CD-19-targeting chimeric antigen receptor

Abstract

The present invention provides compositions and methods that can be used in methods for treating B-cell malignancies, and which exhibit low toxicity and immunogenicity in humans. [Solution] A chimeric antigen receptor (CAR) for CD19, comprising a specific amino acid sequence, is provided. A method for destroying T cells and malignant B cells expressing the CAR is also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This patent application is incorporated by reference to U.S. Provisional Patent Application No. 62 / 006,313, filed on June 2, 2014. They assert the interests of (the people who are born). Statements relating to research or development funded by the federal government This invention was made with the support of the federal government and by the National Institutes of Health and the National Cancer Institute under project number Z01 BC001415. The federal government reserves certain rights in this invention.

[0002] Incorporation by referencing electronically submitted properties A computer-readable nucleotide / amino acid sequence listing, submitted concurrently with this specification and identified below, 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 lymphoma and leukemia, arise when the control of B-cell differentiation and activation is disrupted. Malignancies of mature B cells include follicular lymphoma and mantle cell lymphoma. Burkitt lymphoma, multiple myeloma, diffuse large B-cell lymphoma, Hodgkin's disease This includes lymphoma, lymphoplasmacytic lymphoma, marginal zone lymphoma, and chronic lymphocytic leukemia (Shaffer et al., Nature Reviews Immunology, 2:920-933(2002)). Standard treatment For example, chemotherapy, therapeutic monoclonal antibodies (e.g., Rituximab (RITUXAN) TM )) and allogeneic stem cell transplantation (alloHSCT) are examples of B cell transplantation. It does not cure malignant tumors (e.g., Dreger et al., Leukemia, 21(1):12-17(2007)). ;Gribben, JG, Blood, 109(11):4617-4626(2007);and Armitage, JO, Blood, See 110(1):29-36(2007). In particular, monoclonal antibodies are not therapeutic as monotherapy, and alloHSCT is associated with high levels of mortality and morbidity (e.g., Dreger et al.). See Armitage et al., 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 region and a T cell activation domain (see, for example, Kershaw et al., above, 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 For malignant tumors, adoptive T cell approaches using CD19-targeting CARs have been developed. (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, (See 2011 (epublication ahead of print (2012))). B cell antigen CD19, its expression Since it is limited to normal and malignant B cells, it has been selected as a target for CARs. For example, see 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 the significant toxicity they can induce, 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 that contain mouse 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)). (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] Therefore, there is a need for compositions that can be used in methods for treating B cell malignancies and that have low toxicity and immunogenicity in humans. The present invention provides such compositions and methods. 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 an 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. 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: 1. The present invention further provides an isolated or purified nucleic acid sequence encoding said CARs, a vector comprising such a nucleic acid sequence, an isolated T cell comprising such a vector, and a method of destroying malignant B cells by contacting such an isolated T cell with a malignant CD19-expressing B cell population in vivo or ex vivo.

[0008] The present invention further provides an isolated or purified nucleic acid sequence encoding said CARs, a vector comprising such a nucleic acid sequence, an isolated T cell comprising such a vector, and a method of destroying malignant B cells by contacting such an isolated T cell with a malignant CD19-expressing B cell population in vivo or ex vivo. The present invention further provides an isolated or purified nucleic acid sequence encoding said CARs, a vector comprising such a nucleic acid sequence, an isolated T cell comprising such a vector, and a method of destroying malignant B cells by contacting such an isolated T cell with a malignant CD19-expressing B cell population in vivo or ex vivo.

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

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

[0011] Brief description of each figure of the drawings [Figure 1] FIG. 1 is a graph showing the experimental results explaining the in vitro survival of T cells expressing the indicated CARs as described in Example 2. On 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] FIGS. 2A-2D are FACs plots explaining 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 live CD3+ lymphocytes. [Figure 3] FIGS. 3A and 3B are FACs plots explaining the expression of the 47G4-CD828Z CAR on the surface of T cells (FIG. 3A) compared to a non-transduced control (FIG. 3B). The plots are gated on live CD3+ lymphocytes. [Figure 4]Figures 4A and 4B are graphs illustrating experimental results illustrating TNF production by T cells expressing FMC63-28Z, FMC63-CD828Z, or FMC63-CD8BBZ CARs in CD19+ T cell lines CD19-K562 (Figure 3A) and NALM6 (Figure 3B). 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. The results show the mean and standard error of the mean normalized TNF levels from two different donors. [Figure 5] Figure 5 is a graph illustrating 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 transduced T cells with the indicated CARs degranulated in a CD19-specific manner, as measured by the upregulation of CD107a. [Figure 6-2] Figures 6C and 6D are FACs plots illustrating that transduced T cells with the indicated CARs degranulated in a CD19-specific manner, as measured by the upregulation of CD107a. [Figure 7] Figures 7A–7C are FAC 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 in IL-2-free medium with either the CD19+ cell line CD19-K562 (black curve) or the CD19-negative cell line NGFR-K562 (white curve (open curve)). All plots are gated to viable CD3+ CAR+ lymphocytes. [Figure 8]Figure 8 is a graph illustrating experimental results that demonstrate 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 illustrating experimental results that explain how T cells expressing either FMC63-28Z CAR or 47G4-CD8CD28Z CAR reduce NALM6 tumor size in NSG immunodeficient mice. [Modes for carrying out the invention]

[0012] Detailed description of the invention The present invention provides isolated or purified chimeric antigen receptors (CARs) comprising an antigen recognition moiety and a T cell activation moiety. Chimeric antigen receptors (CARs) are used for T cell signaling. Alternatively, the antigen-binding domain of an antibody linked to a T cell activation domain (e.g., single-strand variable CARs are artificially constructed hybrid proteins or polypeptides containing fragments (scFv). CARs utilize the antigen-binding properties of monoclonal antibodies to achieve MHC-independent binding. This method redirects the responsiveness and specificity of T cells to selected targets. It possesses the ability to do so. MHC-independent antigen recognition confers the ability of T cells expressing CARs to recognize antigens independently of antigen processing, thereby bypassing the major tumor escape mechanism. Furthermore, when expressed in T cells, CARs are advantageous in that they interact with the endogenous T cell receptor (TCR). It does not dimerize with the alpha and beta chains of ).

[0013] "Isolated" means that a substance (e.g., a protein or nucleic acid) is taken out of its natural environment. "Purified" means that a given substance (e.g., a protein or nucleic acid) is either taken out of nature (e.g., genomic DNA and mRNA) or synthesized. Whether it is a synthesized (e.g., cDNA) and / or amplified under laboratory conditions, this means increased purity, 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 may still be isolated for practical purposes. For example, proteins may be used for introduction into cells. When used, it is typically mixed with an acceptable carrier or diluent.

[0014] The CAR of the present invention is effective against CD19 (also known as B lymphocyte antigen CD19, B4, and CVID3). It contains the antigen recognition portion. CD19 is expressed only by hematopoietic B lymphocytes and follicular dendritic cells. It is a cell surface molecule. This is the first of the B-lineage restrictive antigens that are expressed. Most pre-B cells, and most non-T cell acute lymphoblastic leukemia cells and B cells It is present on chronic lymphocytic leukemia cells (Tedder and Isaacs, J. Immun., 143:712-717 (1989)). CD19, along with CD21 and CD81, primarily acts as a B-cell co-receptor (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 end of CD19 is phosphorylated, which leads to binding by Src family kinases and recruitment of PI-3 kinase. CD19 has also been shown to interact with other cellular signaling proteins, such as B These include Lyn tyrosine protein kinase (Fujimoto et al., Immunity, 13:47-57(2000)), CD82 (Imai et al., see above), complement receptor 2 (Bradbury et al., see above; and Horvath et al., see above), and VAV2 (Doody et al., EMBO J., 19(22):6173-6184(2000)), which are major Src kinases in cells.

[0015] The CAR of the present invention comprises a monoclonal antibody against CD19 or an antigen-binding moiety thereof. Includes an antibody recognition moiety. As used herein, the term "monoclonal antibody" means an antibody produced by a single clone of a B cell that binds to the same epitope. This refers to antibodies produced by various B cells. In contrast, "polyclonal antibodies" are produced by various B cells. This refers to a group of antibodies that bind to various epitopes of the same antigen. The antigen recognition portion of the CAR of the present invention This can be the whole antibody or an antibody fragment. The whole antibody is typically composed of four polypeptides. Consists of: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. 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. Includes. Each pair of light and heavy chain variable regions forms the antigen-binding site of the antibody. VH region and VL region The regions have the same general structure, and each region consists of four frames in which the sequence is relatively preserved. This includes the work domain. The framework domain is connected by three complementary decision domains (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of the antibody. This is involved in antibody binding.

[0016] The terms “antibody fragment,” “antibody fragment,” “functional fragment of an antibody,” and “antigen-binding portion” are used interchangeably in this specification. and one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen. This means (see Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005) in general). The antigen recognition portion of the CAR of the present invention may contain any CD19-binding antibody fragment. The antibody fragment is preferably, for example, one or more CDRs, variable regions (or portions thereof) This includes a constant region (or a portion thereof), or a combination thereof. An example of an antibody fragment is (i) the Fab flag, a monovalent fragment consisting of VL, VH, CL, and CH1 domains. (ii) a bivalent fragment F(ab')2 fragment containing two Fab fragments linked by disulfide crosslinks in the hinge region; (iii) a single antibody (iv) The two domains of the Fv fragment (v) Diabody (a dimer of the polypeptide chain) is a monovalent molecule consisting of the two domains (i.e., VL and VH) linked by a synthetic linker that enables the synthesis of these two domains as a single polypeptide chain (see, for example, 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)), as well as (v) Diabody (a dimer of the polypeptide chain) Each polypeptide chain contains VH linked to VL by a peptide linker, and the peptide Examples include, but are not limited to, linkers that are too short to pair between VH and VL on the same polypeptide chain, thereby promoting pairing between complementary domains on different VH-VL polypeptide chains, resulting in a dimer molecule having two functional antigen-binding sites. Antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent Application No. 2009 / 0093024 A1. In a preferred embodiment, the antigen-recognizing portion of the CAR of the present invention comprises an anti-CD19 single-strand Fv(scFv).

[0017] The antigen-binding portion or fragment of a monoclonal antibody may be of any size, as long as it binds to CD19. In this regard, the antigen-binding portion or fragment of a monoclonal antibody against CD19 (hereinafter also referred to as "anti-CD19 monoclonal antibody") preferably contains between about 5 and 18 amino acids (for example, within the range defined by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or any two of the above values). Includes one or more CD-Rs.

[0018] In one embodiment, the CAR of the present invention comprises an anti-CD19 monoclonal antibody containing a variable region. The antigen-recognition portion is included. The anti-CD19 monoclonal antibody may be obtained from or derived from a mammal (including, but not limited to, mouse, rat, or human). Preferably, the antigen-recognition portion includes the variable region of the mouse or human anti-CD19 monoclonal antibody. In this regard, the antigen-recognition portion includes the light chain variable region, the heavy chain variable region, or both the light chain variable region and the heavy chain variable region of the mouse or human anti-CD19 monoclonal antibody. Preferably, the antigen recognition of the CAR of the present invention The recognition portion includes the light chain variable region and heavy chain variable region of a mouse or human anti-CD19 monoclonal antibody. FMC63 antibody (described in Nicholson et al., Molecular Immunology, 34(16-17):1157-1165(1997)) is an example of a mouse 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. (For example, 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.) See Kochenderfer 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 may be used in the present invention.

[0019] In another embodiment, the CAR of the present invention includes a signal sequence. The signal sequence is antigen recognition The signal sequence may be located 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, the CAR of the present invention containing mouse anti-CD19 scFv may include a GM-CSF signal sequence, while the CAR of the present invention containing human anti-CD19 scFv may include a CD8α signal sequence. .

[0020] In another embodiment, the CAR of the present invention includes an extracellular spacer array. - The sequence is a short amino acid sequence that enhances antibody flexibility (see, for example, Woof et al., Nat. Rev. Immunol., 4(2):89-99(2004)), and the antigen recognition region (e.g., anti-CD19 scFv) It may be positioned between the T cell activation region and the extracellular spacer array. It may include all or part of the extracellular region of the protein. 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 includes a transmembrane domain. The transmembrane domain is any known in the art. It may be any transmembrane domain derived from or obtained from the molecule. For example, the transmembrane domain may be obtained from or derived from the CD8α molecule or the CD28 molecule. CD8 is a T molecule It is a transmembrane glycoprotein that acts as a co-receptor for cytoplasmic receptors (TCRs), primarily in the area of ​​cell damage. CD8 is expressed on the surface of harmful T cells. The most common form of CD8 is as a dimer composed of CD8α and CD8β chains. CD28 is expressed on T cells and provides co-stimulatory signals necessary for T cell activation. CD28 is a receptor for CD80(B7.1) and CD86(B7.2). In preferred embodiments, CD8α and CD28 are of human origin.

[0022] The CAR of the present invention includes a T cell activation moiety. The T cell activation moiety includes 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 generates a signal in association with TCRs and includes immunoreceptor tyrosine-based activation motifs (ITAMs). Preferably, the T cell activation moiety includes multiple (i.e., two or more) intracellular T cells. It contains a signaling domain. The intracellular T cell signaling domain is the CD28 molecule, the CD3 zeta (ζ) molecule or a modified version thereof, or the human high-affinity IgE receptor (FcεRI) gamma chain. CD27 molecule, OX40 molecule, 4-1BB molecule, or other intracellular signaling molecules known in this field It can be obtained or derived from. As mentioned above, CD28 is an important T cell marker for T cell costimulation. 4-1BB, also known as CD137, provides a strong costimulatory signal to T cells. It transmits signals, promotes differentiation, and enhances the long-term survival of T lymphocytes. CD27 is a member of the TNF receptor superfamily and is necessary 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 crosslinks. FcεRI is constitutively expressed in mast cells and basophils and is induceable in eosinophils. In preferred embodiments, 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 Includes one or more of the following domains (for example, 1, 2, 3, or 4) in any combination. For example, the CAR of the present invention comprises a CD28 transmembrane domain and intracellular T cell cellularity of CD28 and CD3ζ. It may include a signaling domain. Alternatively, for example, the CAR of the present invention may include a CD8α transmembrane domain and an intracellular T cell signaling domain of CD28, CD3ζ, FcεRI gamma chain and / or 4-1BB. In another embodiment, the CAR of the present invention may include a CD8α transmembrane domain and intracellular T cell signaling domains of CD28, CD3ζ, and CD27. In yet another embodiment, the CAR of the present invention may include a CD28 transmembrane domain and intracellular T cell signaling domains of CD27, 4-1BB, and FcεRI gamma chains.

[0024] The present invention further relates to isolated or purified chimeric antigen receptors (CARs) encoding the chimeric antigen receptors (CARs) of the present invention. This document provides nucleic acid sequences. “Nucleic acid sequences” are intended to encompass polymers of DNA or RNA, i.e., polynucleotides, which may be single-stranded or double-stranded and may include unnatural or modified nucleotides. As used herein, the terms “nucleic acid” and “polynucleotide” mean polymeric forms of nucleotides of any length, which are either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of a molecule and therefore include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides (e.g., methylated and / or capped polynucleotides).

[0025] The CAR of the present invention may 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 mammals, or treat or prevent a disease in mammals). For example, the CAR may contain 50 or more (e.g., The amino acids are 60 or more, 100 or more, or 500 or more, but may include less than 1,000 amino acids (for example, 900 or less, 800 or less, 700 or less, or 600 or less). Preferably, the CAR contains about 50 to about 700 amino acids (for example, about 70, about 80, about 90, about 150, about 200, about 300, about 400, about 550, or The range is defined by approximately 650 amino acids, approximately 100 to approximately 500 amino acids (for example, approximately 125, approximately 175, approximately 225, approximately 250, approximately 275, approximately 325, approximately 350, approximately 375, approximately 425, approximately 450, or approximately 475 amino acids), or any two of the above values.

[0026] The scope of this invention includes the functional portions of the CAR of the present invention as described herein. When used in reference to the CAR, the term “functional portion” means the functional portion of the CAR of the present invention. A part or fragment of the intent, and that part or fragment is CAR (that part or fragment A fragment is a part of this (parent CAR) that retains the biological activity. Functional part For example, the fraction recognizes target cells to a similar, equivalent, or higher degree than the parent CAR. The portion of CAR that retains the ability to do so, or the ability to detect, treat, or prevent disease. The nucleic acid sequence encoding the functional portion of the CAR, relative to the nucleic acid sequence encoding the parent CAR, contains, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR. It can code for proteins.

[0027] The functional part of CAR has an amino terminus or carboxyl terminus or both ends of that part. It may include amino acids that are not found in the amino acid sequence of the parent CAR. Preferably, Furthermore, the additional amino acids do not interfere with the biological function of the functional part (e.g., recognizing target cells, detecting cancer, treating or preventing cancer). More preferably, 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. When used herein, The term "functional variant" refers to a variant of the CAR of the present invention that has a substantial or significant sequence. CARs, polypeptides, or proteins having identity or similarity, and the functional mutation This refers to a body that retains the biological activity of CAR (the functional mutant in question is a mutant of CAR). Functional variants, for example, target similarly, equally, or more highly than the parent CAR. This specification includes variants of the CAR (parent CAR) described herein that retain the ability to recognize cells. With respect to the nucleic acid sequence encoding the parent CAR, the nucleic acid sequence encoding the functional variant of the CAR is, for example, approximately 10%, 25%, 30%, and 50% identical to the nucleic acid sequence encoding the parent CAR. They may be approximately 65% ​​identical, 80% identical, 90% identical, 95% identical, or 99% identical.

[0029] Functional variants may include, for example, the amino acid sequence of the CAR of the present invention having at least one conserved amino acid substitution. The phrase “conserved amino acid substitution” or “conserved mutation” refers to the replacement of one amino acid with another amino acid having common properties. A functional method for defining common characteristics between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins in homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York). (1979)). Through such analysis, groups of amino acids can be defined in which amino acids preferentially exchange with each other and are therefore most similar in their impact on the overall structure of the protein (Schulz, GE and Schirmer, RH, above). Examples of conserved mutations include , amino acid substitutions 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). Examples include: (so that it can be maintained).

[0030] Alternatively, the functional mutant may have at least one non-conservative amino acid substitution. It may contain the amino acid sequence of the parent CAR. A "non-conservative mutation" is an amino acid sequence between different groups. This involves substitutions, such as the substitution of tryptophan with lysine or serine with phenylalanine. In this case, it is preferable that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional mutant. Non-conservative amino acid substitutions result in the biological activity of the functional mutant compared to the parent CAR. The biological activity of functional mutants can be enhanced to increase their physical activity.

[0031] The CARs of the present invention (including their functional parts and functional variants) may contain synthetic amino acids instead 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-hydro Xyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenyl Lalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclo Lohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, 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 CARs of the present invention (including their functional parts and functional variants) are glycosylated and amidated. They can be converted to acid addition salts by carboxylation, phosphorylation, esterification, N-acylation, cyclization (e.g., via disulfide bridges), and / or optionally dimerized, polymerized, or conjugated.

[0033] The present invention also relates to any target molecule of interest to CAR (i.e., any antigen recognition part). (including minutes) and the extracellular spacer, transmembrane domain and intracellular T cell signaling The present invention provides a CAR comprising any combination of one of the signaling domains. For example, the present invention may 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 Sequence ID No. 1). In another embodiment, the present invention provides a CAR comprising (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. It may include a signaling domain (as used in the CAR of SEQ ID NO: 4). In another embodiment, the CAR of the present invention may include (i) an extracellular spacer, (i) a transmembrane domain derived from a human CD8α molecule, and (iii) derived from a human CD28 molecule, a human CD27 molecule, and an FcεRI gamma chain. It may include an intracellular T cell signaling domain (as used in the CAR of SEQ ID NO: 10). In yet another embodiment, the CAR of the present invention may include (i) an extracellular spacer, (iii) a transmembrane domain derived from the human CD8α molecule, and (iii) a human CD28 molecule and an FcεRI gamma chain. It may contain an intracellular T cell signaling domain (as used in CAR of SEQ ID NO: 12).

[0034] In a preferred embodiment, the CAR of the present invention includes 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 CAR of the present invention can be prepared using methods known in the art. For example, nucleic acid sequences, poly Peptides and proteins can be recombinantly produced using standard recombinant DNA methodologies. (e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd See 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). Furthermore, synthetically produced nucleic acid sequences encoding CARs have been found in plants. Nucleic acid sequences can be isolated and / or purified from sources such as bacteria, insects, or mammals (e.g., rats, humans, etc.). Methods for isolation and purification are well known in the art. Alternatively, the nucleic acid sequences described herein can be commercially synthesized. In this regard, nucleic acid sequences can be synthesized, recombinant, isolated and / or purified.

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

[0037] In addition to the nucleic acid sequence encoding the CAR of the present invention, the vector preferably includes a promoter. These include expression regulatory sequences that provide expression of nucleic acid sequences in host cells, such as enhancers, polyadenylation signals, transcriptional terminators, and internal ribosome entry sites (IRESs). Exemplary expression regulatory sequences are known in this field, e.g., Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990). It will be listed.

[0038] Numerous promoters, including constitutive, inducible, and repressive promoters, from various different sources are well known in this art. Typical sources of promoters include, for example, viruses, mammals, insects, plants, yeasts, and bacteria, and suitable promoters from these sources are readily available or can be synthesized based on sequences that are generally available from depositary institutions such as 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. Examples of inducible promoters include the Tet system (US Patent Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci., 93:3346-3351 (1996)), and T-REX. TM (Invitrogen, Carlsbad, CA), LACSWITCH TM This includes the tamoxifen-inducible recombinase 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” means a DNA sequence that increases transcription, such as a nucleic acid sequence that it is operably linked to. Enhancers can be located several 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 various different sources are well known in the art and are available as cloned polynucleotides or within cloned polynucleotides (e.g., from depositary institutions such as ATCC and other commercial or private sources). Many polynucleotides containing promoters (e.g., the commonly used CMV promoter) are also available as enhancer-containing polynucleotides. This includes columns. Enhancers can be located upstream, internally, or downstream of the coding sequence. The term "Ig enhancer" refers to an enhancer element derived from an enhancer region mapped within an immunoglobulin (Ig) locus (such enhancers include, for example, heavy chain (mu) 5' enhancer, light chain (kappa) 5' enhancer, kappa and mu Introduction Examples include 3' enhancers and 3' enhancers (see, in general, 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” means a nucleic acid sequence that enables cells expressing the nucleic acid sequence to be specifically selected or not selected in the presence of a corresponding selector. Appropriate selectable marker genes are known in this field, for example, in international patent applications 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. See also: 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. Patents No. 5,122,464 and 5,770,359.

[0041] In some embodiments, the vector is an “episome expression vector” or “episome,” which is replicable in host cells and, under appropriate selective pressure, persists as an extrachromosomal segment of DNA within the host cell (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available episome expression vectors - This includes Epstein-Barr virus-derived nuclear antigen 1. This includes, but is not limited to, episomal plasmids that utilize (EBNA1) and the Epstein-Barr virus (EBV) origin of replication (oriP). Invitrogen's (Carlsbad, CA) vectors pREP4, pCEP4, pREP7, and pcDNA3.1, as well as Stratagene's (La Jolla, CA) pBK-CMV, represent non-limiting examples of episomal vectors that use the T antigen and SV40 origin of replication instead of EBNA1 and oriP.

[0042] Other suitable vectors may be randomly incorporated into the host cell's DNA or expressed. The invention includes an integrating expression vector which may contain a recombination site that enables specific recombination between the vector and the host cell chromosome. Such an integrating expression vector enables the desired recombination. Endogenous expression regulatory sequences on host cell chromosomes can be utilized to induce protein expression. Examples of site-specific vectors include the flp-in system of Invitrogen (Carlsbad, CA) (e.g., pcDNA). TM 5 / FRT), or the cre-lox system (for example, Stratagene (La Jolla, Examples of components include those that can be found in the pExchange-6 Core Vectors of CA). Examples of vectors randomly incorporated into host cell chromosomes include, for example, Invitrogen (Carlsbad, CA) pcDNA3.1 (when introduced in the absence of the T antigen), and Promega (Madison, WI) pCI or pFN10A(ACT)FLEXI TM These are some examples.

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

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

[0045] As used herein, the term “host cell” means any type that may contain an expression vector. This refers to the host cell. The host cell may be a eukaryotic cell (e.g., plant, animal, fungus, or algae) or a prokaryotic cell (e.g., bacteria or protists). The host cell may be a cultured cell or a primary cell (i.e., directly isolated from an organism such as a human). The host cell may be an adherent cell or a suspension cell (i.e., a cell that grows in a suspension). Suitable host cells are known in this field, for example, DH5α E. coli cells, Chinese hamster cells. This includes ovarian cells, monkey VERO cells, COS cells, HEK293 cells, etc. Recombinant expression vectors For the purpose of amplifying or replicating - the host cell is a prokaryotic cell (e.g., DH5α cell) This may be the case. For the purpose of producing recombinant CARs, the host cell can be a mammalian cell. The host cells are preferably human cells. The host cells may be of any cell type, may originate from any tissue type, and may be at any developmental stage. In one embodiment, the host cells may be peripheral blood lymphocytes (PBLs), peripheral blood mononuclear cells (PMBCs), and natural killer cells (NK cells). ) Or it can be a T cell. Preferably, the host cell is a T cell. Methods for selecting suitable mammalian host cells, as well as methods for cell transformation, culture, amplification, screening and purification, are known in the art.

[0046] The present invention provides an isolated T cell that expresses a nucleic acid sequence encoding the CAR of the present invention described herein. The T cells of the present invention can be any T cell, for example, cultured T cells (such as primary T cells), or T cells derived from a cultured T cell line, or T cells obtained from a mammal. When obtained from a mammal, T cells can be obtained from a number of sources (including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids). T cells can also be enriched or purified. The T cells are preferably human T cells (for example, those isolated from humans). T cells can be of any developmental stage, including CD4 / CD8 double positive T cells, CD4 helper T cells (such as Th1 and Th2 cells), CD8 + / CD8 + double positive T cells, CD4 + helper T cells (such as Th1 and Th2 cells), CD8 + T cells (such as cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, etc., but are not limited thereto. In one embodiment, the T cell is a CD8 T cell or + is a CD4 T cell. 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 derivatives thereof. + T cell. 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 derivatives thereof. are included.

[0047] The nucleic acid sequence encoding the CAR of the present invention is "transfection", "transformation", or It can be introduced into cells by "transduction." As used herein, the terms "transfection," "transformation," or "transduction" mean the introduction of one or more exogenous polynucleotides into a host cell by physical or chemical means. Transfection techniques are known in this field, for example, calcium phosphate DNA coprecipitation. (See, for example, Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; e This includes rectoporation; cationic liposome-mediated transfection; tungsten particle-enhanced microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)). The phage or viral vector is packaged appropriately. After the infection particles have multiplied within Zing cells (many of which are commercially available), they can be introduced into host cells.

[0048] While not bound by any particular theory or mechanism, the CARs of the present invention are thought to provide one or more of the following by inducing an antigen-specific response to CD19: targeting and destruction of CD19-expressing cancer cells, reduction or elimination of cancer cells, and immunity to tumor sites(s). To promote the invasion of disease cells and enhance / expand the anti-cancer response. Therefore, the present invention promotes the invasion of malignant B cells A method of destruction, wherein one or more of the isolated T cells are subjected to a malignant B cell population expressing CD19. The present invention provides a method comprising contacting a group of cells, thereby producing CARs, which bind to CD19 on malignant B cells, and destroying the malignant B cells. As described above, treatment of B-cell malignancies typically involves chemotherapy, therapeutic monoclonal antibodies, and allogeneic stem cell transplantation; however, high recurrence rates are common in patients receiving such treatments. As described above, CD19 is highly expressed by malignant B cells (see, e.g., Nadler et al., above), and the method of the present invention can be used to treat any B-cell malignancy known in the art. Yes, it is possible. Malignant tumors of mature B cells include follicular lymphoma, mantle cell lymphoma, and Burk's disease. Trimphoma, multiple myeloma, diffuse large B-cell lymphoma, Hodgkin lymphoma, This includes, but is not limited to, plasmacytic lymphoma, marginal zone lymphoma, and chronic lymphocytic leukemia (Shaffer et al., see above).

[0049] One or more mononucleotides expressing the nucleic acid sequence encoding the anti-CD19 CAR of the present invention as described herein. The separated T cells can be brought into contact with a population of malignant B cells expressing CD19 ex vivo, in vivo, or in vitro. "Ex vivo" refers to contact in an artificial external environment that minimizes changes to natural conditions. This refers to methods performed within or on cells or tissues. In contrast, the term "in vivo" refers to methods performed within a living organism in a normal, intact state. On the other hand, the "in vitro" method is carried out using biological components isolated from the normal biological environment. The method of the present invention preferably involves ex vivo and in vivo components. In this regard, for example, the isolated T cells described above encode the anti-CD19 CAR of the present invention. Nucleic acid sequences can be cultured ex vivo under conditions for expression and then directly transferred to mammals (preferably humans) suffering from B-cell malignancies. The cell transplantation method is called "adoptive cell transfer (ACT)" in this field. In this transplantation, immune-induced cells are passively transplanted into a new recipient host, and the functionality of the immune-induced donor cells is transferred to the new host. B-cell malignancy Adoptive cell transplantation methods for treating various types of cancer, including hematological malignancies, are known in this field 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 mammals, the cells are allogeneic or autologous to the mammals. It is possible. In the "autologous" administration method, cells (e.g., hematopoietic stem cells or lymphocytes) are taken from a mammal, preserved (and optionally modified), and returned to the same mammal. In the "allogeneic" administration method, the 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] T cells are preferably administered to humans in the form of a composition such as a pharmaceutical composition. Alternatively, The nucleic acid sequence encoding the CAR of the present invention, or a vector containing the nucleic acid sequence encoding the CAR, can be formulated into a composition such as a pharmaceutical composition and administered to humans. The pharmaceutical composition of the present invention may contain a population of T cells expressing the CAR of the present invention. The nucleic acid sequence encoding the CAR of the present invention, or In addition to host cells expressing the CAR of the present invention, the pharmaceutical composition may contain other pharmaceutically active agents or drugs. The substance may include, for example, chemotherapeutic agents such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In a preferred embodiment, the pharmaceutical composition includes isolated T cells expressing the CAR of the present invention, more preferably a population of T cells expressing the CAR of the present invention.

[0052] The T cells of the present invention may be provided in the form of a salt (e.g., a pharmaceutically acceptable salt). Pharmacopoecitable acid addition salts include mineral acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid). This includes salts derived from nitrates and sulfuric acids, 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 aryl sulfonic acids such as p-toluenesulfonic acid).

[0053] The choice of carrier depends on the specific CAR of the present invention, the nucleic acid sequence encoding the CAR, the vector, or the CAR. The host cell expressing the CAR, the nucleic acid sequence encoding the CAR, the vector, or the host cell expressing the CAR, is determined in part by the host cell expressing the CAR. Therefore, there are a variety of suitable formulations of the pharmaceutical composition of the present invention. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives include, for example, methylparaben, p Examples include parabens, sodium benzoate, and benzalkonium chloride. Optionally, a mixture of two or more preservatives may be used. Typical preservatives or mixtures thereof are... It is present in amounts ranging from approximately 0.0001% to approximately 2% by weight of the total composition.

[0054] Furthermore, buffering agents 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 It is typically present in an amount ranging from about 0.001% to about 4% by weight of the total composition.

[0055] Methods for preparing administerable (e.g., parenterally administerable) 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 includes a CAR, a nucleic acid sequence encoding a CAR, a vector, or a host cell expressing a CAR. The composition can be formulated as an inclusion complex (e.g., a cyclodextrin inclusion complex) or liposomes. The liposomes can be used to infect host cells (e.g., T cells or NK cells) in specific tissues. Liposomes can function to target the nucleic acid sequences of the present invention. Liposomes can also be used to increase the half-life of the nucleic acid sequences 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. Patents No. 4,235,871, No. 4,501,728, No. 4,837,028 and No. 5,019,369.

[0057] The compositions may utilize time-released, delayed-release, and sustained-release delivery systems so that the delivery of the compositions of the present invention occurs with sufficient time to induce sensitization before sensitization of the site to be treated. A type of release delivery system is available and known to those skilled in the art. Such a system can avoid repeated administration of the composition, thereby improving the convenience of the subject and the physician, and may be particularly suitable for certain embodiments of the composition of the present invention.

[0058] The composition is preferably in an amount effective for treating or preventing B-cell malignancies, according to the present invention. A host cell expressing a nucleic acid sequence encoding a CAR, or a vector containing such a nucleic acid sequence. -Includes. As used herein, the terms “treatment,” “to treat,” etc., mean obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures a disease and / or adverse symptoms resulting from the disease. For this purpose, the method of the present invention involves host cells expressing the CAR of the present invention, The procedure involves administering a "therapeutably effective amount" of a composition containing a vector that includes a nucleic acid sequence encoding CAR. This includes [details omitted]. “Therapeutic dose” means the effective amount in the required dosage and duration to achieve the desired therapeutic outcome. The therapeutic dose may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of CAR to elicit the desired response in the individual. Example For example, the therapeutically effective amount of CAR of the present invention binds to CD19 on multiple myeloma cells and destroys them. It's the amount that will be destroyed.

[0059] Alternatively, the pharmacological and / or physiological effects may be prophylactic, that is, the effect may completely or partially prevent the disease or its symptoms. In this regard, the method of the present invention includes administering a “prophylactically effective amount” of a composition comprising host cells expressing the CAR of the present invention, or a vector comprising a nucleic acid sequence encoding the CAR, to a mammal susceptible to B-cell malignancies. "Prophylactically effective dose" refers to the amount that is effective in the required dosage and duration to achieve the desired preventive outcome (e.g., prevention of disease onset).

[0060] Typical amounts of host cells administered to mammals (e.g., humans) can range from, for example, 1 million to 100 billion cells; however, amounts less or more than this exemplary range may also be used. It is within the range of clarity. For example, the daily dose of the host cells of the present invention is approximately 1 million From approximately 50 billion cells (for example, approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 50 (100 million cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or a range defined by any two of the above values), preferably approximately 10 million to approximately 100 billion cells (for example, approximately 20 million cells, approximately 30 million cells, approximately 40 million cells, approximately 60 million cells, approximately 70 million cells, approximately 80 million cells, approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or any of the above values) (A range defined by any two of the above), more preferably from about 100 million cells to about 50 billion cells (for example) , about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells This could be 10,0

[0061] The effectiveness of treatment or prevention can be monitored by periodic evaluation of the treated patient. Depending on the condition, treatment may be repeated for several days or longer until the desired suppression of symptoms occurs. However, other administration regimens may be useful and are also within the scope of the present invention. The desired dose 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] A composition comprising host cells expressing the CAR of the present invention, or a vector comprising a nucleic acid sequence encoding the CAR, can be administered to mammals using standard administration techniques (including oral, intravenous, intraperitoneal, subcutaneous, intrapulmonary, transdermal, intramuscular, intranasal, oral, sublingual, or suppository administration). The composition is preferably suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to mammals using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0063] A composition comprising host cells expressing the CAR of the present invention, or a vector containing a nucleic acid sequence encoding the CAR, can be administered together with one or more further therapeutic agents, which are co-administered to mammals. It is possible. "Coadministering" means that the CAR of the present invention is used to treat one or more further therapies. The effect of the agent can be enhanced, or one or more further therapeutic agents can enhance the effect of the CAR of the present invention, by combining one or more further therapeutic agents with the host cells of the present invention or the present invention at a sufficiently close time. This means administering a composition containing a vector. In this regard, the host cells of the present invention or a composition containing a vector of the present invention may be administered first, and one or more further therapeutic agents may be administered second, or one or more further therapeutic agents may be administered first, and the host cells of the present invention or A composition containing the vector of the present invention can be administered secondly. Alternatively, the host cells or the present invention can be administered secondly. A composition containing the vector of the invention and one or more further therapeutic agents can be administered simultaneously. An exemplary therapeutic agent that can be co-administered with a host cell of the present invention or a composition containing the vector of the present invention is IL-2.

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

[0065] Those skilled in the art will readily understand that the CAR of the present invention can be modified in any number of ways to increase the therapeutic or preventive efficacy of the CAR by modification. For example, the CAR can be directly modified The compound (e.g., CAR) can be conjugated to the targeting moiety either directly or indirectly via a linker. The practical aspects are well known in this field. See, for example, Wadwa et al., J. Drug Targeting 3:111 (1995) and U.S. Patent No. 5,087,616.

[0066] The following embodiments further illustrate the present invention, but should not be construed as limiting its scope. [Examples]

[0067] Example 1 This example demonstrates a method for producing anti-CD19 chimeric antigen receptors (CARs) according to the present invention.

[0068] A series of anti-CD19 CARs were designed and synthesized. All CARs were either mouse monoclonal antibody FMC63 (Nicholson et al., Molecular Immunology, 34(16-17):1157-1165(1997)) or fully human monoclonal antibody 47G4 (US Patent Application No. 2010 / 0104509). The CARs contained an antigen recognition domain consisting of a single-stranded variable fragment (scFv) derived from one of the following: ) . CARs are derived from the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor The CARs included signal sequences derived from human CD8 molecules or other human signaling sequences. The CARs included combinations of two or more intracellular T cell signaling domains (or "co-stimulatory domains") derived from human CD3 zeta (CD3ζ) molecules, human CD28 molecules, human 4-1BB molecules, human CD27 molecules, and / or FcεRI gamma chains.

[0069] More specifically, the CAR contains FMC63-derived scFv, GM-CSF receptor signaling sequence, CD8 extracellular and transmembrane components, and the intracellular T cell signaling domains of human CD3ζ and CD28 molecules. The plasmid to be used (indicated as FMC63-CD828Z) is used as the starting material, and plasmid MSGV-FMC63-28Z (Kochenderfer et al., Journal of Immunotherapy, 32(7):689-702(2009) is described It was constructed using (listed). First, the MSGV-FMC63-28Z plasmid was converted to restriction enzyme NotI and BmgBI (New The plasmid was cut at England Biolabs, Ipswich, MA, and the entire CD28 portion was removed. Next, a portion of the extracellular region and all of the transmembrane region of the human CD8 molecule, and the cytoplasmic region of the CD28 molecule. DNA fragments (synthesized by Invitrogen, Carlsbad, CA) encoding the cytoplasmic portion of the CD3ζ molecule were ligated onto a cleaved MSGV-FMC63-28Z plasmid. The sequences of human CD8, CD28, and CD3ζ were obtained from the National Center for Biotechnology Information. The data was obtained from bsite. Guidance on each molecular part to be included in CARs was obtained from Kochenderfer et al., Journal of Immunotherapy, 32(7):689-702 (2009).

[0070] Fully human anti-CD19 CARs were created using the sequence of a fully human 47G4 monoclonal antibody (described in U.S. Patent Application No. 2010 / 0104509). The 47G4 antibody is a human kappa light chain It was produced by vaccinating KM strain mice carrying the transgene and human heavy chain transchromosome. The sequences of the light chain and heavy chain variable region of the 47G4 antibody were obtained from U.S. Patent Application Publication No. 2010 / 0104509. A 47G4 scFv was designed containing the following elements from 5' to 3': CD8 signal sequence, 47G4 antibody light chain variable region, linker peptide containing the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 14) (see Cooper et al., Blood, 101(4):1637-1644 (2003)), and 47G4 The antibody heavy chain variable region. Next, a DNA sequence encoding a CAR containing the following components from 5' to 3' was designed: the 47G4 scFv mentioned above, a portion 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 represented as 47G4-CD828Z and the sequence was synthesized using Invitrogen (Carlsbad, CA).

[0071] Using standard methods, the pRRLSIN.cPPT.MSCV.coDMF5.oPRE lentivirus 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 mentioned above. The resulting plasmid The code is represented as LSIN-47G4-CD8CD28Z.

[0072] A plasmid denoted as MSGV-47G4-CD8BBZ was constructed by modifying the above MSGV-FMC63-CD828Z plasmid using standard methods. The MSGV-47G4-CD8BBZ plasmid encodes a CAR (denoted as 47G4-CD8BBZ) containing the following from 5' to 3': the above 47G4 scFv, part of the extracellular domain and all of the transmembrane domain of the human CD8 molecule, part 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] By replacing the CD28 sequence of plasmid MSGV-FMC63-CD828Z with the same 4-1BB sequence as that contained in MSGV-47G4-CD8BBZ, a CAR is encoded represented as FMC63-CD8BBZ CAR. I constructed a mid-range device (represented as MSGV-FMC63-CD8BBZ).

[0074] The DNA encoding 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 excising the DNA encoding FMC63 scFv to form MSGV-SP6-CD828Z, which recognizes the hapten 2,4,6-trinitrobenzenesulfonic acid and served as a negative control in several experiments.

[0075] All anti-CD19 CARs produced using the above method are listed in Table 1.

[0076] [Table 1]

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

[0078] Example 2 This embodiment demonstrates a method for producing T cells that express nucleic acid sequences encoding the CARs of the present invention. ru.

[0079] The above CARs are encoded by a replication-incompetent gamma retrofit. Viruses or lentiviruses were generated and used to transduce T cells. To temporarily produce gamma retrovirus, plasmids encoding the CARs described in Example 1 were added to 293GP packaging cells (Burns et al., Proc. Natl. Acad. Sci., USA, 90(17):8033-8037(1993)), and plasmids encoding the RD114 envelope protein were added. Together with D (Porter et al., Human Gene Therapy, 7(8):913-919(1996)), LIPOFE CTAMINE TM Transfected using 2000 (Life Technologies, Carlsbad, CA). Transfected cells were incubated in antibiotic-free D10 medium at 37°C for 6-8 hours. Next, the medium to be used for transfection was placed with fresh D10 medium. The cells were changed and incubated for a further 36–48 hours. During and after transfection, 293GP cells were cultured on poly-D-lysine coated dishes (BD Biosciences, San Jose, CA). The supernatant containing the retrovirus was removed from the dishes and centrifuged to remove cell debris. The supernatant was stored at -80°C.

[0080] The supernatants containing lentiviruses encoding each of the CARs described in Example 1 were prepared 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 anti-CD3 monocytoplasmic sperm injection was performed. In T cell medium containing ronal antibody OKT3 (Ortho, Bridgewater, NJ) and 300 IU / mL of IL-2, 1 × 10 6 PBMCs were suspended at a concentration of cells / mL. 20 mL of this supernatant was then transferred to 75 cm. 2 The solution was added to a culture flask (Corning, Corning, NY). The flask was cultured upright at 37°C in 5% CO2 (see, for example, Kochenderfer et al., Journal of Immunotherapy, 32(7):689-702 (2009)).

[0082] Gamma-retroviral transduction of T cells was initially performed using RETRONECTIN. TM This procedure was performed by dissolving (Takara / Clontech Laboratories, Mountain View, CA) in PBS at a concentration of 10 g / 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, RETRONECTIN TM The solution is aspirationed, and a blocking solution (2 mL) consisting of Hanks' balanced salt solution (HBSS) with 2% bovine serum albumin (BSA) is added to each RETRONECTIN TM Co The solution was added to the tingwell. The plate was incubated at room temperature for 30 minutes. The blocking solution was aspirationed and rinsed with HBSS + 2.5% HEPES solution. The gamma retrovirus supernatant was rapidly thawed and diluted 1:1 in T cell medium. Then, the diluted supernatant (2 mL) was added to each RETRONECTIN TM It was added to the coating wells.

[0083] After adding the supernatant, the plate was centrifuged at 32°C for 2 hours at 2000 × g. Then, the supernatant was aspirationed from the wells and cultured with OKT3 and IL-2 for 2 days (2 × 10⁶). 6 T cells were added to each well. When adding the T cells to the retrovirus-coated plate, they were added to T cell medium containing 300 IU / mL of IL-2 at a concentration of 0.5 × 10⁶ per mL. 6 The cells were suspended at their concentration. After adding T cells to each well, the plate was centrifuged at 1000 × g for 10 minutes and incubated overnight at 37°C. After incubation for 24-30 hours, the T cells were removed from the plate and placed in fresh T cell medium with 300 IU / mL of IL-2, at a concentration of 0.5 × 10⁶ per mL. 6 Suspend at the cell concentration and store at 37°C in 5% CO2. It was cultured.

[0084] To induce lentiviral transduction of T cells, activated PBMCs were suspended in lentiviral supernatant supplemented with protamine sulfate and 300 IU / mL of IL-2. The cells were centrifuged at 1200×g for 1 hour. Subsequently, the cells were cultured at 37°C for 3 hours. The supernatant was then analyzed using RPMI (Mediatech, Inc., Manassas, VA). The cells were diluted 1:1 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and IL-2. The cells were cultured overnight in the diluted supernatant, and then returned to the culture medium 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 with FACs buffer (phosphate-buffered saline (PBS) with 0.1% sodium azide and 0.4% BSA) and suspended. To detect FMC63 scFv, biotin-labeled phosphate buffer was used. Liclonal 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 packed into FACs. The samples were suspended in fur and blocked with normal mouse IgG (Invitrogen, Carlsbad, CA). Next, the cells were stained with phycoerythrin (PE)-labeled streptavidin (BD Pharmingen, San Diego, CA), anti-CD4, anti-CD8, and anti-CD3. Flow cytometry was performed using LSR II. The procedure was performed using a flow cytometer (BD Biosciences), and the analysis was carried out using FlowJo software (Treestar, Inc. Ashland, OR). The 47G4 base on transduced T cells was analyzed. The expression of CARs was evaluated 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 anti-Fab antibody or protein. The percentage of T cells in CAR transduced cultures stained with Fab or Protein L was calculated by subtracting the percentage of untransduced T cells from the same donor and cultured in the same manner, stained with Anti-Fab or Protein L.

[0087] Percentage of T cells expressing CARs containing scFv derived from mouse FMC63 antibody on day 7 of culture. The results 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 gamma retroviruses 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 the 47G4-CD828Z CAR.

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

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

[0091] Patient samples and cell lines In the National Cancer Institute (NCI) Surgery Branch, patients with melanoma, chronic lymphocytic leukemia (CLL), or lymphoma who were registered in an Institutional Review Board (IRB) approved protocol were able to obtain non-leukemia. Pathogenic (non-leukemic) PBMC samples were obtained. Cells from five different patients were used. Donor 1 has CLL, Donor 2 is a normal donor, and Donors 3 and 5 both have lymphoma. Donor 4 had melanoma. 90% DMSO (Sigma, St. Louis, MO) was added. PBMCs were cryopreserved during %FBS. In experiments using primary CLL cells as target cells, CLL was... Unmanipulated patient-derived PBMCs were used. The following CD19-expressing immortalized cells were used. Cellular strains used: NALM-6 (acute lymphoblastic leukemia, DSMZ, Braunschweig, Germany) and CD19-K562. The following CD19-negative cell lines were used: A549 (lung cancer, from ATCC), CCRF-CEM (T-cell leukemia, from ATCC), MDA231 (breast cancer, from ATCC), and TC71 (Ewing's sarcoma, provided by Dr. M. Tsokos, National Cancer Institute, Bethesda, MD). All cell lines were maintained in R10 medium. When using CLL PBMCs as the target in the assay, The cells were cultured in R10 medium for 12-18 hours prior to the start of the experiment.

[0092] Enzyme-linked immunosorbent assays (ELISA) of interferon and TNF In clinical trials, the occurrence of hypotension and other toxicities in patients who received T cell injections expressing CAR FMC63-28Z demonstrated the effects of TNF production by T cells expressing the CARs of the present invention and the release of FMC63-28Z. This prompted a comparison with TNF production by expressed T cells.

[0093] The target cells were washed with IL-2-free T cell medium, and 1 × 10⁶ cells were washed per 1 mL. 6 Suspended in cells. Each target cell 100,000 target cells of the cellular type were placed in a 96-well round-bottom plate (Corning, Tewksbury, MA). The solution was added to each of the two wells. Wells containing T cells alone were also prepared. The plates were incubated at 37°C for 18-20 hours. After incubation, IFN was added using a standard method. γ or TNF ELISA assays were performed (Pierce, Rockford, IL). In some experiments, TNF ELISA results were normalized by dividing the TNF level by the percentage of T cells in the overnight culture 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 CAR and FMC63-CD8BBZ CAR, as shown in Figures 4A and 4B. The only difference between FMC63-28Z CAR and FMC63-CD828Z CAR is that FMC63-28Z uses extracellular and transmembrane components derived from human CD28, while FMC63-CD828Z uses extracellular and transmembrane components derived from human CD8 protein. This involved replacing the original component. The significant differences between FMC63-28Z and FMC63-CD828Z in T cell persistence and inflammatory cytokine production led to the subsequent use of CD8 extracellular spacers and transmembrane components in CAR design.

[0095] As shown in Tables 2 and 3 (all units are pg / mL IFNγ), T cells transduced with anti-CD19 CARs, when cultured overnight with the CD19-expressing cell line CD19-K562, produced a large amount of IFN. Although it produced γ, when cultured with a negative control cell line, CAR was phenotypic. The introduced T cells produced only background levels of IFNγ. The results of 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 in 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 together with CD19-negative NGFR-K562 and CCRF-CEM cells... This resulted in the induction of much lower levels of IFNγ. T cells transduced with FMC63-CD827Z 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 contained CD19-K562 cells, one tube contained unprocessed primary CLL cells, and the other... The tubes contained NGFR-K562 cells. In some experiments, the CD19-K562 tubes were omitted. All tubes contained the above anti-CD19 CARs transduced T cells, 1 mL of AIM V TM Culture medium (Life Technologies, Carlsbad, CA) + 5% human serum, titrated concentration The sample contained an anti-CD107a antibody (eBioscience, Inc., San Diego, CA; clone eBioH4A3) and 1 μL of Golgi Stop (BD Biosciences, Franklin Lakes, NJ). All tubes were stored at 37°C. The cells were incubated for 4 hours, and then stained for CD3, CD4, and CD8 expression.

[0100] T cells from different target sources expressing CARs FMC63-CD828Z, FMC63-CD827Z, FMC63-CD8BBZ, 47G4-CD827Z, 47G4-CD82827Z, 47G4-CD827BBZ, or 47G4-CD8BBZ specifically upregulate CD107a in response to stimulation at CD19-expressing target cells. The results of CD107a assays for 47G4-CD827Z, 47G4-CD82827Z, and 47G4-CD827BBZ CARs are shown in Figures 6A-6D. This indicates the occurrence of CD19-specific T cell degranulation, which is a necessary condition for perforin-mediated cytotoxicity (e.g., See Rubio et al., Nature Medicine, 9(11):1377-1382 (2003).

[0101] Growth assay We evaluated the ability of T cells transduced with anti-CD19 CARs to proliferate when stimulated by CD19-expressing target cells. Specifically, 0.5 × 10⁶ 6 Individual irradiated CD19-K562 cells, or 0.5 × 10 6 Irradiated NGFR-K562 cells were transduced with anti-CD19 CAR to 0.75 × 10⁶ cells. 6The cells were co-cultured with whole T cells. The T cells were labeled with carboxyfluorescein diacetate succinimimidyl ester (CFSE) (Life Technologies, Carlsbad, CA) as described in Mannering et al., J. Immunological Methods, 283(1-2):173-183 (2003). The culture medium used for co-culture was AIM V TM Culture medium (Life Technologies, Carlsbad, CA) + 5% human AB serum That was the case. IL-2 was not added to the culture medium. Four days after the start, to eliminate dead cells, Using PanBlue, count the viable cells in each co-culture and perform a flow 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 more than when cultured with negative control NGFR-K562 cells. When cultured with cells, it showed significant dilution of CFSE. These results suggest that anti-CD19 CARs form This study showed that transduced T cells specifically proliferated when stimulated by CD19-expressing target cells. vinegar.

[0103] The results of this example show that T cells expressing the CARs of the present invention produce CD19-specific cytokines. This demonstrates that the organism exhibits degranulation and proliferation.

[0104] Example 4 This embodiment 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 disrupt CD19-expressing, unmodified PBMCs derived from patients with CLL, a cytotoxicity assay was performed. Specifically, the cytotoxicity of target cells was analyzed using, for example, Kochenderfer et al., J. Immunotherapy. 32(7):689-702(2009) and Hermans et al., J. Immunological Methods, 285(1) Using the assay described in 25-40 (2004), etc., negative control CCRF-CEM cells The survival of CD19-expressing target cells (i.e., CLL PBMCs) was measured by comparing their survival to that of other cells.

[0106] CCRF-CEM cells were placed in R10 medium at a rate of 1.5 × 10⁶ 6 Suspend at a concentration of cells / mL and use the fluorescent dye 5-(and -6)- (((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 in 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 placed in PBS + 0.1% BSA at a rate of 1 × 10⁶ 6 Suspended at cell / mL. Fluorescent dye carboxyfluorescein diacetate succinimimidyl ester (CFSE) (Life Technologies, Carlsbad, CA) The substance was added to this cell suspension at a concentration of 1 M. The cells were incubated at 37°C for 10 minutes. After incubation, the labeling reaction is performed by adding an equal volume of FBS to the cell suspension. The process was stopped and the cells were incubated at room temperature for 2 minutes. Then, the cells were placed in cytotoxic medium. It was washed and suspended.

[0107] Approximately 50,000 CD19-expressing CLL PBMCs and 50,000 CCRF-CEM cells were subjected to various numbers of CARs. The transduced T cells and the effector 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 or not transduced with the SP6-28Z control CAR. Co-cultures were constructed in two rows in sterile 5 mL test tubes (BD Biosciences, Franklin Lakes, NJ) with the following T cell:target cell ratios: 20:1, 6.7:1, 2.2, and 0.7:1. The 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 was performed using BD FacsCanto. The analysis was performed using II (BD Biosciences). Analysis was performed using FlowJo Software (Treestar, Inc. Ashland, OR). The analysis was gated to 7AAD-negative (surviving) cells, and the percentages of surviving CLL target cells and surviving CCRF-CEM-negative control cells were measured. This was determined for each T cell + target cell culture.

[0108] For each culture, the survival rate of CLL PBMCs was determined by dividing the proportion of viable CLL PBMCs by the proportion of viable CCRF-CEM negative control cells. The corrected CLL PBMC survival rate was calculated by dividing the CLL PBMC survival rate by the ratio of CLL target cells to CCRF-CEM negative control cells in a tube containing only CLL target cells and CCRF-CEM negative control cells (without any effector T cells). This correction was necessary to account for variability in starting cell numbers and spontaneous target cell death. Cytotoxicity was calculated as CLL PBMC cytotoxicity = 100 - corrected CLL PBMC survival rate. For all effector:target ratios, cytotoxicity was determined in two series and the results were averaged.

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

[0110] Example 5 This embodiment demonstrates that T cells expressing the anti-CD19 CAR of the present 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. After 6 days, palpable tumors formed, and then injected with either the MSGV-FMC63-28Z CAR vector (described in Kochenderfer et al., Journal of Immunotherapy, 32(7):689-702 (2009)) or LSIN-47G4-CD8CD. Human T cells transduced with one of the 28Z CAR vectors (described in Example 1) were subjected to a single IV drip. Mice were treated by applying a solution. Tumors were measured every 3 days, and tumor size was compared to that of untreated mice. It was compared to a ulcer.

[0112] The results of this embodiment shown in Figure 9 show that either FMC63-28Z CAR or 47G4-CD8CD28Z CAR is expressed. This study demonstrates that T cells significantly reduced tumor size in treated mice.

[0113] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as they are incorporated herein in whole, with each reference being individually and specifically indicated as being incorporated herein by reference.

[0114] In relation to the description of the present invention (in particular with respect to the following claims), the terms "a" and "an" The use of "the" and "at least one" and similar referents should be interpreted as covering both singular and plural forms unless otherwise specified herein or clearly inconsistent with the context. The use of the term "at least one" after an enumeration of one or more items (e.g., "at least one of A and B") should be interpreted as covering both singular and plural forms unless otherwise specified herein or clearly inconsistent with the context. Unless otherwise specified, one item (A or B) will be selected from the listed items, or the listed items The term "includes" should be interpreted as meaning any combination of two or more of the above (A and B). "(comprising)", "having", "including", and "containing" are open-ended terms unless otherwise noted (i.e., "~including, but those) It should be interpreted as meaning "not limited to." Unless otherwise specified herein, descriptions of value ranges are intended solely as a way of referring individually to each individual value that falls within that range, and each individual value is incorporated herein as if it were described individually herein. All methods described herein may be carried out in any appropriate order unless otherwise specified herein or if it is clearly inconsistent with the context. The use of any and all example or illustrative terms provided herein (e.g., "such as") is prohibited. This is intended solely to facilitate understanding of the present invention and does not impose any limitations on the scope of the invention unless otherwise claimed. All terms herein should not be construed as indicating any unclaimed element as essential to the practice of the invention.

[0115] Preferred embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art by reading the above description. The inventors anticipate that such variations will be used as appropriate by those skilled in the art, and they intend that the Invention may be carried out in ways different from those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations thereof is encompassed by the Invention unless otherwise specifically noted herein or clearly inconsistent with the context.

Claims

1. A chimeric antigen receptor (CAR) for CD19, wherein the CAR consists of the amino acid sequence of SEQ ID NO:

1.

2. A chimeric antigen receptor (CAR) comprising amino acids 267-502 of the amino acid sequence of SEQ ID NO: 1 and the antigen-binding domain of a human anti-CD19 antibody.

3. A chimeric antigen receptor (CAR) comprising the following: (a) The antigen-binding domain of a human anti-CD19 antibody, comprising amino acids 22-266 of the amino acid sequence of Sequence ID No. 1; (b) Extracellular spacer; (c) Transmembrane domain; and (d) Intracellular T cell signaling domain derived from human CD3ζ molecule.

4. The CAR according to claim 3, wherein the extracellular spacer comprises a human CD8α hinge.

5. The CAR according to claim 4, wherein the human CD8α hinge comprises amino acids 267-321 of SEQ ID NO:

1.

6. The CAR according to any one of claims 3 to 5, wherein the transmembrane domain is derived from a human CD8α molecule.

7. The CAR according to claim 6, wherein the transmembrane domain comprises amino acids 322 to 342 of SEQ ID NO:

1.

8. The CAR according to any one of claims 3 to 7, wherein the intracellular T cell signaling domain derived from the human CD3ζ molecule comprises amino acids 391 to 502 of SEQ ID NO:

1.

9. The CAR according to any one of claims 3 to 8, wherein the CAR further comprises an intracellular T cell signaling domain derived from a human CD28 molecule.

10. The CAR according to claim 9, wherein the intracellular T cell signaling domain derived from the human CD28 molecule comprises amino acids 351-390 of SEQ ID NO:

1.

11. A nucleic acid encoding a CAR according to any one of claims 1 to 10.

12. The nucleic acid according to claim 11, wherein the nucleic acid is present in a vector containing the nucleic acid.

13. A cell comprising the nucleic acid according to claim 11 or 12.

14. The cell according to claim 13, wherein the cell is a peripheral blood lymphocyte (PBL), a peripheral blood mononuclear cell (PMBC), a natural killer (NK) cell, or a T cell.

15. The cell according to claim 13, wherein the cell is a T cell.

16. The cell according to claim 13, wherein the cell is an NK cell.

17. A pharmaceutical composition comprising the cells described in any one of claims 13 to 16 and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17 for preventing or treating B-cell malignant tumors.

19. A method for in vitro producing cells expressing a CAR, wherein the method comprises introducing a vector or nucleic acid encoding a CAR according to any one of claims 1 to 10 into the cells.

20. The method according to claim 19, wherein the cells are T cells or NK cells.

21. An in vitro method for determining the activity of T cells expressing the CAR according to any one of claims 1 to 10, comprising the following steps: (a) A step of bringing the T cells into contact with CD19-expressing cells; (b) A step of measuring the cytotoxicity of CD19-expressing cells that have undergone step (a); and (c) A step of comparing the cytotoxicity of CD19-expressing cells measured in step (b) with the cytotoxicity of negative control CD19-expressing cells measured in the presence of T cells that do not express the CAR.

22. An in vitro method for determining the specificity of T cells expressing the CAR according to any one of claims 1 to 10, comprising the following steps: (a) A step of bringing the T cells into contact with CD19-expressing cells; (b) A step of measuring IFNγ and / or CD107a production by T cells that have undergone step (a); and (c) A step of comparing the IFNγ and / or CD107a production measured in step (b) with the IFNγ and / or CD107a production measured in a negative control in CD19-negative cells.

Images