the same method, except that biotin-labeled protein L (GenScript, Piscataway, NJ) was used instead of biotin-labeled polyclonal goat anti-mouse F(ab)2 antibody.

[0086] The percentage of CAR-expressing (CAR+) T cells was determined in each experiment using either anti-Fab antibody or protein. The percentage of T cells in CAR-transduced cultures stained with anti-Fab or protein L was calculated by subtracting the percentage of similarly cultured non-transduced T cells from the same donor that stained with anti-Fab or protein L.

[0087] Percentage of T cells expressing CARs containing scFv derived from murine FMC63 antibody on day 7 of culture. The survival rates were as follows: FMC63-28Z, 71%; FMC63-CD828Z, 88%; and FMC63-CD8BBZ, 87%. As shown in Figure 1, T cells expressing the FMC63-28Z CAR exhibited shorter in vitro survival in IL-2-containing cultures compared to T cells expressing the FMC63-CD828Z CAR or FMC63-CD8BB CAR. High levels of CAR expression were also detected in T cells transduced with gammaretroviruses encoding FMC63-CD828Z, FMC63-CD8BBZ, and FMC63-CD827Z.

[0088] CARs containing scFv derived from the 47G4 antibody were expressed at high levels on the surface of human T cells. In particular, Figures 2A-2D show the expression of 47G4-based CARs containing the CD27 intracellular signaling domain, while , Figures 3A and 3B show the expression of 47G4-CD828Z CAR.

[0089] The results of this example demonstrate that T cells can be engineered to express the anti-CD19 CARs of the invention.

[0090] Example 3 This example describes a series of experiments used to determine the specificity of the CARs of the invention for CD19.

[0091] Patient samples and cell lines Non-leukemia patients with melanoma, chronic lymphocytic leukemia (CLL), or lymphoma enrolled in an Institutional Review Board-approved protocol at the Surgery Branch of the National Cancer Institute (NCI) were randomly assigned to receive non-leukemia treatment. Non-leukemic PBMC samples were obtained. Cells from five different patients were used. Donor 1 had CLL, donor 2 was a normal donor, and donors 3 and 5 both had lymphoma. Donor 4 had melanoma, and donor 5 had melanoma. PBMCs were cryopreserved in 100% FBS. In experiments using primary CLL cells as target cells, CLL was Unmanipulated PBMCs from patients with the following CD19-expressing immortalized cells were used: Cell lines used were: NALM-6 (acute lymphoblastic leukemia, from DSMZ, Braunschweig, Germany) and and CD19-K562. The following CD19-negative cell lines were used: A549 (lung carcinoma, from ATCC), CCRF-CEM (T-cell leukemia, from ATCC), MDA231 (breast carcinoma, from ATCC), and TC71 (Ewing's sarcoma, kindly provided by Dr. M. Tsokos, National Cancer Institute, Bethesda, MD). All cell lines were maintained in R10 medium. When using CLL PBMCs as targets in the assay, the assay The cells were cultured in R10 medium for 12 to 18 hours before injection.

[0092] Enzyme-linked immunosorbent assays (ELISA) for interferon and TNF In clinical trials, the occurrence of hypotension and other toxicities in patients receiving infusions of T cells expressing CAR FMC63-28Z implicated TNF production by T cells expressing the CARs of the invention and FMC63-28Z. This prompted a comparison with TNF production by T cells expressing IL-1.

[0093] Target cells were washed in IL-2-free T cell medium and diluted to 1 × 10 per mL. 6 Each target cell was suspended in 100,000 target cells of each cell type were plated in a 96-well round-bottom plate (Corning, Tewksbury, MA). Two wells containing T cells were added to each well. Wells containing T cells alone were also prepared. The plates were incubated at 37°C for 18-20 hours. After incubation, IFN-γ was added to each well using standard methods. γ or TNF ELISA assays were performed (Pierce, Rockford, IL). In some experiments, TNF ELISA results were normalized by dividing TNF levels by the percentage of T cells in overnight cultures expressing a given CAR. CAR expression was determined as described in Example 2.

[0094] When normalized for cell surface CAR expression, T cells expressing FMC63-28Z consistently produced more TNF than FMC-CD828Z and FMC63-CD8BBZ CARs, as shown in Figures 4A and 4B. The only difference between FMC63-28Z and FMC63-CD828Z CARs is the use of human CD28 extracellular and transmembrane components in FMC63-28Z, while FMC63-CD828Z uses extracellular and transmembrane components derived from the human CD8 protein. The striking differences between FMC63-28Z and FMC63-CD828Z in T cell persistence and inflammatory cytokine production led to the use of a CD8 extracellular spacer and transmembrane component in subsequent CAR design.

[0095] As shown in Tables 2 and 3 (all units are pg / mL IFNγ), T cells transduced with anti-CD19 CARs produced high amounts of IFNγ when they were cultured overnight with the CD19-expressing cell line CD19-K562. γ, but when cultured with a negative control cell line, CAR was not transduced. The transduced T cells produced only background levels of IFNγ. The results of the IFNγ ELSIA for the 47G4-CD828Z CAR are shown in Figure 5.

[0096] [Table 2]

[0097] [Table 3]

[0098] High background IFNγ secretion was consistently observed with CARs containing the 4-1BB moiety. T cells transduced with the FMC63-CD827Z CAR produced IFNγ in a CD19-specific manner. When 827Z cells were cultured with CD19-negative NGFR-K562 and CCRF-CEM cells, FMC63-CD827Z-transduced T cells also produced TNF in an antigen-specific manner.

[0099] CD107a assay For each T cell culture to be tested, two or three separate tubes were prepared: one tube containing CD19-K562 cells, one tube containing unmanipulated primary CLL cells, and one tube containing unmanipulated primary CLL cells. The tubes contained NGFR-K562 cells. In some experiments, the CD19-K562 tube was excluded. All tubes contained T cells transduced with the anti-CD19 CARs listed above, 1 mL of AIM V. TM Culture medium (Life Technologies, Carlsbad, CA) + 5% human serum, titrated concentration The tubes contained 1 μL of anti-CD107a antibody (eBioscience, Inc., San Diego, CA; clone eBioH4A3), and 1 μL of Golgi Stop (BD Biosciences, Franklin Lakes, NJ). All tubes were incubated at 37°C. The cells were incubated for 4 hours and then stained for expression of CD3, CD4 and CD8.

[0100] T cells from different subjects expressing CARs FMC63-CD828Z, FMC63-CD827Z, FMC63-CD8BBZ, 47G4-CD827Z, 47G4-CD82827Z, 47G4-CD827BBZ, or 47G4-CD8BBZ specifically upregulated CD107a in response to stimulation with CD19-expressing target cells; the results of CD107a assays for 47G4-CD827Z, 47G4-CD82827Z, and 47G4-CD827BBZ CARs are shown in Figures 6A-6D, indicating the occurrence of CD19-specific T cell degranulation (e.g., CD19-specific T cell degranulation), a prerequisite for perforin-mediated cytotoxicity. , see Rubio et al., Nature Medicine, 9(11):1377-1382(2003)).

[0101] Proliferation assay The ability of T cells transduced with anti-CD19 CARs to proliferate when stimulated with CD19-expressing target cells was assessed. 6 irradiated CD19-K562 cells, or 0.5 x 10 6 irradiated NGFR-K562 cells were transduced with anti-CD19 CAR in 0.75 x 10 6The T cells were co-cultured with 1000 total T cells. T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Life Technologies, Carlsbad, CA) as described in Mannering et al., J. Immunological Methods, 283(1-2):173-183 (2003). The medium used for co-culture was AIM V. TM Culture medium (Life Technologies, Carlsbad, CA) + 5% human AB serum IL-2 was not added to the medium. After 4 days from the start, the chicken was cultured to eliminate dead cells. Count viable cells in each co-culture using PBS blue and flow cytometry as described in Example 2. Cytometry was performed.

[0102] As shown in Figures 7A-7C, T cells expressing CARs FMC63-CD8BBZ, FMC63-CD828Z, and 47G4-CD8BBZ all expressed CD19-K562 cells compared to when cultured with negative control NGFR-K562 cells. These results suggest that anti-CD19 CARs are capable of forming CFSE-positive cells. We demonstrated that transduced T cells specifically proliferated when stimulated with CD19-expressing target cells. vinegar.

[0103] The results of this example demonstrate that T cells expressing the CARs of the present invention can induce CD19-specific cytokine production. , degranulation and proliferation.

[0104] Example 4 This example demonstrates that T cells expressing the anti-CD19 CAR of the present invention can destroy chronic lymphocytic leukemia (CLL) cells.

[0105] To determine whether T cells transduced with the FMC63-CD828Z CAR of the present invention can destroy CD19-expressing unmanipulated PBMCs from patients with CLL, a cytotoxicity assay was performed. Specifically, the cytotoxicity of target cells was measured using a method similar to that described, for example, in Kochenderfer et al., J. Immunotherapy, 32(7):689-702(2009) and Hermans et al., J. Immunological Methods, 285(1) :25-40(2004) and other similar assays were used to measure negative control CCRF-CEM cells. The survival of CD19-expressing target cells (i.e., CLL PBMCs) was measured by comparing the survival of CD19-expressing target cells (i.e., CLL PBMCs) to that of CD19-expressing target cells (i.e., CLL PBMCs).

[0106] CCRF-CEM cells were cultured in R10 medium at 1.5 × 10 6 Suspend the cells at a concentration of 5-(and 6-) fluorescent dyes at 1000 x g / mL. (((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR) (Life Technologies, Carlsbad, CA) was added at a concentration of 5 M. The cells were mixed and then incubated at 37°C for 30 minutes. The cells were then washed with cytotoxic medium, suspended, and incubated at 37°C for 60 minutes. The cells were then washed twice with cytotoxic medium and suspended. CLL PBMCs were cultured at 1 x 10 in PBS + 0.1% BSA. 6 Cells were suspended at 1000 cells / mL. Fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) (Life Technologies, Carlsbad, CA) was added to the cell suspension at a concentration of 1 M. The cells were incubated at 37°C for 10 minutes. After incubation, the labeling reaction was initiated by adding an equal volume of FBS to the cell suspension. The reaction was stopped and the cells were incubated at room temperature for 2 minutes. The cells were then soaked in cytotoxic medium. Washed and suspended.

[0107] Approximately 50,000 CD19-expressing CLL PBMCs and 50,000 CCRF-CEM cells were cultured with various numbers of CAR-shaped The transduced T cells were combined in the same tube. In all experiments, the cytotoxicity of effector T cells transduced with the FMC63-CD828Z CAR was compared to the cytotoxicity of negative control effector T cells from the same subject, either transduced with the SP6-28Z control CAR or untransduced. Co-cultures were established in duplicate in sterile 5 mL test tubes (BD Biosciences, Franklin Lakes, NJ) at the following T cell:target cell ratios: 20:1, 6.7:1, 2.2, and 0.7:1. Cultures were incubated at 37°C for 4 hours. Immediately after incubation, 7-amino-actinomycin D (7AAD; BD Biosciences, Franklin Lakes, NJ) was added as recommended by the manufacturer, and flow cytometry acquisition was performed using a BD FacsCanto II (BD Biosciences). Analysis was performed using FlowJo Software (Treestar, Inc. Ashland, OR). Analysis was gated on 7AAD-negative (viable) cells and the percentage of viable CLL target cells and viable CCRF-CEM negative control cells. was determined for each T cell + target cell culture.

[0108] For each culture, CLL PBMC viability was determined by dividing the percentage of viable CLL PBMCs by the percentage of viable CCRF-CEM negative control cells. Corrected CLL PBMC viability was calculated by dividing the CLL PBMC viability in the tube containing only CLL target cells and CCRF-CEM negative control cells without any effector T cells by the ratio of the percentage of CLL target cells to the percentage of CCRF-CEM negative control cells in the tube containing only CLL target cells and CCRF-CEM negative control cells without any effector T cells. This correction was necessary to account for variations in starting cell numbers and natural target cell death. Cytotoxicity was calculated as CLL PBMC cytotoxicity = 100 - corrected CLL PBMC viability. For every effector:target ratio, cytotoxicity was determined in duplicate, and the results were averaged.

[0109] The results of the cytotoxicity assay are shown in Figure 8 and demonstrate that the anti-CD19 CAR of the present invention can be used in a method to destroy malignant B cells.

[0110] Example 5 This example demonstrates that T cells expressing an anti-CD19 CAR of the invention can reduce malignant B cell tumor growth in an animal model.

[0111] Immunodeficient NSG mice were subcutaneously injected with 4 million CD19+ NALM6 tumor cells. Six days later, palpable tumors formed, followed by the development of either the MSGV-FMC63-28Z CAR vector (described in Kochenderfer et al., Journal of Immunotherapy, 32(7):689-702 (2009)) or LSIN-47G4-CD8CD. A single intravenous injection of human T cells transduced with either the 28Z CAR vector (described in Example 1) was administered. Mice were treated by injecting 100 μg of ... compared with tumors.

[0112] The results of this example, shown in Figure 9, show that the expression of either the FMC63-28Z CAR or the 47G4-CD8CD28Z CAR Figure 1 shows that T cells significantly reduced tumor size in treated mice.

[0113] All references cited herein, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each individual reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0114] With respect to the description of the present invention (particularly with respect to the claims that follow), the terms "a" and "an" The use of "at least one" and "the" and "at least one" and similar referents should be construed to cover both the singular and the plural unless otherwise stated herein or clearly contradicted by context. The use of the term "at least one" following a listing of one or more items (e.g., "at least one of A and B") should be construed to cover both the singular and the plural unless otherwise stated herein or clearly contradicted by context. Unless otherwise stated, one item (A or B) selected from the items listed, or The term "includes" should be interpreted as meaning any combination of two or more of the following: "comprising," "having," "including," and "containing" are used in open-ended terms (i.e., "including, but not limited to" and "including"), unless otherwise specified. The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise stated herein, and each separate value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise stated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is to be construed as meaning "within reason." These are intended merely to facilitate understanding of the invention and are not intended to impose limitations on the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0115] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors anticipate that those of skill in the art will employ such variations as appropriate, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. 1. Use of a cell population comprising host cells expressing a chimeric antigen receptor (CAR) against CD19 in the manufacture of a medicament for treating or preventing cancer in a subject, wherein the host cells comprise a nucleic acid encoding a CAR or a vector comprising said nucleic acid, and the CAR comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; The use, wherein the host cell is a T cell or an NK cell.

2. 2. The use of claim 1, wherein the cancer expresses CD19.

3. 3. The use according to claim 1 or 2, wherein the cancer is a B-cell malignancy.

4. 4. The use of claim 3, wherein the B-cell malignancy is leukemia, lymphoma, or multiple myeloma.

5. 5. The use according to claim 3 or 4, wherein the B-cell malignancy is leukemia.

6. 6. The use of claim 5, wherein the leukemia is chronic lymphocytic leukemia.

7. 5. The use according to claim 3 or 4, wherein the B-cell malignancy is lymphoma.

8. 8. The use of claim 7, wherein the lymphoma is follicular lymphoma, mantle cell lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphoma, Hodgkin's lymphoma, lymphoplasmacytic lymphoma, or marginal zone lymphoma.

9. 5. The use according to claim 3 or 4, wherein the B-cell malignancy is multiple myeloma.

10. 1. Use of a cell population comprising host cells expressing a chimeric antigen receptor (CAR) against CD19 in the manufacture of a medicament for eliciting an antigen-specific response against CD19-expressing cells in a subject, wherein the host cells comprise a nucleic acid encoding a CAR or a vector comprising said nucleic acid, and the CAR comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; The use, wherein the host cell is a T cell or an NK cell.

11. The use according to claim 10, wherein the CD19-expressing cells are B cells.

12. The use according to any one of claims 1 to 11, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

1.

13. The use according to any one of claims 1 to 11, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

10.

14. The use according to any one of claims 1 to 13, wherein the host cell is a eukaryotic cell.

15. The use according to any one of claims 1 to 14, wherein the host cell is a T cell.

16. The use according to any one of claims 1 to 14, wherein the host cells are NK cells.

17. 16. The use of claim 15, wherein the T cells are cytotoxic to CD19-expressing cells.

18. 1. A method for generating an antigen-specific response against a CD19-expressing cell in vitro, the method comprising contacting a T cell with a CD19-expressing cell, wherein the T cell comprises a nucleic acid encoding a CAR or a vector comprising the nucleic acid, and the CAR comprises the amino acid sequence of SEQ ID NO:

1.

19. 1. Use of one or more T cells in the manufacture of a medicament for generating an antigen-specific response against a CD19-expressing cell in a subject, wherein the T cells comprise a nucleic acid encoding a CAR against CD19, and the CAR comprises the amino acid sequence of SEQ ID NO:

1.

20. 1. A T cell comprising a nucleic acid encoding a CAR against CD19, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

1.

21. 1. A method for producing a T cell that expresses a CAR, the method comprising introducing into the T cell a vector encoding a CAR comprising the amino acid sequence of SEQ ID NO:1.

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